©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Proteolytic Pathway That Recognizes Ubiquitin as a Degradation Signal (*)

(Received for publication, March 27, 1995; and in revised form, May 11, 1995)

Erica S. Johnson (§) Philip C. M. Ma (¶) Irene M. Ota (**) Alexander Varshavsky (§§)

From the Division of Biology, California Institute of Technology, Pasadena, California 91125

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous work has shown that a fusion protein bearing a ``nonremovable'' N-terminal ubiquitin (Ub) moiety is short-lived in vivo, the fusion's Ub functioning as a degradation signal. The proteolytic system involved, termed the UFD pathway (Ub fusion degradation), was dissected in the yeast Saccharomyces cerevisiae by analyzing mutations that perturb the pathway. Two of the five genes thus identified, UFD1 and UFD5, function at post-ubiquitination steps in the UFD pathway. UFD3 plays a role in controlling the concentration of Ub in a cell: ufd3 mutants have greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants can be restored by overexpressing Ub. UFD2 and UFD4 appear to influence the formation and topology of a multi-Ub chain linked to the fusion's Ub moiety. UFD1, UFD2, and UFD4 encode previously undescribed proteins of 40, 110, and 170 kDa, respectively. The sequence of the last 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation involves E6AP. UFD5 is identical to the previously identified SON1, isolated as an extragenic suppressor of sec63 alleles that impair the transport of proteins into the nucleus. UFD5 is essential for activity of both the UFD and N-end rule pathways (the latter system degrades proteins that bear certain N-terminal residues). We also show that a Lys Arg conversion at either position 29 or position 48 in the fusion's Ub moiety greatly reduces ubiquitination and degradation of Ub fusions to -galactosidase. By contrast, the ubiquitination and degradation of Ub fusions to dihydrofolate reductase are inhibited by the Ub but not by the Ub moiety. ufd4 mutants are unable to ubiquitinate the fusion's Ub moiety at Lys, whereas ufd2 mutants are impaired in the ubiquitination at Lys. These and related findings suggest that Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains involve not only the previously identified Lys but also Lys of Ub, and that structurally different multi-Ub chains have distinct functions in Ub-dependent protein degradation.


INTRODUCTION

In eukaryotes, a large fraction of intracellular proteolysis is mediated by pathways whose common feature is the covalent conjugation of ubiquitin (Ub)()to short-lived proteins prior to their degradation by the proteasome, a multisubunit, multicatalytic protease. The coupling of Ub to proteins is catalyzed by a family of Ub-conjugating (E2) enzymes. At least some of these enzymes function in complexes with proteins of a distinct class, termed recognins, E3s, or ubiquitin ligases (reviewed by Pickart(1988), Rechsteiner(1991), Finley(1992), Jentsch(1992), Goldberg and Rock (1992), Hochstrasser(1992), Varshavsky(1992), Hershko and Ciechanover(1992), Vierstra(1993), Parsell and Lindquist(1993), and Ciechanover(1994)). The substrate-conjugated Ub acts as an accessory (or ``secondary'') degradation signal (degron), in that its post-translational coupling to the substrate protein is mediated by sequence and conformational features of the substrate that act as an initial (or ``primary'') degradation signal.

A Ub-protein conjugate contains an isopeptide bond between the C-terminal Gly of Ub and the -amino group of a lysine in the substrate protein. Ubiquitination of a substrate often yields a substrate-linked multi-Ub chain, in which the C-terminal glycine of one Ub moiety is conjugated to an internal lysine of the adjacent Ub moiety, resulting in a chain of Ub-Ub conjugates. In the initially characterized multi-Ub chains, only Lys of a Ub moiety was found to be linked to another Ub moiety within a chain (Chau et al., 1989; Finley, 1992). More recently, Ub-Ub linkages mediated by other (Lys and Lys) lysines of Ub have been described as well (Spence et al., 1995; Arnason and Ellison, 1994; Haas et al., 1991).

Unlike branched Ub-protein conjugates, which are formed post-translationally, linear Ub adducts are formed as the translational products of natural or engineered Ub gene fusions (Bachmair et al., 1986; Finley et al., 1987, 1989). In eukaryotes, such fusion proteins, for example, Ub-X--galactosidase (Ub-X-gal), are rapidly cleaved at the Ub-protein junction by Ub-specific processing proteases, unless the junctional residue X is proline (P), in which case the rate of deubiquitination is greatly reduced (Bachmair et al., 1986). The X-gal proteins produced in vivo by deubiquitination of the other 19 Ub-X-gals are either long-lived or metabolically unstable, depending on the identity of their N-terminal residue X (Bachmair et al., 1986; Gonda et al., 1989; Tobias et al., 1991). The relation between the in vivo half-life of a protein and the identity of its N-terminal residue is referred to as the N-end rule (reviewed by Varshavsky(1992)). The N-end rule-based degradation signal, termed the N-degron, comprises a destabilizing N-terminal residue and an internal lysine (or lysines) of a substrate (Bachmair and Varshavsky, 1989; Johnson et al., 1990; Hill et al., 1993; Dohmen et al., 1994). This Lys residue is the site of formation of a multi-Ub chain (Chau et al., 1989).

Deubiquitination of a Ub fusion is completely inhibited if the C-terminal Gly of its Ub moiety is converted into another residue, for example, Ala or Val (Butt et al., 1988; Johnson et al., 1992). In the yeast Saccharomyces cerevisiae, a Ub fusion such as Ub-P-gal or Ub-V-gal (Fig. 1) is rapidly degraded (t of 4 min at 30 °C) by a Ub-dependent system termed the UFD pathway (Ub fusion degradation). The targeting of a Ub fusion by the UFD pathway results in multiubiquitination of the fusion's ``nonremovable'' Ub moiety, a step required for the fusion's subsequent degradation by the proteasome (Johnson et al., 1992). At least the initial steps of the UFD pathway are distinct from those of the N-end rule pathway: mutational elimination of N-recognin, the recognition component of the latter pathway, abolishes the degradation of N-end rule substrates but does not impair the degradation of Ub fusions such as Ub-V-gal (Bartel et al., 1990; Johnson et al., 1992). Physiological substrates of the UFD pathway are unknown. It is also unknown whether these substrates are largely proteins that bear Ub-like domains, or whether the targeting of a Ub moiety by the UFD pathway is an epiphenomenon, the consequence of a step in a Ub-dependent pathway that involves recognition of Ub moieties within a post-translationally added multi-Ub chain. If so, physiological substrates of the UFD pathway may lack Ub-like domains.


Figure 1: Test proteins. Fusions used in this work contained some of the following elements (see ``Experimental Procedures''): (i) the wt Ub moiety (constructs I and XI); (ii) a mutant Ub moiety bearing Val instead of Gly at the C-terminal position 76 (constructs II, VI, and X); (iii) a mutant Ub moiety bearing Val and Arg instead of wt Gly and Lys (constructs III and VII); (iv) a mutant Ub moiety bearing Val and Arg instead of wt Gly and Lys (constructs IV and VIII); (v) a mutant Ub moiety bearing Val, Arg, and Arg instead of wt Gly, Lys, and Lys (constructs V and IX); (vi) a Pro residue at the junction between Ub and the rest of the fusion (construct I); (vii) a Val residue at the same junction (constructs II-X); (viii) an Arg residue at the same junction (construct XI); (ix) a 45-residue, E. coli Lac repressor-derived sequence, termed e, between Ub and the reporter part of a fusion (constructs I, X, and XI); (x) an e-derived sequence, termed e (see ``Experimental Procedures''), in which Lys and Lys were replaced with Arg residues (constructs II-IX); (xi) the E. coli -galactosidase (gal) moiety (see Bachmair and Varshavsky, 1989) (constructs I-V and XI); (xii) DHFRha, a mouse dihydrofolate reductase moiety extended at the C terminus by a sequence containing the hemagglutinin epitope (constructs VI-IX); (xiii) Ura3ha, the S. cerevisiae Ura3p moiety bearing a sequence containing the hemagglutinin epitope at the N terminus of Ura3p (construct X). Amino acid residues are indicated by their single-letter abbreviations. Residues that vary among constructs are boxed in the sequence at the top of the diagram.



We began dissection of the S. cerevisiae UFD pathway by analyzing mutations that perturb the pathway, and by cloning the corresponding genes. Among the UFD genes characterized thus far (UFD1, UFD2, UFD4 and UFD5), all but one were unknown previously. We also probed the structure and substrate attachment points of multi-Ub chains produced by the UFD pathway.


EXPERIMENTAL PROCEDURES

Strains, Media, Genetic Techniques, and gal Assay

S. cerevisiae strains are listed in Table 1. Escherichia coli strain JM101 (propagated in Luria broth, LB) was used as a host for plasmids and phage M13 derivatives. Synthetic yeast media (Sherman et al., 1986) contained 0.67% yeast nitrogen base without amino acids (Difco) and either 2% glucose (SD medium), 2% galactose (SG medium), or 2% galactose and 2% raffinose (SGR medium) as a carbon source. X-gal plates contained synthetic media supplemented with 0.1 M KHPO-KHPO (pH 7.0) and 40 µg/ml 5-bromo-4-chloro-3-indolyl -D-galactoside (X-gal) (Rose et al., 1981). Synthetic media lacking appropriate nutrient(s) were used to select for (and maintain) specific plasmids. Yeast mating, sporulation, and tetrad analysis were performed as described (Sherman et al., 1986). Transformation of S. cerevisiae was carried out according to Baker(1991). Strains were cured of URA3-expressing plasmids using 5-fluoroorotic acid (5-FOA) (Boeke et al., 1984). Enzymatic activity of gal in extracts from mid-exponential cultures growing in synthetic media was measured using o-nitrophenyl--D-galactoside (Ausubel et al. 1989) or chlorophenol red--D-galactopyranoside (Eustice et al., 1991). Protein concentration was determined using the Bradford assay (Bio-Rad).



Growth at 37 °C was assayed by streaking wt and mutant cells pregrown on YPD plates (Sherman et al., 1986) at 23 °C onto YPD plates and incubating at 37 °C. Canavanine sensitivity was estimated by streaking cells pregrown on YPD plates at 23 °C onto SD plates lacking arginine and containing 1.5 µg/ml canavanine. Sensitivity to UV light was estimated by exposing a few hundred cells on a YPD plate to a UV source for increasing amounts of time, followed by incubation in the dark at 30 °C.

DNA Manipulation and Sequencing

Standard procedures were used (Ausubel et al., 1989). The polymerase chain reaction (PCR) was performed using the GeneAmp kit (Perkin Elmer). Oligonucleotide-directed mutagenesis was carried out using the Mutagene kit (Bio-Rad). DNA was sequenced by subcloning restriction fragments into M13- mp18 or M13mp19 (Yanisch-Perron et al., 1985), by generating sets of deletions using the method of Dale et al. (1985), and by utilizing oligonucleotide primers hybridizing to regions of the DNA that had already been sequenced. Single-stranded M13 DNA was sequenced using the Sequenase kit (U. S. Biochemical Corp.). Supercoiled plasmids were prepared for sequencing as described by Hattori and Sakaki(1986). For Southern analysis, DNA was isolated from S. cerevisiae as described by Hoffman and Winston(1987), and processed for hybridization as described by Bartel et al.(1990). DNA probes were labeled using [-P]dCTP and the method of Feinberg and Vogelstein (1986). A Southern blot of yeast chromosomes fractionated by pulse-field gel electrophoresis (Chromoblot) was purchased from Clontech. PrimeClone filters (Olson et al., 1986) were a gift from L. Riles and M. Olson and were probed according to their directions.

Computer-assisted sequence analysis was performed using the Genetics Computer Group (GCG) Sequence Analysis Software Package (Devereux et al., 1984). DNA and predicted amino acid sequences were compared to data bases (GenBank, EMBL, PIR, SWISS-PROT) using either the FastA or Blast algorithms (Altschul et al., 1990).

Plasmid Constructs

The URA3-marked, 2µ-based plasmids pUb-P-e-gal, pUb-R-e-gal, and pUb-V-e-gal, which expressed, respectively, Ub-P-e-gal, Ub-R-e-gal, and Ub-V-e-gal (Fig. 1) from the P promoter in S. cerevisiae, have been described elsewhere (Bachmair et al., 1986; Johnson et al., 1992). Other plasmids utilized the same vector background and were constructed as follows. pUb-V-e-gal, which expressed Ub-V-e-gal (Fig. 1), was produced using oligonucleotide-directed mutagenesis of a HindIII-BamHI fragment of pUb-V-e-gal to convert Lys of Ub into Arg. The mutagenized XhoI-BamHI fragment was ligated into the vector-containing XhoI-BamHI fragment of pUb-V-e-gal. The plasmid pUb-V-e-gal, which expressed Ub-V-e-gal (Fig. 1), was produced using PCR. The template pUb-V-e-gal was initially amplified in two sets of reactions. One PCR utilized primer A (5`-GAAAGTTCCAAAGAGAAGG-3`), which annealed 5` to the XhoI site in these plasmids, and primer B (5`-TGAATTCTCGACTTAACG-3`), whose sequence encoded Arg instead of Lys. The second PCR utilized primer C (5`-AAGTCGAGAATTCAAGAC-3`), whose sequence also encoded Arg, and primer D (5`-CAAGCTCCGGATCCGTG-3`), which annealed to the lacI-derived DNA 3` to the BamHI site. The products of these reactions were isolated and used together as a template in another round of amplification, using primers A and D. The XhoI/BamHI-cut PCR product was ligated to the vector-containing XhoI-BamHI fragment of pUb-V-e-gal, yielding pUb-V-e-gal. The plasmid pUb-V-e-gal, which expressed Ub-V-e-gal (Fig. 1), was constructed similarly to pUb-V-e-gal, except that pUb-V-e-gal was used as the initial template. pUb-V-e-DHFRha encoded Ub-V-e-DHFRha (Fig. 1), a fusion of Ub-V-e to mouse dihydrofolate reductase (DHFR) bearing a 14-residue C-terminal extension (GTYPYDVPDYAAFL) derived in part from hemagglutinin of influenza virus. The construction of this plasmid is partially described in Johnson et al. (1992); other details are available on request. pUb-V-e-DHFRha, pUb-V-e-DHFRha, and pUb-V-e-DHFRha, which expressed, respectively, Ub-V-e-DHFRha, Ub-V-e-DHFRha, and Ub-V-e-DHFRha (Fig. 1), were constructed by ligating the small XhoI-BamHI fragments of the corresponding Ub-gal-encoding plasmids to the vector-containing XhoI-BamHI fragment of pUb-V-e-DHFRha. All final constructs were verified by sequencing.

``e'' denotes a 45-residue E. coli Lac repressor-derived sequence; e indicates an e-derived sequence in which Lys and Lys have been replaced with Arg residues (Fig. 1). In e-gal fusions (constructs II-V in Fig. 1), e contained Gly-Ser at positions 39 and 40, instead of Leu-Ala in the otherwise identical e regions of e-gal fusions (Johnson et al., 1992). The e sequence in e-DHFRha fusions (constructs VI-IX in Fig. 1) contained the sequence Arg-Ser-Gly-Ile-Met in place of residues 39-45 of e. The e region is required for the degradation of gal- or DHFR-based N-end rule substrates (Bachmair and Varshavsky, 1989; Johnson et al., 1990), but there is little if any difference between the degradation rates of e- versus e-containing gal or DHFR fusions such as Ub-P-gal and Ub-V-gal or their DHFR counterparts (Johnson et al., 1992).

The plasmid pUb-V-e-Ura3ha::TRP1, which expressed Ub-V-e-Ura3ha, bearing the hemagglutinin tag between the e and Ura3p moieties (Fig. 1), was constructed from pBA2 (provided by B. Andrews, K. Madura, and J. Dohmen), a pRS314-derived (Sikorski and Hieter, 1989) TRP1-marked CEN plasmid expressing Ub-M-e-Ura3ha from the P promoter. A 200-bp BstXI-BamHI fragment (derived from pUb-V-e-gal (Johnson et al., 1992)) encoding the C terminus of Ub and the junction, was used to replace a homologous BstXI-BamHI fragment in pBA2. pCUP1-Ub::LEU2, which expressed Ub from the P promoter, was produced from pRB238 (provided by R. Baker), a YEplac195-based plasmid containing a BamHI-ClaI fragment of YEp96 (Hochstrasser et al., 1991). The small BamHI-PstI fragment of pRB238 was ligated to BamHI/PstI-cut YEplac181 (Gietz and Sugino, 1988).

Isolation of ufd Mutants

The strain BWG1-7a (Table 1), carrying pUb-P-e-gal and growing in SG medium, was mutagenized with ethyl methanesulfonate (Sherman et al., 1986) to 18% survival and plated on SG plates lacking uracil. After 4 days at 23 °C, plates containing a total of 2.5 10 colonies were replica-plated onto SG/X-gal plates and incubated at 23 or 37 °C. About 400 blue colonies were observed; 63 of them, which remained blue upon replating on SG/X-gal plates, were tested for gal activity using the o-nitrophenyl--D-galactoside assay (Ausubel et al., 1989). The 22 mutants that passed this test were cured of pUb-P-e-gal using 5-FOA (Boeke et al., 1984), retransformed with plasmids expressing Ub-M-gal, Ub-R-gal, or Ub-P-gal (Bachmair et al., 1986), and tested by carrying out pulse-chase analyses and additional gal activity assays. The mating type of several mutants was switched by a transient expression of HO endonuclease (Sherman et al., 1986) (using HO expressed from the P promoter; plasmid provided by J. Brill), and the mutants were assigned to complementation groups by crossing the mutants to each other and by testing the resulting diploids (expressing Ub-P-gal) on SG/X-gal plates. Eleven of the thus characterized mutations were assigned to five complementation groups, termed ufd1-ufd5 (Ub fusion degradation). The ufd1-ufd5 strains listed in Table 1were chosen for further analysis.

Isolation of mTn3 Insertion-bearing ufd Mutants

S. cerevisiae JD52 (Table 1) expressing Ub-V-e-Ura3ha was transformed with NotI-digested DNA from a yeast genomic DNA library bearing quasi-random insertions of the mTn3 (LEU2 lac) minitransposon (Burns et al., 1994) (a gift from M. Snyder, Yale University), and plated on SD plates lacking tryptophan and leucine. After 2 days of growth at 23 °C, the colonies were replica-plated onto SD plates lacking uracil and tryptophan, to select for mTn3 insertion-bearing cells with elevated steady-state levels of Ub-V-e-Ura3ha (see ``Results''). About 100 Trp Leu Ura colonies (out of the 4 10 colonies screened) were picked and retested for growth on medium lacking uracil. 24 of these putative mutants were cured of pUb-V-e-Ura3ha::TRP1 (expressing Ub-V-e-Ura3ha) by selection with 5-FOA, and thereafter retransformed with plasmids expressing Ub-R-e-gal or Ub-V-e-gal. These isolates were then screened for blue colonies on SG/X-gal plates, or examined by pulse-chase analysis. Based on these and other tests, the insertion ufd mutants (impaired in the degradation of Ub-V-e-gal) were sorted into five classes. In the mutants of one class, Ub-V-e-gal was metabolically stabilized while the degradation of R-gal was unaffected; in addition, the pattern of ubiquitinated Ub-V-gal derivatives was similar to that seen with the ufd4-1 mutant (Fig. 2, lanes 13-15). These mutants were confirmed to contain insertions in the UFD4 gene (see below). The other classes of insertion ufd mutants remain to be assigned to specific UFD genes.


Figure 2: Pulse-chase analysis of Ub fusions in S. cerevisiae ufd mutants. Cells were labeled with TransS-label for 5 min at 30 °C, followed by a chase for 0, 10, and 30 min, extraction, immunoprecipitation of test proteins, and SDS-polyacrylamide gel electrophoresis (see Fig. 1for the structure of test proteins, Table 1for the strain's genotypes, and ``Experimental Procedures'' for other details). A, Ub-V-gal in the wild-type strain BWG1-7a (lanes 1-3), in the ufd1 strain PM373 (lanes 4-6), in the ufd2 strain PM211 (lanes 7-9), in the ufd3 strain PM164 (lanes 10-12), in the ufd4 strain PM642 (lanes 13-15), and in the ufd5 strain PM381 (lanes 16-18). B, same as in A but the strains also bore a plasmid overexpressing free Ub (see ``Experimental Procedures''). C, same as in A but with the test protein Ub-V-DHFRha. Times of chase, strain genotypes, and approximate half-lives of the test proteins (see main text) are indicated below the lanes. Also indicated are the bands of Ub-V-gal and Ub-V-DHFRha. An arrow indicates a 90-kDa, long-lived gal cleavage product characteristic of a fraction of short-lived gal-based proteins (Bachmair et al., 1986). Half-open square brackets denote bands of multiubiquitinated Ub-V-gal; three of these bands are also indicated by the numbers 1, 2, and 4. One ubiquitinated derivative of Ub-V-DHFRha is indicated by an arrowhead. An asterisk denotes a cleavage product of Ub-V-DHFRha. The ratios of S in each of the bands 1, 2, and 4 to S in the band of the initial Ub-V-gal are indicated below the lanes in A and B.



The UFD1 Gene

The strain PM373 (ufd1; Table 1), carrying a derivative of pUb-P-e-gal in which the URA3 marker had been replaced with LEU2 (Bartel et al., 1990), was transformed with a S. cerevisiae genomic DNA library carried in the URA3, CEN4-based vector YCp50 (Rose et al., 1987), and plated on SD plates lacking uracil and leucine. After 3 days at 30 °C, these plates, containing 8 10 colonies, were replica-plated on SG/X-gal plates lacking leucine and uracil to screen for white colonies (low levels of gal). Cells from 25 white colonies thus obtained were cured of their library-derived plasmids by selection on 5-FOA plates lacking leucine, and retested on X-gal plates, where 5 isolates formed blue colonies. Plasmid DNA (Hoffman and Winston, 1987) from these isolates was used to transform E. coli to ampicillin (amp) resistance. The YCp50 library-derived plasmids were distinguished from the plasmid expressing Ub-P-gal by picking white E. coli transformants on LB/amp plates containing X-gal. Four of the resulting plasmids contained identical inserts, and the fifth contained an overlapping insert. The smaller of these two plasmids, pUFD1, contained an 8-kb insert; a 3.7-kb HindIII fragment of this insert also complemented ufd1 (Fig. 4A), while an overlapping 2.5-kb EcoRI fragment failed to complement. Sequencing of the HindIII fragment revealed two ORFs, one of which was completely contained within the noncomplementing EcoRI fragment. The other ORF (UFD1; see ``Results'') was contained within a PvuII-NsiI fragment, which did complement the ufd1 proteolytic defect. The entire 3.7-kb HindIII fragment was sequenced on at least one strand, and the PvuII-NsiI fragment containing UFD1 was sequenced on both strands. The mutant ufd1-1 allele was isolated from the PM373 genomic DNA using PCR and primers that hybridize to the 5` and 3` termini of the UFD1 ORF (Johnson, 1992). Two amplification products from each of two independent PCR reactions were sequenced and found to contain a single missense mutation (see ``Results'').


Figure 4: Organization, restriction maps, subcloning strategies and deletion alleles of UFD1, UFD2, UFD4, and UFD5. A, UFD1 and its vicinity on chromosome VII. B, UFD2 and its vicinity on chromosome IV. C, UFD4 and its vicinity on chromosome XI. D, UFD5 and its vicinity on chromosome IV. ORFs are named as described in the main text, and are shown as arrow-shaped boxes indicating the direction of transcription. Incompletely sequenced ORFs are shown as jagged-end boxes. Subcloned regions are indicated below the restriction map, with (+),(-), or (±) denoting the subclone's ability, lack of ability, or partial ability, respectively, to complement a defect in degradation of Ub-V-gal by a corresponding ufd mutant. Dashed lines indicate the regions of UFD genes that were replaced by a marker gene in deletion constructs (LEU2 in the case of UFD1, UFD2, and UFD5, and TRP1 in the case of UFD4. Arrow-shaped boxes representing the deletion markers LEU2 and TRP1 are not to scale. Arrowheads indicate the sites of mTn3 (LEU2 lac) insertions that conferred null ufd4 phenotypes (see ``Experimental Procedures''). In B, a putative intron in SOS1 is also indicated. Restriction sites: B, BamHI; Bg, BglII; C, ClaI; E, EcoRI; H, HindIII; K, KpnI; N, NsiI; P, PstI; Pv, PvuII; S, SalI; Sc, SacI; Sm, SmaI; Sp, SphI; Xb, XbaI; Xh, XhoI. Not all restriction sites are shown in all maps. The nucleotide sequences of UFD1 and UFD2 have been submitted to the GenBank/EMBL data base with accession numbers U22153 and U22154, respectively.



The ufd1-1 allele was constructed by inserting a HindIII-XmnI fragment containing the 5`-flanking sequence and the 5`-proximal coding region of the UFD1 ORF into SalI/HindIII-cut pUC/LEU#3, with the SalI end filled out with Klenow polymerase I. The pUC/LEU#3 plasmid (a gift from R. Baker) contained a SalI-XhoI fragment (bearing LEU2) inserted into the SalI site of pUC9 (Vieira and Messing, 1982). A 1.3-kb BamHI-BglII fragment containing the 3`-flanking sequence of UFD1 was inserted into the BamHI site of the resulting plasmid to yield pufd1::LEU2. This plasmid, cut with HindIII and SmaI, was introduced into the diploid strain DF5 (Table 1), and Leu transformants were selected (Rothstein, 1991). The ufd1-1 deletion was confirmed by Southern analysis of EcoRI-digested yeast DNA, using a 1.5-kb NsiI fragment from the 3` flank of UFD1 as a probe. To verify that the locus of the lesion in the ufd1-1 strain PM373 was the cloned UFD1 gene, PM373 carrying a LEU2-marked plasmid that expressed Ub-P-gal was crossed to strain EJY158 (Table 1), which bore the HIS3-marked ufd1-2 deletion allele (construction details available on request) and carried a URA3-marked plasmid that expressed wt UFD1. The resulting diploid, when cured of the UFD1-expressing plasmid by growth on 5-FOA, formed blue colonies on X-gal plates, indicating that PM373 and EJY158 contained mutations in the same gene (see also the main text).

The UFD2 Gene

UFD2 was cloned using the YCp50-based S. cerevisiae DNA library, the ufd2-1 strain PM211 (Table 1), and a protocol similar to that used for cloning UFD1. None of the initially constructed subclones of the complementing plasmid pUFD2-33 fully complemented the ufd2 proteolytic defect. In particular, a 6.5-kb EcoRI fragment that encompassed the putative UFD2 gene yielded partial complementation. One end of this subclone, near the ARF1 gene (Fig. 4B), was not present in one of the fully complementing library plasmids, while the other end encompassed only the 5`-proximal region of another ORF (ORFb; Fig. 4B). That ORF could not have been UFD2 because it was fully contained within a noncomplementing BamHI fragment. Thus, the putative UFD2 ORF was the one responsible for complementation, with more than 1 kb of 5`-flanking sequence required for full complementation (data not shown). About 6 kb of DNA, from the HindIII site at the 3` end of the putative UFD2 ORF to beyond the SacI site in PPH22, was sequenced on at least one strand, and the UFD2 ORF was sequenced on both strands. By joining this sequence to the sequences in GenBank, the region of chromosome IV from ARF1 to beyond PPH22 could be read without gaps.

The ufd2-2 allele was produced by purifying a 6.5-kb EcoRI fragment encompassing UFD2, circularizing it by ligation, digesting the product with BamHI and KpnI, and ligating the resulting fragment to the KpnI/BamHI-cut, vector-containing fragment of a variant of YIplac128 (Gietz and Sugino, 1988) that lacked the EcoRI site. The resulting plasmid, pufd2::LEU2, was cut with EcoRI and introduced into the diploid JD51 (Table 1), selecting for Leu transformants (Rothstein, 1991). The deletion was verified by Southern analysis of XbaI-digested genomic DNA from the Leu diploid and from its Leu and Leu haploid segregants, using a BamHI-EcoRI fragment from the 3` flank of UFD2 as a probe (data not shown). To verify that the locus of the lesion in the ufd2-1 strain PM211 was the cloned UFD2 gene, PM211 bearing pUb-P-e-gal was crossed to EJY129 (Table 1) and analyzed as described under ``Results.''

UBI4 as a Suppressor of ufd3-1

Attempts to clone UFD3 from the YCp50-based S. cerevisiae genomic library by complementation of the proteolytic defect in the ufd3-1 strain PM164 (Table 1) repeatedly yielded the UBI4 gene. To determine whether the lesion in PM164 was in UBI4, this strain was crossed to SUB63, a ubi4 strain (Finley et al., 1987). The PM164/SUB63 diploid expressing Ub-P-gal produced white colonies on X-gal plates, and sporulated well, indicating that UBI4 and UFD3 were different genes (see also ``Results'').

The UFD4 Gene

Partial clones of UFD4 were obtained by rescuing mTn3 insertions and flanking DNA from mTn3-produced ufd4 mutants of S. cerevisiae (see above). These mutants were transformed with the PvuI-cut integration plasmid YIp5 (Struhl et al., 1979) and plated on SD medium lacking uracil, in order to select for plasmid integrants in the transposon's amp gene. Genomic DNA isolated from YIp5-containing strains was digested with NsiI, ligated to circularize the resulting fragments, and used to transform E. coli (by electroporation) to ampicillin resistance. YIp5 contained one NsiI site, and the other NsiI site was provided by the yeast DNA flanking the transposon. Only the fragment bearing the flanking region near the transposon's lacZ sequence contained both the E. coli replication origin and the amp gene, and therefore could be rescued by this technique (Burns et al., 1994). The cloned plasmids were sequenced using the primer 5`-AAACGACGGGATCCCCC-3`, which hybridized to the 5` region of the transposon's lacZ sequence. Using this method, six independent insertions in UFD4 were isolated from three of the seven sets of mutants, with each set derived from one pool of insertion-mutagenized library plasmids. The mTn3 insertion points were located at the following sites of the S53418 locus (Pascolo et al., 1992): 23-1 at bp 6599; 23-2 at bp 4669; 26-1 at bp 5981; 26-3 at bp 5760; 26-7 at bp 6103; and 31-3 at bp 5734 (Fig. 4C). All insertions except 26-1 contained the lacZ reading frame oriented in the direction opposite to that of the UFD4 ORF.

A UFD4-containing plasmid that complemented the proteolytic defect of the ufd4-1 strain PM642 was constructed by first amplifying a DNA fragment from a ufd4 insertion mutant (26-1) using the UFD45` primer (see below) and the sequencing primer described above. This fragment was digested with BglII and SacI, and ligated to BamHI/SacI-cut pRS313 (Sikorski and Hieter, 1989). The resulting plasmid was cut with SacI and ligated to the SacI fragment containing the 3`-proximal coding and 3`-flanking region of the UFD4 gene.

The ufd4-1 allele (see ``Results'') was produced using PCR. Initially, two sets of reactions with S. cerevisiae genomic DNA were performed, one with the primers UFD45` (5`-GGCAGATCTAACATCTTCCTCAGCTGG-3`) and UFD4N (5`-GCCGGGACGTCGTATGGGTACATTGTTTAAACGGTACCGCTTGGCTACTCTTCTATTTTAAATG-3`) to amplify 800 bp of the 5`-flanking region of UFD4, and another with the primers UFD4C (5`-GCCGGTACCCGGCCGGCAGGAGCTTTTCTACTTTCC-3`) and UFD43` (5`-CCGAGATCTTCCTATGAACTCGACCAG-3`) to amplify 1.0 kb of the 3`-flanking region of UFD4. The products were digested with BglII and ligated together. The resulting fragment was used as a template in a second round of PCR, with primers UFD4N and UFD4C. The product was digested with KpnI and ligated to KpnI-cut pRS304 (Sikorski and Hieter, 1989), yielding pufd4::TRP1. This plasmid was digested with BglII and introduced into the diploid JD51 (Table 1), selecting for Trp transformants. The deletion was confirmed by Southern analysis of EcoRI-digested genomic DNA from the ufd4-1/UFD4 diploid and from both Trp and Trp haploid segregants, using a fragment containing the 3`-flanking region of UFD4 as a probe (data not shown). To verify that the locus bearing the lesion in the ufd4-1 strain PM642 was the cloned UFD4 gene, PM642 (carrying pUb-P-e-gal) was crossed to EJY150 (Table 1). The resulting diploid formed blue colonies on X-gal plates, indicating that PM642 and EJY150 contained mutations in the same gene.

The UFD5 Gene

UFD5 was cloned using the YCp50-based S. cerevisiae DNA library, the ufd5-1 strain PM381 (Table 1), and a protocol similar to that used for cloning UFD1 and UFD2. The initial complementing plasmid contained a 20-kb yeast DNA insert. Both a 5-kb PstI fragment and a 2.8-kb SmaI-PstI fragment of this insert also complemented the proteolytic defect of PM381. The region containing the UFD5 ORF was sequenced on one strand. The ufd5-1 allele was produced by isolating the 5-kb PstI fragment containing UFD5, circularizing it by self-ligation, and using the product as a template in a PCR reaction with primers 5`-CCAAGCTTGCTTGAAAATTCTAG-3` and 5`-GGAAGCTTGTTAATTAAGGTTAC-3`. The product was digested with ScaI and HindIII, and then ligated to SmaI/HindIII-cut pRS305 (Sikorski and Hieter, 1989), yielding pufd5:::LEU2. This plasmid was cut with PstI and introduced into the diploid JD51 (Table 1), selecting for Leu transformants (see ``Results''). To verify that the locus of the lesion in the ufd5-1 strain PM381 lesion was the cloned UFD5 gene, PM381 (carrying pUb-P-e-gal) was crossed to EJY141 (Table 1). The resulting diploid formed blue colonies on X-gal plates, indicating that PM381 and EJY141 contained mutations in the same complementation group.

Pulse-Chase and Immunoblot Analyses

Pulse-chase experiments, including the extraction, immunoprecipitation, electrophoretic fractionation, detection, and quantitation of S-labeled, gal- or DHFRha-based test proteins, were carried out as described by Bartel et al.(1990) and Johnson et al.(1992), with yeast cultures grown in SGR medium, and with 50 mMN-ethylmaleimide present during extraction to inhibit Ub-specific proteases (Johnsson and Varshavsky, 1994). SDS-polyacrylamide gel electrophoreis of gal-based proteins was carried out in 5% polyacrylamide-SDS gels. DHFRha-based proteins were immunoprecipitated with the 12CA5 monoclonal antibody to the hemagglutinin epitope (Johnsson and Varshavsky, 1994), and were analyzed in 12% polyacrylamide-SDS gels (Ausubel et al., 1989) or in 7.5% polyacrylamide SDS-Tricine gels (Schägger and Jagow, 1987). Electrophoretic patterns were quantified using a PhosphorImager (Molecular Dynamics). In experiments where S. cerevisiae also carried pCUP1-Ub::LEU2, which expressed Ub, growth media contained 0.1 mM CuSO.

For immunoblot analysis, extracts were made from mid-exponential (A of 1) S. cerevisiae cultures grown in rich (YPD) medium at 30 °C. Cells were collected by centrifugation, washed twice in cold lysis buffer (50 mM Na-HEPES (pH 7.5), 5 mM Na-EDTA, 0.15 M NaCl) containing 50 mMN-ethylmaleimide, resuspended in 0.8 ml of cold lysis buffer containing 1% Triton X-100, protease inhibitors, and 50 mMN-ethylmaleimide (Johnson et al., 1992), and vortexed with 0.5-mm glass beads for 3 min at 4 °C, with intermittent cooling on ice. After centrifugation at 12,000 g for 10 min, samples of supernatant containing 15 µg of total protein were mixed with SDS-containing electrophoretic sample buffer, heated at 100 °C for 3 min, and electrophoresed in a 7.5% polyacrylamide SDS-Tricine gel (Schägger and von Jagow, 1987), followed by electroblotting of separated proteins onto ProBlott polyvinylidine difluoride membrane (Applied Biosystems). Ub was detected on the polyvinylidine difluoride membrane using a polyclonal anti-Ub antibody (East Acres Biologicals, Southbridge, MA) and the ECL system (Amersham).


RESULTS

Isolation of ufd Mutants and Pulse-Chase Analysis of Ub-V-gal

Yeast expressing a short-lived gal-based test protein form colonies that are white on plates containing the chromogenic gal substrate X-gal. Mutants that cannot degrade the test protein accumulate higher steady-state levels of gal and therefore form blue colonies on X-gal plates. We used this screen (Bartel et al., 1990) to identify mutants that are impaired in their ability to degrade Ub-P-gal. Putative ufd mutants were retested by measuring gal activity in cell extracts, and also directly by pulse-chase analysis. Eleven of the mutants that passed these tests were assigned to five complementation groups, termed ufd1-ufd5 (see ``Experimental Procedures''). This round of screening was far from exhaustive, as three of the complementation groups (ufd1, ufd2, and ufd5) were represented by only one mutation each. In addition, no mutations were isolated in UBC4 or in several other genes (PRE1, SUG1 (CIM3), CIM5, and DOA4 (UBP4)) whose mutant alleles are known to stabilize Ub-P-gal (Johnson et al., 1992; Seufert and Jentsch, 1992; Ghislain et al., 1993; Papa and Hochstrasser, 1993).

In wild-type (wt) cells, Ub-V-gal was multiubiquitinated and rapidly degraded (t of 4 min; Fig. 2A, lanes 1-3). (Ub-V-containing gal and DHFRha fusions bore the e module between V and the gal or DHFRha moiety (see the legend to Fig. 1).) The in vivo degradation of short-lived proteins analyzed in this and earlier studies did not obey first-order kinetics (Bachmair et al., 1986; Baker and Varshavsky, 1991; Tobias et al., 1991), apparently because newly formed proteins are targeted and degraded more efficiently than their conformationally mature counterparts. Since a single half-life is inapplicable to a non-first-order decay, the t values cited below for short-lived proteins are ``initial'' half-lives, determined by assuming first-order kinetics between the earliest times of chase (0 and 10 min) (Baker and Varshavsky, 1991). The first two bands in a ``ladder'' of multiubiquitinated Ub-V-gal derivatives were the species containing one and two conjugated Ub moieties (``1'' and ``2'' in Fig. 2A) (Johnson et al., 1992). These species were of about equal intensity, and each contained 30% as much S as the band of Ub-V-gal (Fig. 2A). These were followed by at least three larger species, the middle one (band ``4'' in Fig. 2A) being of greater intensity than the other two.

In the ufd1 mutant, Ub-V-gal was long-lived (t> 2 h; Fig. 2A, lanes 4-6). In some experiments, a small amount of the 90-kDa gal cleavage product, characteristic of short-lived gal-based proteins (Bachmair et al., 1986) (see Fig. 2A, lanes 1-3), could be detected in ufd1 cells (data not shown). The distribution of multiubiquitinated derivatives was mildly affected in this mutant, with species 1 and 2 containing less than wt amounts of S at the end of the pulse (``0 min''), but nearly wt amounts after 30 min of chase (Fig. 2A, lanes 4-6).

In the ufd2 mutant, Ub-V-gal was also long-lived (t > 2 h) (Fig. 2A, lanes 7-9). In addition, a greater proportion of S was present at 0 min in the first ubiquitinated derivative of Ub-V-gal (band 1; 35% compared to 28% in wt cells; Fig. 2A, lane 7, compare to lane 1), but there was markedly less S in the larger ubiquitinated species (for example, 8% in band 2, versus 33% in wt cells).

In the ufd3 mutant, the degradation of Ub-V-gal was strongly decreased but still detectable (t of 1 h), and the 90-kDa gal cleavage product (see above) accumulated to a significant level during the chase (Fig. 2A, lanes 10-12). The pattern of ubiquitinated Ub-V-gal derivatives was also affected in this mutant; for example, bands 1 and 2 contained at 0 min about half the amount of S as in wt cells.

In the ufd4 mutant, Ub-V-gal was long-lived (t > 2 h); the bulk of ubiquitinated Ub-V-gal derivatives was confined to band 1, and the species beyond band 2 were present in trace amounts (Fig. 2A, lanes 13-15). While not apparent from Fig. 2A, whose samples were fractionated in a 5% polyacrylamide gel, the patterns obtained using a 6% gel showed closer spacing between bands 1 and 2 in the ufd4 mutant than in wt cells (Johnson, 1992), suggesting that ubiquitinated derivatives of Ub-V-gal in this mutant may be structurally different from those formed in wt cells (see below).

In the ufd5 mutant, Ub-V-gal was long-lived (t > 2 h); its ubiquitinated derivatives comprised a smaller than wt fraction of the substrate (Fig. 2A, lanes 16-18).

Overexpression of Ub Suppresses the ufd3 Defect in Ub-V-gal Degradation

To determine whether the metabolic stabilization of Ub-V-gal in the ufd1-ufd5 mutants could be reversed by increasing the concentration of Ub, we carried out pulse-chase analyses in cells containing a high copy plasmid that expressed Ub from the induced P promoter (Fig. 2B). Overexpression of Ub led to a slight decrease in the rate of Ub-V-gal degradation in wt cells (t of 13 versus 4 min in the absence of Ub overexpression), and also to a significant increase in the content of ubiquitinated Ub-V-gal derivatives (Fig. 2, B, lanes 1-3versusA, lanes 1-3). Overexpression of Ub in the ufd1, ufd2, ufd4, and ufd5 mutants also increased the relative content of multiubiquitinated Ub-V-gal; however, it did not reverse the mutant's inability to degrade Ub-V-gal (Fig. 2B).

By contrast, overexpression of Ub in the ufd3 mutant did reverse the metabolic stabilization of Ub-V-gal, which was degraded only slightly more slowly in ufd3 cells overexpressing Ub than in wt cells that also overexpressed Ub (t of 18 and 13 min, respectively; Fig. 2B, lanes 1-3 and 10-12; note also the accumulation of the 90-kDa gal cleavage product characteristic of short-lived gal-based proteins (Bachmair et al., 1986)). In addition, the overexpression of Ub in ufd3 cells increased the amounts of multiubiquitinated Ub-V-gal derivatives to nearly wt levels, suggesting that a primary defect in ufd3 cells is in the control of the Ub concentration rather than in ubiquitination or proteolysis per se. Indeed, immunoblotting analysis with cell extracts and an antibody to Ub showed that ufd3 was the only ufd mutant in which the concentration of free Ub was considerably (at least 10-fold) lower than in wt cells (Fig. 3A). Thus, the ufd3 mutation appears to affect the degradation of Ub-V-gal indirectly, through a decrease in the level of free Ub.


Figure 3: Effects of ufd mutations on the concentration of free Ub and activity of the N-end rule pathway. A, immunoblot analysis of free Ub levels in wild-type S. cerevisiae and ufd mutants (see ``Experimental Procedures''). The strains were the same as those in Fig. 2. The band of free Ub is indicated. B, pulse-chase analysis of Ub-R-gal (R-gal) in wild-type (lanes 1-3) and ufd5 cells (lanes 4-6). See Fig. 2for procedures, band designations and other notations.



Degradation of Other Proteins in ufd Mutants

The effect of ufd mutations was also tested using Ub-V-DHFRha, a counterpart of Ub-V-gal in which the 114-kDa gal-based moiety (whose oligomerization yields the 460-kDa Ub-V-gal tetramer) was replaced by the monomeric mouse DHFR bearing a 14-residue C-terminal extension containing the ``hemagglutinin'' epitope (Fig. 1) (Johnson et al., 1992; Field et al., 1988). Similarly to Ub-V-gal, Ub-V-DHFRha was a short-lived protein in wt cells (t of 4 min). Fewer ubiquitinated derivatives of Ub-V-DHFRha could be detected than in analogous experiments with Ub-V-gal, and these derivatives contained a much smaller fraction of the labeled material (Fig. 2C, lanes 1-3). The ufd1, ufd3, ufd4, and ufd5 mutations stabilized Ub-V-DHFRha (t of 30 min, 1 h, 30 min, and 30 min, respectively, in comparison to a t of 4 min in wt cells) (Fig. 2C, lanes 1-6 and 10-18). The ufd1, ufd3, and ufd5 mutations also reduced the relative levels of monoubiquitinated Ub-V-DHFRha, similarly to their effects on Ub-V-gal, while the ufd4 mutation had little effect on the amount of monoubiquitinated Ub-V-DHFRha (Fig. 2C compare to Fig. 2A). Remarkably, the ufd2 mutation had no effect on either ubiquitination or degradation of Ub-V-DHFRha, in contrast to its strong effect on the degradation of Ub-V-gal (Fig. 2, C, lanes 7-9versusA, lanes 7-9).

We also tested the ufd mutants for their ability to degrade R-gal (derived from Ub-R-gal), a substrate of the N-end rule pathway (Varshavsky, 1992). Prior to degradation, R-gal is multiubiquitinated at one of the two Lys residues in a 45-residue E. coli Lac repressor-derived extension between the N-terminal residue (R) of R-gal and the gal moiety (Fig. 1) (Chau et al., 1989). Two proteins that are not required for the degradation of Ub-V-gal have been shown to be essential for the degradation of N-end rule substrates such as R-gal: Ubr1p (called N-recognin or E3), which binds to destabilizing N-terminal residues such as R (Arg) (Bartel et al., 1990), and Ubc2p, one of at least 11 Ub-conjugating (E2) enzymes in S. cerevisiae (Dohmen et al., 1991; Madura et al., 1993). Thus, the N-end rule and UFD pathways must differ at least by their initial steps.

The degradation of R-gal was not affected in the ufd1-ufd4 mutants (data not shown). However, R-gal was metabolically stable in the ufd5 mutant (t > 1 h; Fig. 3B, lanes 4-6). In contrast to ubr1 or ubc2 mutants, where R-gal is neither degraded nor ubiquitinated (Bartel et al., 1990; Dohmen et al., 1991), R-gal was multiubiquitinated in the ufd5 mutant (Fig. 3B). Thus, Ufd5p appears to function at a post-ubiquitination step that is common to the UFD and N-end rule pathways. (Note that these experiments could not establish whether the ufd3 mutation affects R-gal degradation, because R-gal was expressed as Ub-R-gal; deubiquitination of this fusion would have been sufficient to suppress the ufd3 defect through an overproduction of free Ub, as described above.)

The UFD1 Gene

UFD1 was isolated from a YCp50-based S. cerevisiae genomic DNA library (Rose et al., 1987) by complementation of the proteolytic defect in the ufd1 strain PM373 (see ``Experimental Procedures''). Sequencing of the complementing DNA region (Fig. 4A) revealed an ORF encoding a 361-residue (40 kDa) protein (Fig. 5A). The position of the start (ATG) codon was inferred so as to yield the longest ORF. When this ORF was expressed in S. cerevisiae from the P promoter, a protein of the expected size was overproduced (data not shown). Computer-assisted comparisons of Ufd1p to other proteins detected no similarities to sequences in data bases, except for a sequence encoded by a partial cDNA from rice (EMBL accession no. D23997). The Codon Adaptation Index (Sharp and Li, 1987) of UFD1 is 0.125, characteristic of weakly expressed yeast genes. A probe derived from the HindIII fragment encompassing UFD1 and SCM4 (Fig. 4A) hybridized to clone 6247 of the PrimeClone Blots (Olson et al., 1986), positioning UFD1 on the right arm of chromosome VII, between CEN7 and SPT6. The UFD1 gene is located between TFC4, which encodes a subunit of transcription factor IIIC (Marck et al., 1993), and SCM4, isolated as a high copy suppressor of a cdc4 mutant (Smith et al., 1992) (Fig. 4A).


Figure 5: Deduced amino acid sequences of Ufd proteins and sequence comparisons. A, Ufd1p. Amino acid residues are numbered on the left. The Val residue that is altered by the mutation which produced ufd1-1 is boxed. B, Ufd2p. C, alignment of the C-terminal domain of Ufd4p with the sequences of other members of the E6AP protein family. E6AP denotes the E6-associated protein (Huibregtse et al., 1993a); rat 100 kDa denotes the product of a cloned rat cDNA (Müller et al., 1992). Sequences are shown using single-letter amino acid abbreviations, with residue numbers indicated on the right. Identities among at least two of the sequences are denoted by shading. Periods indicate gaps introduced to maximize alignments (generated using the GCG program PileUp), and asterisks denote termination codons. D, comparisons of zinc finger-like domains in Ufd5p to similar domains. Ufd5p-1 and Ufd5p-2 denote two such domains in Ufd5p (Son1p) (Nelson et al., 1993); TfIIIA-1, -2, and -3 denote three domains of S. cerevisiae TFIIIA (Archambault et al., 1992; Woychik and Young, 1992); Krox 20-1 and -2 denote two domains from the mouse Krox 20 protein (Chavrier et al., 1988); su(hw)-1 and -2 denote two domains of the D. melanogaster hairy wing suppressor protein (su(hw)) (Parkhurst et al., 1988); Rme1p denotes one domain of S. cerevisiae Rme1p (Covitz et al., 1991); Mig1p-1 and -2 denote two domains of S. cerevisiae Mig1p (Nehlin and Ronne, 1990). Identities among at least five sequences are denoted by shading. The Cys and His residues involved in zinc binding are shown in white on a black background.



The ufd1-1::LEU2 allele was constructed by deleting 85% of the UFD1 ORF and replacing it with LEU2 (Fig. 4A). A linear DNA fragment containing ufd1-1::LEU2 was introduced into a [leu2/leu2 UFD1/UFD1] diploid. Leu transformants were analyzed by Southern hybridization, and a strain with a restriction fragment profile diagnostic for the UFD1 replacement (Rothstein, 1991) was selected for further study (Johnson, 1992; data not shown). When this ufd1-1/UFD1 diploid was sporulated and dissected, each tetrad contained two Leu spores that formed colonies and two spores that germinated but formed only 20 misshapen cells before growth arrest. When this experiment was repeated with the same diploid carrying UFD1 on a URA3-marked plasmid, the ufd1-1 spores that carried the plasmid germinated and grew well; however, these cells were unable to form colonies on 5-FOA, which selects against Ura3-expressing cells (Boeke et al., 1984), indicating that S. cerevisiae requires UFD1 for vegetative growth.

A complementation test was performed to verify that the cloned gene was in fact UFD1. A ufd1 strain carrying a URA3-marked UFD1 plasmid was crossed to the original ufd1-1 mutant, and the diploid obtained was cured of the plasmid by growth on 5-FOA (see ``Experimental Procedures''). The resulting diploid strain grew much more slowly than haploid ufd1-1 strains of either parental background, and its colonies contained mostly interconnected, abnormally elongated cells, suggesting that a single copy of the ufd1-1 allele does not fully complement the growth defect of the ufd1 mutant in a diploid (ufd1-1/ufd1) background. A derivative of this diploid that expressed Ub-V-gal formed blue colonies on X-gal plates, indicating that the cloned gene was indeed UFD1. This conclusion was independently confirmed by a genetic mapping experiment showing that the locus bearing the ufd1-1 lesion and the putative UFD1 gene were tightly linked (Johnson, 1992). The ufd1-1 allele was isolated from the ufd1-1 mutant by PCR and sequenced, revealing a single missense mutation (a Val Asp substitution at position 94) (Fig. 5A). It has not been demonstrated directly that this mutation alone confers the ufd1-1 phenotype.

The ufd1-1 mutant was at most weakly hypersensitive to UV light; it grew more slowly than wt cells, and homozygous ufd1-1 diploids failed to sporulate unless they carried a plasmid expressing the wt UFD1 (data not shown). A UFD1-ha allele encoding Ufd1p-ha (which bore a C-terminal hemagglutinin epitope tag (Field et al., 1988)) was constructed and expressed from the P or the Ppromoter. In either case, Ufd1p-ha complemented the inviability of a ufd1-1 strain nearly as efficiently as the wt Ufd1p. A 110-kDa protein of unknown identity could be specifically coimmunoprecipitated with Ufd1p-ha from S. cerevisiae extracts (data not shown).

The UFD2 Gene

An approach similar to the one described above for UFD1 was used to isolate a gene that complemented the ufd2-1 proteolytic defect. A partially complementing subclone contained an ORF encoding a 961-residue (110 kDa) protein (Fig. 5B). Despite the partial complementation by this subclone, it encoded the relevant protein (see ``Experimental Procedures''). The start codon of UFD2 was inferred so as to yield the longest ORF. No ATGs occur in any of the three forward reading frames upstream of the putative start codon until position -386. The Codon Adaptation Index (Sharp and Li, 1987) of the putative UFD2 gene (it is shown below to be, in fact, UFD2) is 0.178, characteristic of weakly expressed yeast genes. The deduced amino acid sequence of the Ufd2p protein bears no significant similarities to sequences in the data bases.

Sequencing of the regions flanking UFD2 positioned this gene within a stretch of chromosome IV that appears to be partially duplicated (Ronne et al., 1991). UFD2 is located between ARF1, which encodes an ADP-ribosylation factor (Sewell and Kahn, 1988), and PPH22, which encodes a type 2A serine/threonine protein phosphatase (Sneddon et al., 1990; Ronne et al., 1991) (Fig. 4B). The other copy of the proposed duplication contains ARF2 and PPH21, which are separated by 2.5 kb (Ronne et al., 1991), in contrast to 7 kb separating ARF1 and PPH22 in the UFD2-containing copy of duplication (Fig. 4B). No gene similar to UFD2 is present between ARF2 and PPH21. An ORF (ORFb) between PPH22 and UFD2 (Fig. 4B) encodes a 457-residue protein whose sequence bears no similarities to sequences in data bases. The 5`-untranslated region abutting ORFb contains perfect matches to the consensus sequences of S. cerevisiae RNA splicing sites (Padgett et al., 1986). SOS1, the gene between ARF1 and UFD2, contains an intron between its start and second codons (Fig. 4B), and encodes a 120-residue protein whose sequence is similar to that of the rat ribosomal protein L35 (Zhong and Arndt, 1993).

The ufd2-2::LEU2 allele lacking 80% of the putative UFD2 ORF (Fig. 4B) was constructed and used to replace one of two UFD2 copies in a diploid strain, as described above for UFD1 (Fig. 4B). When the resulting diploid was sporulated and the tetrads dissected, the Leu phenotype segregated 2:2, and all segregants grew at wt rates, indicating that UFD2 is not required for vegetative growth. ufd2 mutants also grew well at 37 °C, and were not hypersensitive to either UV light or the arginine analog canavanine (data not shown). Ub-P-gal and Ub-V-gal were stabilized to the same extent in the ufd2-2 strain as in the original ufd2-1 mutant, as determined by measuring gal activity in cell extracts (data not shown). In addition, both the ufd2-2 and ufd2-1 alleles did not stabilize the normally short-lived Ub-V-DHFRha, as determined by pulse-chase analysis (Fig. 2C, lanes 7-9, and data not shown). A diploid (expressing Ub-V-gal) produced by a cross between the ufd2-2 strain and the original ufd2-1 mutant formed blue colonies on X-gal plates, indicating that the putative UFD2 was indeed the UFD2 gene.

High-stringency Southern analysis of DNA from the ufd2-1 strain, using a probe contained within the region of UFD2 that had been deleted to yield the ufd2-1 allele, indicated the presence of at least one, and more likely two, loci that cross-hybridized with UFD2 in S. cerevisiae DNA (Johnson, 1992; data not shown). Thus, a functional homolog of UFD2 might be the reason for viability of ufd2 mutants.

Suppression of the ufd3 Defect by the UBI4 Gene

Attempts to isolate UFD3 by complementation of the proteolytic defect in the ufd3-1 mutant yielded exclusively clones containing UBI4, the S. cerevisiae polyubiquitin gene (zkaynak et al., 1987). Although the ubi4-2::LEU2 strain SUB63 (Finley et al., 1987; Table 1) carrying a plasmid expressing Ub-P-gal did form blue colonies on X-gal plates (indicating that ubi4 mutants could be among those identified by our initial screen), additional tests showed that the ufd3-1 lesion was not in UBI4. Specifically, a ufd3-1/ubi4-2 diploid expressing Ub-P-gal formed white colonies on X-gal plates; it also sporulated well, in contrast to ubi4/ubi4 diploids (Finley et al., 1987). Furthermore, the LEU2 marker of the ubi4-2::LEU2 allele and the ufd3-1 phenotype segregated independently upon sporulation and tetrad dissection of the ufd3-1/ubi4-2::LEU2 diploid (data not shown). Finally, ufd3-1 spores did not lose viability after 4 days in sporulation media (data not shown), in contrast to ubi4-2::LEU2 spores (Finley et al., 1987). Thus, one or a few extra copies of UBI4, expressed from its own promoter on a low copy plasmid can suppress the ufd3-1 proteolytic defect; this result is consistent with a greatly reduced level of free Ub in the ufd3-1 mutant (Fig. 3A).

UFD4 Encodes a Member of the E6AP Family

The UFD4 gene was isolated using mutants bearing transposon insertions in UFD4. S. cerevisiae strain JD52 (Table 1) expressing Ub-V-Ura3ha (Fig. 1) from a high copy plasmid was transformed with restriction fragments from a yeast genomic DNA library carrying random insertions of mTn3 (LEU2 lac), a Tn3-derived minitransposon bearing the yeast LEU2 gene and the E. coli lacZ gene lacking its start codon. In this approach (Burns et al., 1994), transposons from the library are inserted into chromosomal copies of S. cerevisiae genes through homologous recombinations between a library plasmid's yeast DNA flanking the transposon and yeast chromosomal DNA (see ``Experimental Procedures''). Leu (insertion-bearing) transformants were screened for the Ura phenotype (Ub-V-Ura3ha is short-lived in wt cells, which are therefore phenotypically Ura). Leu Ura mutants were cured of the Ub-V-Ura3ha-expressing plasmid by growth on 5-FOA, then retransformed with plasmids expressing either Ub-V-gal or R-gal (Ub-R-gal), and tested for their ability to degrade these substrates by determining colony color on X-gal plates, or by pulse-chase analysis (data not shown). One class of insertion mutations strongly stabilized Ub-V-gal but not R-gal; the pattern of ubiquitinated Ub-V-gal derivatives in these mutants was similar to the one observed with the ufd4-1 mutant (Fig. 7B, lanes 1-3; compare to Fig. 2A, lanes 13-15; and data not shown). That the above mutations indeed resided in UFD4 was confirmed by crossing a strain deleted for the gene containing these mTn3 insertions (see below) to the ufd4-1 strain. The resulting diploid (expressing Ub-P-gal) formed blue colonies on X-gal plates, indicating that the two mutations were in the same complementation group.


Figure 7: Effects of mutations in UBC and UFD genes on ubiquitination of fusions bearing Ub and Ub moieties. Pulse-chase analysis was performed as described in the legend to Fig. 2(see also ``Experimental Procedures,'' Fig. 1, and Table 1). A, in the ubc4ubc5 strain EJY108: lanes 1-3, Ub-V-gal; lanes 4-6, Ub-V-gal; lanes 7-9, Ub-V-gal. B, same as A but in the ufd4 strain EJ23-1. C, same as A but in the ufd2 strain EJY130. For designations, see the legend to Fig. 2. Times of chase, test proteins, and ratios of the amount of S in ubiquitinated species to that in a band of the unmodified Ub-gal are indicated below the lanes.



Several partial clones of UFD4 were isolated from the insertion mutants by rescuing plasmids containing a portion of the mTn3 transposon and flanking S. cerevisiae DNA (see ``Experimental Procedures''). This flanking DNA was sequenced and found to be a previously sequenced ORF of chromosome XI (Pascolo et al., 1992). Insertions at six sites within this ORF (UFD4) were identified among several libraries of mTn3 insertions (Fig. 4C). UFD4 encodes a 1,483-residue (170 kDa) protein. Comparisons to proteins in data bases revealed that the sequence of a 280-residue C-terminal region of Ufd4p is similar to sequences in C-terminal regions of several other proteins, among which the E6-associated protein (E6AP) is the most extensively studied species (Fig. 5C). Outside of its conserved C-terminal region, Ufd4p is also similar to one other member of the E6AP family, a 1,992-residue protein encoded by a human cDNA (GenBank accession no. D28476).

E6AP is a 100-kDa mammalian protein that interacts with the E6 protein of high-risk (oncogenic) human papilloma viruses such as HPV16 and HPV18 (Huibregtse et al., 1991). A complex of E6 and E6AP binds to the tumor suppressor protein p53, accelerating the Ub-dependent degradation of p53 in reticulocyte extract and apparently also in vivo (in papilloma virus-infected cells). In vitro, a set of proteins sufficient for ubiquitination of p53 comprises the purified E6E6AP complex, Ub-activating (E1) enzyme, a specific Ub-conjugating (E2) enzyme, and Ub (Scheffner et al., 1993, 1995). In the presence of E1 and certain E2s, E6AP enhances ubiquitination of proteins other than p53 as well (Scheffner et al., 1993). Deletion analysis has shown that the E6AP's region of similarity to S. cerevisiae Ufd4p (Fig. 5C)) is required for the ability of E6AP to stimulate ubiquitination of p53; however, this region is not required for the binding of either E6 or p53 to E6AP (Huibregtse et al., 1993b). Thus, E6AP (and also, by inference, Ufd4p) is likely to be a member of a class of proteins called recognins, E3s or Ub ligases (Varshavsky, 1992; Ciechanover, 1994). The only other member of this class that has been analyzed genetically is N-recognin, the UBR1-encoded recognition component of the N-end rule pathway in S. cerevisiae (Bartel et al., 1990; Madura et al., 1993). Ubr1p and the analogous mammalian N-recognins function in association with specific E2 enzymes. Through binding to proteins that bear destabilizing N-terminal residues, N-recogninE2 complexes mediate ubiquitination and the subsequent (proteasome-dependent) degradation of these proteins (Varshavsky, 1992). The sequence of S. cerevisiae Ubr1p bears no significant similarities to E6AP, Ufd4p, or other E6AP-like proteins (Huibregtse et al., 1993a; data not shown).

Among the members of the E6AP family are a rat protein (Müller et al., 1992) and the hyperplastic discs gene product of Drosophila melanogaster (Mansfield et al., 1994). These proteins also contain regions of sequence similarity to RNA-binding proteins. Two other members of the E6AP family, the product of the mouse NEDD-4 cDNA and an S. cerevisiae ORF on chromosome V (accession numbers D10714 (EMBL) and L11119 (GenBank), respectively), contain three copies of a distinct 35-residue sequence.

UFD4 is located on the right arm of chromosome XI, between an ORF encoding a protein similar to the S. cerevisiae ribosomal protein L10 (Fig. 4C; YKL160) and the CCE1 gene, which encodes a cruciform-cutting endonuclease (Pascolo et al., 1992). Interestingly, an ORF (YKL165 in Fig. 4C) beyond CCE1 encodes a protein containing three copies of the 35-residue sequence that is also present (in three copies) in two members of the E6AP family but not in Ufd4p (see above).

The ufd4-1::TRP1 allele was constructed by replacing the entire UFD4 ORF save for its last 21 bp with a TRP1 marker (Fig. 4C). This allele was used to replace one of two UFD4 copies in a Trp diploid strain, as described above for UFD1. When the resulting ufd4-1::TRP1/UFD4 diploid was sporulated and the tetrads dissected, the Trp phenotype segregated 2:2, and all segregants grew at wt rates (data not shown), indicating that UFD4 is not essential for vegetative growth. ufd4 mutants also grew well at 37 °C, and were not hypersensitive to either UV light or canavanine. The results of pulse-chase analyses of Ub-V-gal in either the ufd4-1::TRP1 strain or the above ufd4 insertion mutants were similar to those obtained with the original ufd4-1 mutant (Fig. 2A, lanes 13-15, and data not shown). Thus, the C terminally truncated insertion alleles of Ufd4p (among them an allele encoding all but a 300-residue C-terminal region of Ufd4p, the region of similarity to E6AP) have the phenotype of the null allele. Using the same mTn3-containing library (see above), Burns et al.(1994) have isolated a different mTn3 insertion in UFD4 (YKL162) that yielded a fusion of the first 1,268 residues of Ufd4p to gal. Immunofluorescence experiments with an antibody to gal showed that the bulk of this fusion was present as 50 to 100 discreet spots in the cytoplasm (Burns et al., 1994).

The UFD5 Gene Is Identical to SON1

An approach similar to the one described above for UFD1 was used to isolate a gene that complemented the ufd5-1 proteolytic defect. Sequencing of the complementing region revealed that the putative UFD5 is identical to the previously described S. cerevisiae gene SON1 (Nelson et al., 1993). SON1 (UFD5) is located on the left arm of chromosome IV, adjacent to an ORF (ORFc, Fig. 4D) encoding a protein whose sequence is similar to that of phosphoglycerate mutase. Mutations in SON1 (UFD5) were originally isolated as extragenic suppressors of a ts growth defect of certain sec63 alleles; these suppressor mutations did not reverse another phenotype of sec63 alleles, mislocalization of proteins bearing nuclear localization signals (NLSs) (Nelson et al., 1993). son1 mutants were cold-sensitive and were themselves (in the SEC63 background) partially defective in the nuclear transport of proteins at low temperature (Nelson et al., 1993). The 531-residue Son1p (Ufd5p) contains an NLS and is located largely in the nucleus (Nelson et al., 1993). The sequence of Son1p (Ufd5p) contains two regions that are similar to sequences in some members of the Cys-His class of zinc finger domains (Fig. 5D), except that neither of these sequences in Son1p (Ufd5p) contains the conserved Cys residues at the consensus positions. However, these regions of Son1p (Ufd5p) do contain cysteines several residues N terminally to the consensus positions (see Nelson et al., 1993).

The ufd5-1::LEU2 allele was constructed by replacing the entire UFD5 (SON1) ORF and a short 5`-flanking sequence with a LEU2 marker (Fig. 4D). This allele was used to replace one of two copies of UFD5 (SON1) in a Leu diploid strain, as described above for UFD1. When the resulting ufd5-1::LEU2/ UFD5 diploid was sporulated and the tetrads dissected, the Leu phenotype segregated 2:2 and, as also found by Nelson et al.(1993), Leu segregants (containing the ufd5-1::LEU2 allele) formed smaller colonies at 30 °C than did Leu (UFD5) segregants (data not shown). The ufd5-1 strain was at most weakly hypersensitive to UV light or canavanine. The ufd5-1::LEU2 allele had the same effects on degradation of Ub-V-gal, R-gal, and Ub-V-DHFRha as did the original ufd5-1 allele (data not shown). A diploid (expressing Ub-P-gal) was produced by crossing the ufd5-1 strain to a ufd5 (son1) strain. This diploid formed blue colonies on X-gal plates, indicating that the cloned SON1 gene was indeed UFD5.

Lysine Residues of Ub That Are Required for Degradation of Ub-V-gal

The Lys residue of Ub is a major site of the Ub-Ub isopeptide bonds in substrate-linked multi-Ub chains (Chau et al., 1989; Gregori et al., 1990; Chen and Pickart, 1990; Glotzer et al., 1991; Hochstrasser et al., 1991; van Nocker and Vierstra, 1993). When Lys of the N-terminal Ub moiety in Ub-V-gal was converted into Arg, which cannot be ubiquitinated, the resulting Ub-V-gal (Fig. 1) was no longer short-lived, but it was still monoubiquitinated at an unknown lysine residue of its Ub moiety; a much smaller amount of a putative diubiquitinated species was also observed (Johnson(1992); see also Fig. 6A, lanes 7-9). To address the nature of these Ub conjugates, we used N-terminal Ub moieties with multiple Lys Arg substitutions.


Figure 6: Degradation of a Ub fusion is influenced by the presence of Lys and Lys in the fusion's Ub moiety. A: lanes 1-3, Ub-V-gal; lanes 4-6, Ub-V-gal; lanes 7-9, Ub-V-gal; lanes 10-12, Ub-V-gal. B: lanes 1-3, Ub-V-DHFRha; lanes 4-6, Ub-V-DHFRha; lanes 7-9, Ub-V-DHFRha; lanes 10-12, Ub-V-DHFRha. For designations, see the legend to Fig. 2. Times of chase, test proteins, and approximate half-lives are indicated below the lanes. Also indicated (below the lanes in A) are the ratios of S in each of the bands 1 and 2 to S in the Ub-V-gal band, and the steady-state levels of gal (its enzymatic activity, in nanomoles of CPRG hydrolyzed per min per mg of protein). The apparent discrepancy between the half-lives of Ub-V-gal and Ub-V-DHFRha in these experiments, in comparison to those in Fig. 2, stems from differences between the two S. cerevisiae strains used. The test proteins had reproducibly longer half-lives in JD52 (this Fig.) than in BWG1-7a (Fig. 2; see also Table 1).



Plasmids expressing Ub-V-gal, Ub-V-gal, and Ub-V-gal, whose N-terminal Ub moieties bore Lys Arg substitutions at the indicated positions (Fig. 1), were constructed and introduced into S. cerevisiae strain JD52 (Table 1). Pulse-chase analysis of the corresponding gal fusions showed them to be long-lived, with specific and reproducible patterns of ubiquitination (Johnson, 1992; data not shown). In particular, the ubiquitination patterns of Ub-V-gal and Ub-V-gal were indistinguishable from that of Ub-V-gal, indicating that Lys, Lys, and Lys were not among the major ubiquitination sites in Ub-V-gal. By contrast, at most trace amounts of ubiquitinated species were observed with Ub-V-gal, suggesting that one or more lysines among Lys, Lys, and Lys was the ubiquitination site(s) in Ub-V-gal. Arnason and Ellison(1994) have constructed Ub-peptide fusions in which the Ub moiety bears just one of the seven lysines of Ub. By expressing these fusions in S. cerevisiae, they have detected Ub-Ub linkages at only Lys, Lys, and Lys (Arnason and Ellison, 1994), suggesting that the ubiquitination site in the Ub moiety of Ub-V-gal (Fig. 6A, lanes 7-9) was Lys. Indeed, Ub-V-gal was long-lived and nearly devoid of ubiquitination, similarly to Ub-V-gal (Fig. 6A, lanes 10-12; data not shown). These results are consistent with the conjecture that Lys of Ub is the major ubiquitination site in Ub-V-gal. However, since we did not alter Lys and Lys individually, it was still possible (but quite unlikely, given especially the findings by Arnason and Ellison(1994)) that the Lys Arg substitution at position 29 affected ubiquitination at position 27 or 33.

Altering exclusively Lys of the Ub moiety had a dramatic effect: Ub-V-gal was long-lived and largely monoubiquitinated (t > 2 h; Fig. 6A, lanes 4-6). However, S. cerevisiae expressing either Ub-V-gal or Ub-V-gal had significantly lower steady-state levels of gal (as determined by measuring gal activity in extracts from mid-exponential cultures) than did a strain expressing Ub-V-gal (Fig. 6A, and data not shown). Thus, neither Ub-V-gal nor Ub-V-gal was as long-lived as Ub-V-gal, suggesting that Lys and Lys of the N-terminal Ub moiety make at least partially independent contributions to the degradation of Ub-V-gal.

Remarkably, analogous experiments in which the tetrameric (114 kDa per subunit) gal moiety was replaced with the monomeric, 22-kDa DHFRha moiety (bearing the hemagglutinin epitope tag) yielded different results. Specifically, Ub-V-DHFRha and Ub-V-DHFRha were long-lived in comparison to the initial test protein, Ub-V-DHFRha (Fig. 6B, lanes 1-6 and 10-12), similarly to the findings with gal counterparts of these fusions (Fig. 6A, lanes 1-6 and 10-12). Ub-V-DHFRha was strongly monoubiquitinated; as in the case of Ub-V-gal, the major ubiquitination site of Ub-V-DHFRha is likely to be Lys, because ubiquitination of Ub-V-DHFRha was barely detectable (Fig. 6B, lanes 4-6 and 10-12). However, Ub-V-DHFRha, in which only Lys was converted into Arg, was both ubiquitinated and rapidly degraded (Fig. 6B, lanes 7-9), in contrast to the result with Ub-V-gal (Fig. 6A, lanes 7-9).

Effects of Mutations in UFD Genes on the Structure of Multi-Ub Chains

Previous work (Johnson et al., 1992) has shown that a ubc4 mutant, which lacks the Ub-conjugating enzyme Ubc4p, is unable to degrade Ub-P-gal and analogous Ub fusions. In a ubc4 ubc5 mutant, which also lacks Ubc5p, a close homolog of Ubc4p (Seufert and Jentsch, 1990), Ub-V-gal was long-lived as well, and only two ubiquitinated derivatives of this fusion were observed (Fig. 7A, lanes 1-3). With Ub-V-gal in the same genetic background, the ubiquitination pattern was similar to the one observed with Ub-V-gal (Fig. 7A, lanes 4-6), indicating that Ubc4p/Ubc5p were not required for ubiquitination at the Lys position of Ub. By contrast, virtually no ubiquitination of Ub-V-gal was observed in the ubc4 ubc5 mutant, similarly to the findings with Ub-V-gal in wt cells (Fig. 7A, lanes 7-9). We conclude that Ubc4p/Ubc5p are required for ubiquitination at the Lys position of Ub.

Similar results (ubiquitination of Ub-V-gal but not of Ub-V-gal) were obtained when these experiments were carried out in the ufd4 background (Fig. 7B, lanes 1-9), indicating that Ufd4p is required for ubiquitination at the Lys but not at the Lys position of Ub. Ub-V-gal was ubiquitinated in the ufd4 mutant to a slightly lower extent than Ub-V-gal, suggesting, among other possibilities, that a functional homolog of Ufd4p may exist that (inefficiently) ubiquitinates the N-terminal Ub moiety at the Lys position in ufd4 cells.

The amount of multiubiquitinated derivatives of Ub-V-gal was also reduced in the ufd2 background, but to a lesser extent than in the ubc4 ubc5 or ufd4 mutants (Fig. 7C, lanes 1-3). Ubiquitination of Ub-V-gal in the ufd2 mutant was indistinguishable from that in wt (Fig. 7C, lanes 7-9; compare to Fig. 6A, lanes 7-9). By contrast, Ub-V-gal was ubiquitinated to a significantly lower extent in the ufd2 strain than in wt (8% in band 1 at 0 min of chase increasing to only 10% at 30 min of chase in ufd2, versus 11% in band 1 at 0 min increasing to >20% at 30 min in wt cells; Fig. 7C, lanes 4-6; compare to Fig. 6A, lanes 4-6). Thus, Ufd2p enhances ubiquitination at the Lys position of Ub but is not required for ubiquitination at Lys, the converse of the pattern observed with Ufd4p. The finding that Ufd2p is involved in ubiquitination at only the Lys position of Ub is likely to account for the apparently paradoxical observation that the ufd2 background impairs the degradation of Ub-V-gal but not of Ub-V-DHFRha (Fig. 2, A, lanes 7-9 and C, lanes 7-9), since degradation of Ub-V-DHFRha (unlike the degradation of Ub-V-gal) does not require ubiquitination at Lys (Fig. 6B, lanes 7-9).


DISCUSSION

We isolated S. cerevisiae mutants in the UFD pathway, a Ub-dependent proteolytic system that degrades Ub fusions such as Ub-V-gal. A common feature of these fusions is their nonremovable N-terminal Ub moiety (Johnson et al., 1992). We also cloned and analyzed four genes (UFD1, UFD2, UFD4, and UFD5) encoding components of the UFD pathway, and carried out tests with Ub fusions whose Ub moiety lacked certain Lys residues.

1) The UFD1 gene is essential for viability of vegetative cells. It encodes a 40-kDa protein that lacks significant sequence similarities to proteins of known function. The initially isolated missense allele of UFD1 stabilized the normally short-lived Ub-V-gal but did not significantly alter its post-translational ubiquitination, indicating that Ufd1p functions at a post-ubiquitination step in the UFD pathway.

2) The UFD2 gene is not essential for cell viability and encodes a 110-kDa protein whose sequence lacks significant similarities to sequences in data bases. The absence of Ufd2p stabilized the normally short-lived Ub-V-gal and significantly decreased the proportion of Ub-V-gal derivatives bearing multi-Ub chains. Strikingly, Ub-V-DHFRha, a counterpart of Ub-V-gal that contained the monomeric 22-kDa DHFRha reporter instead of the tetrameric (114 kDa per subunit) gal reporter, remained short-lived in the ufd2 mutant. A likely explanation of this result is given below (item 9).

3) In the ufd3 mutant, the degradation of Ub-V-gal was strongly reduced but still detectable. The formation of multiubiquitinated Ub-V-gal derivatives was also inhibited in this mutant. Attempts to clone UFD3 by complementation yielded the polyubiquitin gene UBI4, which was shown to be an extragenic suppressor of the ufd3 proteolytic defect. Consistent with this result, ufd3 mutants had greatly reduced levels of free Ub, and the degradation of Ub fusions in these mutants could be restored by overexpressing Ub. The UFD3 gene has recently been isolated in a screen unrelated to the screens of the present work.()

4) The UFD4 gene is not essential for cell viability and encodes a 170-kDa protein. The sequence of the last 280 residues of Ufd4p is similar to that of E6AP, a human protein that binds to both the E6 protein of oncogenic papilloma viruses and the tumor suppressor protein p53, whose Ub-dependent degradation requires E6AP (Huibregtse et al., 1993a; Scheffner et al., 1993). E6AP and, by inference, S. cerevisiae Ufd4p, belong to a class of proteins called recognins, E3s or Ub ligases (see Introduction). While the exact function of Ufd4p in the UFD pathway remains unknown, it has been found that Ufd4p is required for ubiquitination at the Lys but not at the Lys position of the N-terminal Ub moiety in Ub fusions such as Ub-V-gal (see item 9).

5) The UFD5 gene is not essential for cell viability but is required for normal growth rates. UFD5 is identical to the previously identified gene SON1 (Nelson et al., 1993). Mutations in SON1 were originally isolated as extragenic suppressors of a ts growth defect of certain sec63 alleles; these suppressor mutations did not reverse another phenotype of the above sec63 alleles, mislocalization of proteins bearing nuclear localization signals. The 60-kDa Son1p (Ufd5p) is a nuclear protein (Nelson et al., 1993). When ufd1-ufd5 mutants were tested for their ability to degrade substrates of the N-end rule pathway, short-lived proteins bearing certain (destabilizing) N-terminal residues (see Introduction), only UFD5 was found to be required for the degradation of N-end rule substrates such as R-gal. While the absence of Ufd5p stabilized both Ub-V-gal and R-gal, it did not strongly affect their multiubiquitination, indicating that Ufd5p functions at a post-ubiquitination step that is common to the UFD and N-end rule pathways.

6) Previous work (Johnson, 1992) has shown that the Lys Arg substitution at position 48 in the Ub moiety of Ub-V-gal rendered the resulting Ub-V-gal long-lived and precluded the formation of the bulk of multiubiquitinated Ub-V-gal derivatives. However, Ub-V-gal was still monoubiquitinated at an unknown lysine residue of its Ub moiety. To address the location of isopeptide bonds that form between the N-terminal Ub of Ub-V-gal and the post-translationally conjugated Ub, we employed N-terminal Ub moieties with multiple Lys Arg substitutions, and identified Lys of Ub as the likely major ubiquitination site in Ub-V-gal.

7) We also found that altering exclusively Lys of Ub in Ub-V-gal had the same dramatic effect as the previously examined alteration at Lys: Ub-V-gal was long-lived and largely monoubiquitinated. This, along with other evidence (see ``Results'') indicated that both Lys and Lys of the N-terminal Ub moiety contribute to the formation of multi-Ub chains required for the degradation of Ub-V-gal.

8) Unlike Ub-V-gal, which was long-lived (see item 7), the similarly designed Ub-V-DHFRha, which contained the monomeric 22-kDa DHFRha reporter instead of the tetrameric (114 kDa per subunit) gal reporter, remained short-lived in wt S. cerevisiae. Thus, the Lys Arg substitution at position 48 of Ub stabilized a gal-based fusion but not its DHFR-based counterpart. By contrast, the Lys Arg substitution at position 29 that yielded, respectively, Ub-V-gal and Ub-V-DHFRha, stabilized both of these fusions. We conclude that the formation of a Ub-Ub isopeptide bond at Lys is essential for the degradation of an oligomeric, gal-based Ub fusion but is not required for the degradation of an analogous but much smaller and monomeric, DHFR-based Ub fusion. Thus, the conformation and/or the overall size of a target protein evidently influences the requirements for distinct multi-Ub chains in targeting the protein for degradation by the UFD pathway. The relevant specific difference between the DHFRha and gal reporters is unknown.

9) Analyses of ubiquitination patterns with Ub-V-gal (and its derivatives lacking Lys and/or Lys) in different genetic backgrounds, ufd2, ufd4, and ubc4 ubc5, have shown that Ufd2p (see item 2) enhances ubiquitination at the Lys position of the N-terminal Ub moiety, but is not required for ubiquitination at Lys. Conversely, Ufd4p (see item 4) is required for ubiquitination at the Lys position of the N-terminal Ub moiety, but not for ubiquitination at Lys. Analogous to Ufd4p, the nearly identical Ub-conjugating enzymes Ubc4p and Ubc5p are also required in the UFD pathway for ubiquitination of the N-terminal Ub moiety at Lys. Ub-dependent systems that require Ubc4p/Ubc5p include, but are not limited to, the UFD pathway, inasmuch as the phenotype of ubc4 ubc5 strains (Jentsch, 1992) is much more severe than the phenotype of ufd4 strains. The fact that Ufd2p is involved in ubiquitination only at the Lys position of Ub is likely to account for the observation (see item 2) that the ufd2 background does not affect the degradation of Ub-V-DHFRha: indeed, the degradation of this test protein has been shown not to require ubiquitination at Lys of Ub (see item 8).

This work initiated systematic analysis of the UFD pathway, but leaves unanswered several key questions. For example: what are physiological UFD substrates? Since mutational impairment of the UFD pathway is phenotypically inconspicuous (see ``Results''), it is likely that, similarly to the N-end rule pathway, the UFD system targets a relatively small fraction of short-lived intracellular proteins. It is unknown whether physiological UFD substrates are largely proteins that bear Ub-like nonremovable domains (e.g. Banerji et al., 1990; Linnen et al., 1993; Watkins et al., 1993). An alternative possibility is that the multiubiquitination and degradation of Ub fusions such as Ub-V-gal by the UFD pathway is the consequence of a step that involves recognition of Ub moieties within a post-translationally added multi-Ub chain. In this interpretation, a nonremovable N-terminal Ub moiety of an engineered Ub fusion is recognized by a component of the UFD pathway that normally recognizes a post-translationally conjugated Ub within a multi-Ub chain. If so, physiological substrates of the UFD pathway may lack Ub-like domains.

The mechanistic differences between multi-Ub chains bearing isopeptide bonds at Lysversus Lys remain unknown. Recent identification of multi-Ub chains that involve Lys of Ub (Spence et al., 1995; Arnason and Ellison, 1994), and the finding that these chains are essential for certain aspects of DNA repair that also require the Ubc2p Ub-conjugating enzyme (Spence et al., 1995) have added yet another aspect to the problem of multi-Ub chains. One unanswered question in this area is whether physiologically relevant multi-Ub chains are of unmixed topology (i.e. either Lys, or Lys, or Lys), or whether multi-Ub chains of mixed topology (including, possibly, branching chains) also exist and have distinct functions.

Our data are neutral as to the possibility of a substrate-linked multi-Ub chain of mixed topology (both Lys and Lys). Since the N-terminal Ub moiety of Ub-V-gal can be conjugated to Ub through either Lys or Lys (see ``Results''), it is not excluded that this N-terminal moiety is actually linked to more than one Ub moiety (at both Lys and Lys), forming a branched multi-Ub chain. (This possibility is compatible with the relevant aspect of Ub structure, because Lys and Lys are located on opposite sides of the folded Ub globule (Vijay-Kumar et al., 1987).

While it is clear that a multi-Ub chain of at least the Lys topology has affinity for a specific component of the 26 S proteasome (Rechsteiner et al., 1993), the mechanistic functions of multi-Ub chains remain unknown. Two mutually nonexclusive classes of models for the function of a substrate-linked multi-Ub chain are first, that such a chain, through its binding to a component of the proteasome, reduces the rate of dissociation of a substrate-proteasome complex, and second, that a multi-Ub chain conformationally destabilizes a substrate to which it is covalently linked by interacting with the substrate noncovalently as well, in a manner analogous to that of chaperonins. Further dissection of the UFD pathway should help answering some of the questions posed above.


FOOTNOTES

*
This work was supported in part by National Institutes of Health Grants GM31530 and DK39520 (to A. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) U22153 [GenBank Link]and U22154[GenBank Link].

§
Supported by a Whitaker Health Sciences Fund predoctoral fellowship. Present address: The Rockefeller University, 1230 York Ave., New York, NY 10021-6399.

Present address: McKinsey & Co., Suite 2900, One First National Plaza, Chicago, IL 60603-2900.

**
Present address: Dept. of Chemistry & Biochemistry, University of Colorado, Boulder, CO 80309-0215.

§§
To whom correspondence should be addressed: Div. of Biology, 147-75, Caltech, 391 S. Holliston Ave., Pasadena, CA 91125. Tel.: 818-395-3785; Fax: 818-440-9821; varshavskya{at}starbase1.caltech.edu

The abbreviations used are: Ub, ubiquitin; ORF, open reading frame; wt, wild-type; kb, kilobase pairs; gal, E. coli -galactosidase; DHFRha, mouse dihydrofolate reductase bearing a 14-residue C-terminal extension containing the ``hemagglutinin'' epitope (see ``Experimental Procedures''); NLS, nuclear localization signal; PCR, polymerase chain reaction; UFD, Ub fusion degradation; bp, base pair(s); kb, kilobase pair(s); X-gal, 5-bromo-4-chloro-3-indolyl -D-galactoside; 5-FOA, 5-fluoroorotic acid; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; E6AP, E6-associated protein.

M. Ghislain and A. Varshavsky, unpublished results.


ACKNOWLEDGEMENTS

We thank M. Snyder for the yeast mTn3 insertion library; colleagues whose names are cited in the paper, especially J. Dohmen, R. Baker, and K. Madura, for providing strains and/or plasmids; A. Choy for technical assistance; and M. Ellison and D. Finley for sharing their data before publication. We also thank former and present members of this laboratory, especially J. Dohmen and M. Ghislain, for discussions and comments on the manuscript. E. S. J. thanks O. Lomovskaya and J. Ashkenas for helpful suggestions.


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