(Received for publication, March 27, 1995; and in revised form, May 11, 1995)
From the
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
In eukaryotes, a large fraction of intracellular proteolysis is
mediated by pathways whose common feature is the covalent conjugation
of ubiquitin (Ub) A Ub-protein
conjugate contains an isopeptide bond between the C-terminal Gly 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- Deubiquitination of a Ub fusion is completely inhibited if the
C-terminal Gly
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
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.
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.
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).
``e The
plasmid pUb
Figure 2:
Pulse-chase analysis of Ub fusions in S. cerevisiae ufd mutants. Cells were labeled with
Trans
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
The ufd1-
The ufd2-
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-
For immunoblot analysis,
extracts were made from mid-exponential (A
In wild-type (wt) cells, Ub In the ufd1 mutant, Ub In the ufd2 mutant,
Ub In the ufd3 mutant, the degradation of
Ub In the ufd4 mutant, Ub In the ufd5 mutant,
Ub
By contrast,
overexpression of Ub in the ufd3 mutant did reverse the
metabolic stabilization of 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-
We also
tested the ufd mutants for their ability to degrade R- The degradation of
R-
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- A complementation test was performed to
verify that the cloned gene was in fact UFD1. A ufd1 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
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 The ufd2- High-stringency Southern analysis of DNA from the ufd2-
Figure 7:
Effects of mutations in UBC and UFD genes on ubiquitination of fusions bearing Ub
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 ( E6AP is a 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 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 The ufd4-
The ufd5-
Figure 6:
Degradation of a Ub fusion is influenced
by the presence of Lys
Plasmids expressing Ub Altering exclusively Lys Remarkably, analogous experiments in
which the tetrameric (
Similar results (ubiquitination of
Ub The amount of multiubiquitinated derivatives
of Ub
We isolated S. cerevisiae mutants in the UFD
pathway, a Ub-dependent proteolytic system that degrades Ub fusions
such as Ub 1) The UFD1 gene is essential
for viability of vegetative cells. It encodes a 2) The UFD2 gene is not essential for cell
viability and encodes a 3) In the ufd3 mutant, the degradation of
Ub 4) The UFD4 gene is not essential for cell viability and encodes a
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 6) Previous work (Johnson, 1992) has shown that the Lys
7) We also found that altering
exclusively Lys 8) Unlike
Ub 9)
Analyses of ubiquitination patterns with Ub 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 The mechanistic differences between multi-Ub chains
bearing isopeptide bonds at Lys Our data are neutral as to the
possibility of a substrate-linked multi-Ub chain of mixed topology (both Lys While it is clear that a multi-Ub chain of at least the Lys
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].
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
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.
(
)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.
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).
-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).
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.
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.
Strains, Media, Genetic Techniques, and
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
KHgal
Assay
PO
-K
HPO
(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).
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.
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.
'' 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).
-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.
S-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'').
-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.
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.
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.
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
.
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).
Isolation of ufd Mutants and Pulse-Chase Analysis of
Ub
Yeast expressing a
short-lived -V-
gal
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).
-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.
-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).
-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).
-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.
-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).
-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
To determine
whether the metabolic stabilization of Ub-V-
gal Degradation
-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).
-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.
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).
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.
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).
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.
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.
or the
P
promoter. 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.
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).
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.
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.
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 ubc4
ubc5
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.
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).
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
E6
E6AP 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-recognin
E2
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).
35-residue
sequence.
35-residue sequence that is also
present (in three copies) in two members of the E6AP family but not in
Ufd4p (see above).
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).
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
The Lys-V-
gal
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.
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).
-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.
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.
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.
-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.
-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).
-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.
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.
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).
-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.
(
)
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).
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.
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.
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.
-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.
-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).
-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.
versus 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.
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).
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.
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.
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.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.