Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205
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Abstract |
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Maturation of the Saccharomyces cerevisiae
a-factor precursor involves COOH-terminal CAAX
processing (prenylation, AAX tripeptide proteolysis,
and carboxyl methylation) followed by cleavage of an
NH2-terminal extension (two sequential proteolytic processing steps). The aim of this study is to clarify the
precise role of Ste24p, a membrane-spanning zinc metalloprotease, in the proteolytic processing of the a-factor precursor. We demonstrated previously that Ste24p
is necessary for the first NH2-terminal processing step
by analysis of radiolabeled a-factor intermediates in
vivo (Fujimura-Kamada, K., F.J. Nouvet, and S. Michaelis. 1997. J. Cell Biol. 136:271-285). In contrast,
using an in vitro protease assay, others showed that
Ste24p (Afc1p) and another gene product, Rce1p,
share partial overlapping function as COOH-terminal
CAAX proteases (Boyartchuk, V.L., M.N. Ashby, and
J. Rine. 1997. Science. 275:1796-1800). Here we resolve
these apparently conflicting results and provide compelling in vivo evidence that Ste24p indeed functions at
two steps of a-factor maturation using two methods. First, direct analysis of a-factor biosynthetic intermediates in the double mutant (ste24 rce1
) reveals a previously undetected species (P0*) that fails to be COOH
terminally processed, consistent with redundant roles
for Ste24p and Rce1p in COOH-terminal CAAX processing. Whereas a-factor maturation appears relatively normal in the rce1
single mutant, the ste24
single
mutant accumulates an intermediate that is correctly
COOH terminally processed but is defective in cleavage of the NH2-terminal extension, demonstrating that
Ste24p is also involved in NH2-terminal processing. Together, these data indicate dual roles for Ste24p and a
single role for Rce1p in a-factor processing. Second, by
using a novel set of ubiquitin-a-factor fusions to separate the NH2- and COOH-terminal processing events
of a-factor maturation, we provide independent evidence for the dual roles of Ste24p. We also report here
the isolation of the human (Hs) Ste24p homologue, representing the first human CAAX protease to be
cloned. We show that Hs Ste24p complements the mating defect of the yeast double mutant (ste24
rce1
)
strain, implying that like yeast Ste24p, Hs Ste24p can
mediate multiple types of proteolytic events.
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Introduction |
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MANY proteins are synthesized as precursors that
undergo one or more maturation steps to attain
their full activity, to acquire proper localization,
or to facilitate protein-membrane or protein-protein interactions. The Saccharomyces cerevisiae mating pheromone a-factor provides an excellent model for dissecting
several distinct types of posttranslational modification
events. Fully mature a-factor (M)1 is a prenylated, carboxyl-methylated dodecamer that is initially synthesized
as a precursor encoded by the functionally redundant genes, MFA1 and MFA2. The a-factor precursor (P0) consists of the mature a-factor (12 residues) flanked by an
NH2-terminal extension (21 residues for Mfa1p) and a
COOH-terminal CAAX motif (C, cys; A, an aliphatic residue; X, one of several residues) (Michaelis and Herskowitz, 1988). A goal of our laboratory is to define the biosynthetic intermediates and cellular components necessary
for each step of a-factor biogenesis. Our current view of
a-factor maturation, derived from pulse-chase and SDS-PAGE analysis of a-factor biosynthetic intermediates, is
summarized in Fig. 1. Our studies have shown that a-factor biogenesis occurs in three ordered stages: (a) COOH-terminal CAAX processing, (b) NH2-terminal proteolysis
comprised of a pair of successive cleavage events, and (c)
export (Chen et al., 1997b
).
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In addition to a-factor, the COOH-terminal CAAX motif is present on a number of eukaryotic proteins. Examples include nuclear lamins of multicellular organisms, the
subunit of a heterotrimeric G protein, and most notably
Ras proteins and the Ras-related Rho proteins (for reviews see Clarke, 1992
; Schafer and Rine, 1992
; Zhang and
Casey, 1996
). The CAAX motif directs three sequential
posttranslational modification steps: (a) prenylation of the
cysteine by farnesyl or geranylgeranyl, (b) proteolytic cleavage to remove the AAX residues (herein referred to
as AAXing), and (c) carboxyl methylation of the newly exposed prenyl cysteine (Clarke, 1992
; Schafer and Rine,
1992
; Zhang and Casey, 1996
). The CAAX modifications
confer distinctive properties to the processed protein. For
Ras, the prenyl group is required for its activity and more
importantly, for its transforming activity (Casey et al.,
1989
; Schafer et al., 1989
; Kato et al., 1992
). Likewise for
a-factor, the farnesyl group facilitates membrane association before export and is necessary for promoting growth
arrest and mating activity by extracellular a-factor. Removal of the AAX tripeptide is required for methylation,
and in turn, methylation of a-factor is important for its intracellular stability, export, and receptor interaction (He
et al., 1991
; Marcus et al., 1991
; Sapperstein et al., 1994
).
In S. cerevisiae, the genetic analysis of mutants defective
in a-factor biogenesis has facilitated the identification and
characterization of the yeast CAAX processing components. These enzymes include (a) the farnesyl transferase
complex (Ram1p/Ram2p); (b) the CAAX prenyl proteases that carry out AAXing (Rce1p or Ste24p [see below]); and (c) the carboxyl methyltransferase (Ste14p) (Hrycyna and Clarke, 1990; Schafer et al., 1990
; He et al.,
1991
; Boyartchuk et al., 1997
). The genes encoding mammalian CAAX processing enzymes that have been cloned
to date are the rat, bovine, and human farnesyltransferases; the rat and human geranylgeranyltransferase; and
the human prenyl protein carboxylmethyltransferase
(Kohl et al., 1991
; Andres et al., 1993
; Zhang, 1994 [no.
1610]; Dai et al., 1998
). In contrast, no mammalian CAAX
protease genes have as yet been identified.
For a-factor, COOH-terminal CAAX processing is only
the first stage of its maturation. CAAX processing of the
a-factor precursor (P0) produces an intermediate (P1) that
is completely modified at the COOH terminus (farnesylated, AAXed, and carboxyl methylated) but contains an
intact NH2-terminal extension (see Fig. 1). The NH2-terminal extension is removed in two sequential and obligatorily ordered NH2-terminal proteolytic cleavages, the first
one yielding the partially processed precursor (P2), and the second cleavage generating fully mature a-factor (M). We recently showed that Ste24p is required for the first
NH2-terminal proteolytic step (P1 P2 processing) (Fujimura-Kamada et al., 1997
), as discussed below. The second step (P2
M) is mediated by Axl1p and can also be
carried out redundantly by Ste23p (Adames et al., 1995
).
Upon completion of the NH2-terminal processing steps,
mature a-factor is exported from the cell via the Ste6p
transporter, a member of the ATP-binding cassette superfamily (Kuchler et al., 1989
; McGrath and Varshavsky,
1989
; Michaelis, 1993
).
The aim of this study is to clarify the role of Ste24p, a
predicted multiple membrane-spanning zinc metalloprotease, in the biogenesis of a-factor. Interestingly, the
STE24 gene was identified by two independent genetic
screens that assigned different functions for Ste24p in
a-factor maturation (Boyartchuk et al., 1997; Fujimura-Kamada et al., 1997
). Our laboratory isolated STE24 as a
mating-defective mutant (hence the designation ste) in a
screen specifically aimed at identifying mutants with reduced mating efficiency (Fujimura-Kamada et al., 1997
).
A ste24 mutant accumulates the a-factor intermediate P1
in vivo. Since P1 is fully COOH terminally modified but its
NH2-terminal extension is not proteolytically removed in
the ste24 mutant, we concluded that Ste24p is required for
the first NH2-terminal processing step (P1
P2) of a-factor
maturation. In a separate screen using a mutant version of
a-factor with an altered CAAX motif (CAMQ instead of
CVIA), Boyartchuk et al. (1997)
also identified STE24
(called AFC1 in their study, a-factor converting enzyme).
Using an in vitro assay for release of the AAX tripeptide, ste24 mutants showed reduced AAXing activity. Boyartchuk et al. (1997)
concluded that Ste24p and a second
functionally redundant protein, Rce1p, share overlapping
roles in the COOH-terminal AAXing step of a-factor maturation. Rce1p, which is predicted to contain multiple
membrane spans, bears no sequence similarity to Ste24p,
and lacks any known protease motifs. Although STE24 was identified in genetic screens based on defective extracellular a-factor production, the two reports reached surprisingly different conclusions regarding the role of Ste24p
in a-factor maturation. Likely explanations for the divergent findings are that Boyartchuk et al. (1997)
examined
only COOH-terminal processing in their in vitro AAXing
assay and our study did not detect an AAXing defect in
vivo for the single ste24 mutant because AAXing can be
carried out redundantly by Rce1p.
In this study, we reconcile the apparently conflicting data for the roles of Ste24p in a-factor processing. We examine both NH2- and COOH-terminal processing of a-factor in vivo in strains deleted for STE24 and RCE1. By directly analyzing the a-factor biosynthetic intermediates produced by the mutant strains, we provide evidence that Ste24p indeed participates in both NH2- and COOH-terminal processing steps. In contrast, Rce1p is involved only in CAAX processing, not NH2-terminal cleavage of a-factor. We also use an independent method employing ubiquitin (Ubi)-a-factor fusions that uncouple the NH2- and COOH-terminal processing steps to demonstrate the dual roles for Ste24p in a-factor maturation. Finally, we report here the cloning of the human Ste24p homologue, the first mammalian CAAX prenyl protease. We show that Hs Ste24p can complement the yeast double deletion (ste24 rce1) strain for mating.
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Materials and Methods |
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Strains and Media
The yeast strains used in this study are listed in Table I. The rce1-1::
TRP1 deletion allele, referred to as rce1
, replaces codons 116 to the stop
codon 315 with TRP1. Strains (SM3613, SM3614, SM3689, and SM3691)
harboring the rce1
allele were constructed using one-step gene disruption by transforming SM1058, SM3103, SM2331, and SM3375, respectively, with a BamHI-XhoI fragment from pSM1285 bearing rce1-
1::
TRP1, and selecting for Trp+ transformants. The ste24
::LEU2 deletion
allele, referred to as ste24
, is a
disruption of ste24 that eliminates nearly
the entire coding sequence (codons 1-444 of 453 total). Strains (SM3375
and others) harboring this allele were constructed by transformation of
SM2331 with linearized pSM1072 and selection of Leu+ transformants, as
described (Fujimura-Kamada et al., 1997
). All deletion strains were confirmed by Southern analysis. MAT
strains bearing the single (rce1
) and
the double (ste24
rce1
) deletions were constructed by mating-type
switching of MATa strains SM3613 and SM3614, respectively, using the
HO endonuclease as described (Herskowitz and Jensen, 1991
). Yeast
transformations were performed by the Elble method (Elble, 1992
). All
strains were grown at 30°C in complete yeast extract/peptone/dextrose (YPD) media, synthetic complete drop-out (SC-URA, TRP, LEU), or synthetic minimal media (SD) (Michaelis and Herskowitz, 1988
; Kaiser et al.,
1994
).
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Patch Mating Test and a-Halo Assay
To assay mating, we used the semiquantitative patch mating test as described previously (Fujimura-Kamada et al., 1997). In this assay, master
plates containing patches of strains to be tested are replica plated onto a
mating tester lawn of the opposite mating type that has been spread on an
SD plate. The lawn is prepared by resuspending the mating tester strain in
YPD and spreading 0.3 ml of this suspension onto an SD plate. The stringency for the patch mating test was increased by resuspending the mating tester lawns in decreasing concentrations of YPD diluted in sterile water
(100% YPD is permissive, 1% YPD is stringent) since limiting nutrients
decrease plate mating efficiency, and thereby increase the mating test sensitivity. The strains tested are Trp
, Leu
, or His
auxotrophs, whereas
the mating testers (SM1067 and SM1068) are Lys
. Only cells that mate to
generate prototrophic diploids are able to grow on SD minimal media after incubation for 2 d at 30°C.
To compare the level of extracellular a-factor for wild-type and mutant
strains, we performed the a-factor spot halo assay as described previously
(Nijbroek and Michaelis, 1998). Serial dilutions of concentrated a-factor
(2 µl) were spotted onto a lawn of supersensitive halo tester cells
(SM1086) spread on a YPD plate. Plates were incubated for 1 d at 30°C.
Plasmid Constructions
The plasmids used in this study are listed in Table II. The single-step gene
disruption plasmid, pSM1285, used to generate rce1-1::TRP1 alleles, was
constructed as follows: A HindIII-MluI fragment containing the RCE1
open reading frame (ORF) from pHY01 (provided by A. Toh-e, University of Tokyo, Tokyo, Japan) (Yashiroda et al., 1996
) was rendered blunt-ended with Klenow and subcloned into the EcoRV-SmaI sites of pBluescriptIISK (Stratagene, La Jolla, CA) to yield pSM1284. Plasmid
pSM1284 was digested with EcoRI to remove a fragment corresponding
to the last 200 codons of the RCE1 ORF, which was replaced with a TRP1
EcoRI fragment from pUC18-TRP1 (Sapperstein et al., 1994
) to generate
pSM1285.
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Ubi-a-factor fusion constructs encode chimeric proteins consisting of
ubiquitin (76 residues) fused either to the full-length a-factor precursor
encoded by MFA1, to the NH2 terminally truncated a-factor (P2) (see Fig.
1), or to mature a-factor (M). All of these fusions contain the intact
COOH-terminal CAAX motif and are expressed under the control of the
MFA1 promoter. These constructs are designated Ubi-P1, Ubi-P2, and
Ubi-M, where the a-factor segments correspond to codons 1-36 (full-length), codons 8-36, and codons 21-36 of MFA1-encoded a-factor, respectively. The fusion constructs were generated by recombination-mediated PCR cloning, a method in which a linearized or gapped acceptor plasmid serves as the target for homologous recombination directed by a
donor PCR fragment in yeast (Muhlrad et al., 1992; Oldenburg et al.,
1997
). The PCR fragments were amplified with oligonucleotides encoding
precise fusion junctions between the COOH terminus of ubiquitin and the
NH2 terminus of the various species of a-factor.
The linear target vector and the donor PCR fragment were cotransformed into a strain deleted for the chromosomal a-factor genes mfa1
and mfa2
(SM2331). Subsequently, candidate plasmids were screened by
yeast colony PCR. In brief, crude yeast extracts were prepared by incubating a small amount of yeast cells in 60 µl of lysis buffer (0.45% NP-40,
0.45% Tween 20, 50 mM KCl, 10 mM Tris, pH 8.3, 1.5 mM MgCl2, 0.1%
gelatin, 0.3 mg/ml zymolyase) for 90 min at 37°C. The cleared lysate (5 µl)
was used as the template for the PCR screening. Plasmids were recovered
from yeast as described (Robzyk and Kassir, 1992
). Ubi-P1, Ubi-P2, and
Ubi-M are encoded by pSM1368, pSM1369, and pSM1366, respectively.
All Ubi-a-factor fusion plasmids were amplified in Escherichia coli strain
DH5
, prepared by alkaline lysis, analyzed by restriction digests, and then
confirmed by DNA sequencing.
Metabolic Labeling, Immunoprecipitation, and SDS-PAGE
To examine a-factor biosynthetic intermediates, extracts were prepared
from metabolically labeled cells and radiolabeled proteins were immunoprecipitated with anti-a-factor antiserum and analyzed by SDS-PAGE, essentially as previously described (Chen et al., 1997b; Fujimura-Kamada et al.,
1997
). In brief, 5 OD600 U log phase cells were harvested and resuspended
in 250 µl of SD media supplemented with the appropriate amino acids. Cells were pulse labeled with 150 µCi [35S]cysteine for the indicated times,
and the labeling was stopped on ice by the addition of an equal volume of
2× azide buffer (40 mM methionine, 40 mM cysteine, 20 mM NaN3, 500 mg/ml BSA). For pulse-chase experiments, chases were initiated by addition of 10 µl of 1 M cysteine per time point. Intracellular (I) and extracellular (E) fractions were processed as described (Chen et al., 1997b
; Fujimura-Kamada, et al., 1997). Radiolabeled a-factor was immunoprecipitated with anti-a-factor antiserum 9-137 or 9-497, and the immunoprecipitated material was subjected to SDS-PAGE and PhosphorImager
(Molecular Dynamics, Inc., Sunnyvale, CA) analysis as described (Chen
et al., 1997b
). To maximize the resolution between partially processed
a-factor precursors, we used 16% polyacrylamide separating gels that
were 13 cm in length. The half-life of each a-factor precursor (P0* or P1)
was determined by quantitation of the [35S] counts corresponding to the
a-factor signal using ImageQuant software (Molecular Dynamics, Inc.).
The 0 min time point was used as the 100% reference value for each time
course experiment.
a-Factor Carboxyl Methylation Assay
The carboxyl methylation levels of the immunoprecipitated a-factor intermediates that had been cut out of a dried polyacrylamide gel were measured (Fujimura-Kamada et al., 1997). In general, the procedure involves
generating double-labeled a-factor with [3H] at the carboxyl methyl group
and with [35S] at the prenyl cysteine. For each a-factor species (P0*, P1,
P2, or M), the ratio of [3H]/[35S] cpm reflects the absolute methylation
level. The relative methylation level is then determined by dividing the
absolute methylation level of the mutant strain by that of the wild-type
strain and converting this number to a percentage value.
Specifically, this procedure was carried out in two steps. First, cells were radiolabeled, immunoprecipitated, and subjected to SDS-PAGE, as described in the previous section, with the following changes: cells were double-labeled with 50 µCi S-adenosyl-L-[3H-methyl]methionine and 150 µCi [35S]cysteine for 6 min. For this assay, labeled cells were not lysed by base treatment since base hydrolysis could release the methyl esters which we wish to detect in this assay (see below). Instead, protein extracts were prepared by vortexing labeled cells at 4°C with zirconium beads in breaking buffer (50 mM potassium phosphate buffer, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 1 mM 2-mercaptoethanol, 1 mM PMSF). After separation by SDS-PAGE, the radiolabeled and immunoprecipitated a-factor intermediates were visualized by autoradiography.
Second, to determine the extent of carboxyl methylation for each a-factor intermediate, we measured the levels of [3H] and of [35S] incorporation
by scintillation counting. The a-factor gel bands were excised from dried
gels. The [3H] labeling was determined by the vapor-phase diffusion assay, which detects volatile [3H]methyl esters (i.e., carboxyl methyl) cleaved by
base hydrolysis (Xie et al., 1990; Hrycyna et al., 1991
). Gel slices (P0*, P1, P2, and M) were placed in an open microfuge tube containing 150 µl of 1 M
NaOH. The microfuge tube was placed inside a tightly capped 20 ml scintillation vial that contained 5 ml of scintillation fluid such that the contents
of the microfuge tube did not mix with the scintillation fluid. After incubation at 37°C for 24-36 h, the released volatile [3H]methanol was measured
by scintillation counting. The total amount of [35S] incorporated into each
a-factor species was then determined. First, the contents of the microfuge
tube (including the gel slice) were neutralized with 100 µl of glacial acetic
acid and then dissolved in 1 ml of Solvable (New England Nuclear Research Products, Boston, MA), a tissue solubilizer, in capped microfuge
tubes at 65°C for 6 h. The [35S] counts of the entire sample were then measured by liquid scintillation counting. The ratio of [3H] cpm to [35S] cpm
was calculated for each a-factor species to give the absolute methylation
level. The relative methylation level was determined by dividing the absolute methylation level of the mutant strain by the absolute methylation
level of the corresponding wild-type a-factor species.
Human STE24 Cloning
A BLAST search (Altschul et al., 1990) of the database of expressed
sequence tags (ESTs) revealed several human ESTs with high amino acid
similarity to S. cerevisiae (Sc) Ste24p. These ESTs represented cDNAs
from many cell types. A 703-bp HindIII fragment from EST N76181
National Center for Biotechnology Information (NCBI) (no. 69928) was
used to screen ~3 × 106 clones from a human B cell cDNA library (provided by S. Elledge, Baylor College of Medicine, Houston, TX) by standard methods (Ausubel, 1987). Nine positive clones were obtained and
sequenced, each of which encoded partial ORFs corresponding to the 3'
end of human STE24. The most complete Homo sapiens (Hs) clone
contained an ORF encoding 383 residues and included the poly A tail. To
obtain the remaining 5' Hs STE24 ORF, this clone was used in 5' rapid amplification of cDNA ends (RACE) PCR procedure with a fetal brain library according to the Marathon-Ready cDNA kit (Clontech, Palo Alto,
CA). The full-length STE24 ORF was subcloned by recombination-mediated PCR cloning into a yeast plasmid such that the coding sequence of Sc
STE24 was precisely replaced with that of Hs STE24. The resulting plasmid (pSM1468) contains the 5' upstream and 3' downstream sequences of
the yeast STE24 ORF flanking the human STE24 ORF.
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Results |
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Mating Phenotype and Extracellular a-Factor
Production by ste24, rce1
, and ste24
rce1
Mutants
To examine the roles of Ste24p and Rce1p in a-factor maturation, we first compared the extracellular a-factor produced from strains deleted for STE24 and RCE1. We used
the spot halo dilution assay, which provides a semiquantitative measure of the amount of mature a-factor secreted
by these strains. In this assay, extracellular a-factor causes
the growth arrest of a lawn of supersensitive MAT sst2
cells, resulting in a clear zone (Michaelis and Herskowitz,
1988
; Nijbroek and Michaelis, 1998
). The relative amounts
of a-factor produced by the different strains are compared
by determining the highest dilution that yields a clear spot
(Fig. 2 A). Whereas the ste24
mutant produces significantly decreased levels of extracellular a-factor (eightfold
less than wild-type), the rce1
mutant is virtually indistinguishable from wild-type (Fig. 2 A). In contrast, we detect
no extracellular a-factor for the double ste24
rce1
mutant. This result is consistent with the notion that Ste24p and Rce1p play redundant roles in the COOH-terminal
AAXing step of a-factor maturation, such that only one
gene must be intact for AAXing to occur, as proposed by
Boyartchuk et al. (1997)
. However, the more severe phenotype of the ste24
single mutant versus the rce1
single
mutant most likely reflects the additional role of Ste24p in
the NH2-terminal processing of a-factor.
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A comparison of the MATa strains in a plate mating assay indicates that their mating efficiencies parallel the results of the a-factor spot dilution assay. As shown in Fig. 2 B, the rce1 mutant mates like wild-type as indicated by
the confluent growth of diploids, whereas the ste24
mutant has a leaky mating defect as indicated by growth of
fewer diploids. The double mutant fails to mate altogether, consistent with its complete lack of extracellular
a-factor production (Fig. 2 B). Importantly, the mating
defects exhibited by these mutants is MATa cell type-specific, since MAT
strains bearing either the single (ste24
) or double (ste24
rce1
) mutations did not have any mating defects (Fig. 2 B). Because MATa and MAT
cells differ mainly by pheromone and receptor expression, the
MATa mating defects seen in the ste24
and ste24
rce1
mutants are likely to reflect specific effects on a-factor biogenesis.
A Novel a-Factor Intermediate (P0*) Is Observed in the
Double ste24 rce1
Mutant
To directly examine the roles of STE24 and RCE1 in a-factor maturation, we compared the a-factor biosynthetic intermediates produced in strains deleted for STE24 and
RCE1. Cells were metabolically labeled, and the a-factor
biosynthetic intermediates were immunoprecipitated and
separated by SDS-PAGE. For the wild-type strain, the
typical profile of the intracellular (I) forms of a-factor includes the partially processed precursor species (P1 and
P2) and mature (M) a-factor (Fig. 3 A, lane 1; refer to Fig.
1 for a description of each band). The band migrating
slightly faster than M is the a-factor-related peptide
(AFRP), whose biogenesis involves mechanisms and machinery distinct from those used to generate mature a-factor (Chen et al., 1997a). The extracellular (E) fraction contains exported mature (M) a-factor (Fig. 3 A, lane 1). For
the rce1
mutant, the biosynthetic profile is similar to
wild-type (Fig. 3 A, lane 2). Because the stepwise removal
of the NH2-terminal extension appears to be normal in
the rce1
mutant, Rce1p does not play a major role in
NH2-terminal processing of a-factor. Furthermore, this apparently normal processing in the rce1
mutant is expected if Ste24p and Rce1p play redundant functions in
COOH-terminal AAXing of a-factor (see below), since
Ste24p would compensate for the lack of Rce1p. In contrast, the ste24
mutant has a dramatic phenotype of P1
accumulation (Fig. 3 A, lane 3), as we have also shown previously (Fujimura-Kamada et al., 1997
). In the ste24
mutant, COOH-terminal AAXing is complete but the cleavage of the NH2-terminal extension is defective, indicating
that Ste24p is necessary for the first NH2-terminal cleavage step (P1
P2) in a-factor maturation (see also Fig. 3
B). Notably, only a tiny amount of mature a-factor produced by the ste24
mutant is detected in the extracellular
fraction by SDS-PAGE and autoradiography after a long
exposure (Fujimura-Kamada et al., 1997
); the more sensitive spot halo assay detects this low level of extracellular
mature a-factor (refer to Fig. 2 A).
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If Ste24p does indeed function redundantly in COOH-terminal AAXing with Rce1p, then we predict that the
double mutant (ste24 rce1
) would be completely defective for a-factor AAXing. Such a processing block should
produce a new intermediate (P0*) that is prenylated but
retains its AAX and thus cannot be methylated (refer to
Fig. 1). In the double mutant (ste24
rce1
), we observe a
single band, designated P0*, that shows a subtle mobility
shift compared with P1 (Fig. 3 A, compare lane 4 with 3).
Since this slight mobility difference is difficult to detect
consistently, we used a methylation assay, described below, to show that the intermediate generated by the double mutant is truly distinct from P1.
Methylation can serve as an indirect AAXing assay,
since AAXing is required to expose the methylation substrate, the free carboxyl of prenyl cysteine (Hrycyna and
Clarke, 1992; Ashby and Rine, 1995
; Hrycyna et al., 1995
).
We measured the degree of carboxyl methylation for each
intracellular a-factor species made in wild-type and mutant strains. Cells were metabolically double-labeled with S-adenosyl-L-[3H-methyl]methionine and [35S]cysteine,
which label the COOH-methyl and prenyl cysteine of a-factor, respectively. Methylation levels were first normalized to protein levels ([3H]/[35S]) and then calculated as a
fraction of the corresponding a-factor intermediate from
the wild-type strain (refer to Materials and Methods). As
we have previously shown, the P1 intermediate produced by
the ste24
mutant is completely methylated relative to the methylation levels of the wild-type strain (Fig. 3 B)
(Fujimura-Kamada et al., 1997
), indicating that this strain
has AAXing activity, presumably mediated by Rce1p. In
the rce1
mutant, we also detect a significant amount of
methylation of P2 and M, although the levels are somewhat
reduced, and the slowest migrating species (Fig. 3 B, top
band) is not detectably methylated (see below). The methylation that occurs in the rce1
mutant most likely reflects the AAXing activity mediated by Ste24p. In contrast to the
single mutants, the double ste24
rce1
mutant produces a
single intermediate that is completely unmethylated (Fig.
3 B), and thus represents P0*, a prenylated but unAAXed
biosynthetic intermediate of a-factor. We previously have
not detected P0* probably because it is converted to P1
very quickly. Unexpectedly, the rce1
mutant produces a precursor species (Fig. 3 A, lane 2, top band) that is likely to be P0* because it is unmethylated (Fig. 3 B). In this
case, and in contrast to P0* of the double ste24
rce1
mutant, P0* in the rce1
mutant is converted to P2 and then
to M. A likely explanation is that when Ste24p recognizes
P0* in the rce1
mutant, the dual roles of Ste24p permit it
to cleave P0* at both the NH2 and COOH termini to generate a P2-like intermediate that is subsequently methylated. Our conclusions from this analysis of a-factor intermediates generated in vivo are consistent with AAXing
roles for both Ste24p and Rce1p, and an additional NH2-terminal processing role for Ste24p only.
The Biosynthetic Intermediate, P0*, Is Metabolically Unstable
We have shown elsewhere that a defect in the carboxyl
methylation of a-factor correlates with the metabolic instability of a-factor biosynthetic intermediates (Sapperstein et al., 1994). We compared the metabolic stability of
P0* and P1 generated by the double (ste24
rce1
) and
single (ste24
) mutants, respectively, by pulse-chase analysis (Fig. 4 A). This experiment reveals that the methylated
P1 intermediate that accumulates in the ste24
mutant is
metabolically stable with a half-life greater than 60 min
(Fig. 4, A, lanes 7-9 and B). In contrast, the half-life of the
unmethylated P0* species present in the double ste24
rce1
mutant is dramatically less (half-life of ~9 min) (Fig.
4, A, lanes 10-12 and B). The biosynthetic profile and metabolic stability of the a-factor intermediates in the single
rce1
mutant does not appear to be significantly different
from that of the wild-type strain (Fig. 4 A, compare lanes
4-6 with 1-3).
|
Taken together, the experiments carried out in Figs. 3
and 4 indicate that although the gel mobility difference between P0* and P1 is small, they can be discriminated by a
large difference in their methylation level and metabolic
stability. P0* is unstable and unmethylated whereas P1 is
metabolically stable and methylated. The CAAX processing of a-factor in the double mutant (ste24 rce1
) fails to
proceed beyond P0*, and thus is consistent with redundant
roles for Ste24p and Rce1p in COOH-terminal AAXing. In contrast, the single ste24
mutant accumulates the stable, methylated P1 intermediate, indicating that COOH-terminal AAXing can be carried out via Rce1p and that
Ste24p is needed to mediate the first NH2-terminal P1
P2
processing step in a-factor biogenesis.
Characterization of a-Factor Species Produced by Ubiquitin-a-Factor Fusions
Since Ste24p appears to have dual roles in a-factor maturation involving both NH2- and COOH-terminal steps, we
sought a method to uncouple the two processing events.
This goal necessitated a way to produce mature active
a-factor independent of the normal NH2-terminal processing steps. The expression of NH2-terminally truncated
MFA1 to directly generate the desired a-factor intermediates posed two problems: (a) translation requires an initiator methionine and thus would yield abnormal versions of the a-factor intermediates and (b) such MFA1 truncation mutants were expressed poorly (Nouvet, F., and S. Michaelis, unpublished data). Instead, we used a method
for expressing polypeptides that does not require the initiator methionine (Bachmair et al., 1986). In this system, the
ubiquitin structural gene is fused to the gene of interest,
a-factor in our case. During translation, the ubiquitin moiety is recognized by ubiquitin-specific proteases (Ubp),
which cleave precisely after the COOH-terminal residue of
ubiquitin (Tobias and Varshavsky, 1991
; Baker et al., 1992
), and thus should yield the desired a-factor intermediates.
For this study, we made three ubiquitin-a-factor fusion constructs, designated here as Ubi-a-factor fusions (Fig. 5 A). We fused the ubiquitin gene to the full-length MFA1 gene (Ubi-P1), and to the truncated MFA1 corresponding either to the P2 intermediate (Ubi-P2) or to mature a-factor (Ubi-M). Ubi-P2 lacks the first seven residues of the MFA1 precursor and Ubi-M lacks the entire NH2-terminal extension. As shown in Fig. 5 B, Ubi-P1, Ubi-P2, and Ubi-M directly generate the respective a-factor species that are depicted diagrammatically in Fig. 5 A.
|
These Ubi-a-factor fusions show normal production of mature exported a-factor, as determined by pulse-chase metabolic labeling, immunoprecipitation, and SDS-PAGE. As expected, the biogenesis profiles of Ubi-P1 and wild-type MFA1 are indistinguishable from one another (Fig. 5 B, compare lanes 5-8 with lanes 1-4). The ubiquitin moiety appears to be rapidly and effectively cleaved. Likewise, Ubi-P2 produces P2 directly, since it lacks the first seven residues of the NH2-terminal extension. P2 is then processed correctly to M, which is exported (Fig. 5 B, lanes 9-12). The Ubi-M construct directly yields the predicted mature a-factor, which is exported normally (Fig. 5 B, lanes 13-16). However, the intracellular mature species derived from Ubi-M is metabolically unstable (Fig. 5 B, lanes 13-16). By the a-factor spot dilution assay and by mating assays carried out under stringent conditions, a strain bearing Ubi-M produces somewhat reduced extracellular a-factor activity compared with Ubi-P1 and Ubi-P2 (Fig. 5, C and D). Presumably this is due to the lower steady-state amount of exported a-factor, which in turn possibly reflects inefficient methylation or perhaps a protective role normally played by the NH2-terminal extension (see Discussion). Since the Ubi-P1 and Ubi-P2 constructs produce mature a-factor that is stable and supports mating (Fig. 5), we used these two constructs to study specific processing steps in a-factor biogenesis.
Ubi-a-Factor Fusions Provide Independent Evidence that Ste24p Plays a Critical Role in the NH2-terminal Processing of a-Factor
We used the Ubi-a-factor fusion constructs to confirm the
role of Ste24p in the first NH2-terminal cleavage step of the a-factor precursor. To do so, we compared the processing of Ubi-P1 versus Ubi-P2 in the ste24 mutant (Fig. 6).
As expected, MFA1 and Ubi-P1, which both possess the
Ste24p NH2-terminal cut site (P1
P2), are not properly
processed to mature a-factor in the ste24
mutant (Fig. 6,
lanes 4 and 5). In contrast, mature a-factor expressed from
the Ubi-P2 construct bypasses the requirement for STE24
in the ste24
mutant (Fig. 6, lane 6). The STE24 requirement in the production of mature a-factor by Ubi-P1, but
not by Ubi-P2, provides strong independent evidence for
the role of Ste24p in the first NH2-terminal cleavage step of a-factor. Interestingly, these data also verify the obligatory sequential order of the two NH2-terminal processing steps of the a-factor precursor, reinforcing the notion that
the first seven residues of the NH2-terminal extension
must be removed before Axl1p can act in the second NH2-terminal processing step.
|
Ubi-P2 Provides Independent Evidence for the Overlapping Roles for Ste24p and Rce1p in COOH-terminal AAXing of a-Factor
Since Ubi-P2 can bypass the need for STE24 in NH2-terminal processing, we could study the involvement of
Ste24p in the COOH-terminal AAXing of a-factor in a
system where its NH2-terminal processing activity was not
required to generate mature a-factor. Thus, we were able
to probe the individual contributions of Ste24p and Rce1p
solely with respect to the AAXing of a-factor. We examined the amount of extracellular a-factor derived from
Ubi-P2 in wild-type and mutant strains by the spot halo assay. As shown in Fig. 7 A, compared with the wild-type,
the single mutants harboring Ubi-P2 show reduced but
equivalent levels of a-factor activity. This result with Ubi-P2
contrasts with our findings with MFA1 where the single
ste24 and rce1
mutants differ markedly in extracellular
a-factor levels (refer to Fig. 2 A). Apparently this difference is due to the additional role of Ste24p in NH2-terminal processing of the full-length MFA1-encoded a-factor.
The use of Ubi-P2 thus allows us to dispense with the need
for the NH2-terminal processing activity and permits us to
conclude that Ste24p and Rce1p can play roughly equivalent roles in contributing to the AAXing of a-factor. As
expected, the double mutant fails to produce any extracellular a-factor from Ubi-P2, confirming the redundant roles
for Ste24p and Rce1p in a-factor AAXing.
|
We also examined the production of mature a-factor
from Ubi-P2 by pulse-chase analysis in wild-type and mutant strains (Fig. 7 B). As predicted by the spot dilution
test, extracellular a-factor is generated by the single mutants at a reduced level and is absent altogether for the
double mutant (Fig. 7 B). Notably, in the intracellular fractions of all strains including the double mutant, we observe
the conversion of P2 to M (from Ubi-P2), indicating that
Axl1p processing is intact. A reasonable explanation for
the presence of intracellular but not extracellular a-factor
in the double mutant is that AAXing fails to occur and
consequently methylation is blocked. The lack of methylation would in turn prevent recognition by the Ste6p transporter and therefore block export (Sapperstein et al.,
1994). To ascertain if AAXing is defective for Ubi-P2 in
the double mutant, we compared the methylation levels of
a-factor when the wild-type and mutant strains are expressing Ubi-P2 (Fig. 8). For the double mutant, we detect
no methylation for the slowest migrating band and thus
conclude that this band is P2*, not P2 (Fig. 7 C). This verifies the overlapping roles of Ste24p and Rce1p in a-factor AAXing. The single mutants bearing Ubi-P2 show slightly
different patterns of methylation from one another. The
methylation profile for Ubi-P2 in the ste24
mutant is similar to that of the wild-type strain (Fig. 8), indicating that
Rce1p can mediate AAXing very efficiently. The rce1
mutant strain also shows a methylation profile similar to
wild-type, but only 30 min after labeling. At the early time
point, the level of methylation is about half that of wild-type (Fig. 8). Thus, the AAXing mediated by Ste24p in the
rce1
mutant may be somewhat less efficient than that
mediated by Rce1p in the ste24
mutant. Nevertheless, either Ste24p or Rce1p can mediate sufficient AAXing to
produce a substantial amount of steady-state mature a-factor.
|
The Human Ste24p Homologue Complements the
S. cerevisiae ste24 Mutants
CAAX processing is evolutionarily conserved. Multicellular organisms as well as yeast express and process CAAX
proteins, such as Ras. Of the trio of human CAAX-processing enzymes, only the CAAX prenyl protease(s) has
not been cloned to date. Towards this end, we searched for
homologues of Ste24p in the database of expressed sequence tags and identified multiple ESTs with amino acid
similarity to S. cerevisiae Ste24p (Boyartchuk et al., 1997,
Fujimura-Kamada et al., 1997
). Using a fragment from one
of these ESTs, N76181, we screened a human B cell cDNA
library and identified several positive clones encoding the
3' partial ORFs with sequence similarity to S. cerevisiae
Ste24p. The most complete clone encoded an ORF of 383 residues and contained the poly A tail. We obtained the remaining 5' ORF sequence using the longest clone for 5'
RACE PCR with a human fetal brain library.
The human clone, which we designate Hs STE24, encodes a protein of 475 amino acids with 36% identity and
51% similarity to the S. cerevisiae Ste24p (Fig. 9 A). Like
Ste24p of S. cerevisiae and S. pombe, Hs Ste24p has a characteristic zinc metalloprotease motif (HEXXH: H, histidine; E, glutamate; X, any amino acid), several corresponding transmembrane spans as predicted by
hydropathy analysis (Fig. 9 B), and regions I, II, and III
that are conserved among the homologues of this subfamily of zinc metalloproteases (Fig. 9 A) (Fleischman et al.,
1995; Fujimura-Kamada et al., 1997
; Kornitzer et al.,
1991
). Interestingly, Hs STE24 has a short stretch of residues within region I that is not present in either yeast homologue (Fig. 9 A, dashed lines). Hs Ste24p does not possess a consensus dilysine ER localization signal at its
COOH terminus but does contain lysines at positions
3
and
6 from the COOH terminus (Fig. 9 A), which may
reflect a degenerate ER localization signal.
|
We expressed the human STE24 clone in S. cerevisiae
strains deleted for STE24 and RCE1 to examine whether
it can complement their mating defects. We find that Hs
STE24 partially corrects the mating defect of the double
ste24 rce1
mutant strain (Fig. 9 C) and fully complements the modest mating defect of the single ste24
mutant under stringent mating conditions (data not shown). These data suggest that Hs Ste24p can carry out both the
NH2-terminal processing and the COOH-terminal AAXing steps in a-factor maturation.
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Discussion |
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Ste24p Is Involved in Two Distinct
Proteolytic Maturation Steps of the a-Factor
Precursor: NH2- (P1 P2) and COOH-terminal
(CAAX) Processing
The analysis of biosynthetic intermediates in a-factor biogenesis has facilitated the characterization of processing activities required for several posttranslational modifications, including the CAAX farnesyltransferase (Ram1p/
Ram2p), the prenyl cysteine carboxyl methyltransferase
(Ste14p), and the a-factor P2 M protease (Axl1p) (Hrycyna and Clarke, 1990
; Schafer et al., 1990
; He et al., 1991
;
Adames et al., 1995
). The present study focuses on the
most recently discovered a-factor processing component,
Ste24p, a transmembrane protease that has been previously assigned distinct, and seemingly disparate roles in
a-factor processing, by us and others. We showed that
Ste24p is required for the first NH2-terminal cleavage step
in a-factor biogenesis, the P1
P2 cleavage, by characterizing the a-factor intermediate that accumulates in a ste24
mutant (Fujimura-Kamada et al., 1997
). In contrast, Boyartchuk et al. (1997)
provided evidence that Ste24p and
a dissimilar, but functionally redundant gene product
(Rce1p) are involved at a different step, COOH-terminal
CAAX processing of a-factor, using a biochemical assay.
Here we investigate and present compelling evidence for
the unifying hypothesis that Ste24p mediates dual steps in
the maturation of the a-factor precursor: COOH-terminal
CAAX processing and NH2-terminal (P1
P2) processing.
We show that Ste24p functionally overlaps with Rce1p
only for CAAX processing. Our evidence for the roles of Ste24p and Rce1p in a-factor maturation is summarized
below.
We demonstrate that Ste24p and Rce1p are functionally redundant for COOH-terminal CAAX processing of a-factor by the characterization of biosynthetic intermediates generated in both the ste24 and rce1 single and double mutants. A significant amount of AAXing occurs (indicated by methylation) in each of the single mutants (ste24 or rce1) but completely fails to occur in the double mutant (ste24 rce1) (refer to Fig. 3 B) (see below for a discussion of P0*). Since either STE24 or RCE1 is sufficient for AAXing, and deletion of both genes results in a complete defect in AAXing, we conclude that Ste24p and Rce1p share redundant roles in a-factor AAXing and appear to be the only CAAX proteases for a-factor in yeast.
A key finding here is the identification of the novel a-factor biosynthetic intermediate, P0*, in the double mutant (ste24 rce1). P0* is an early a-factor intermediate that is incompletely COOH terminally processed (prenylated but not AAXed or methylated). P0* is formed before P1, from which it shows only a subtle difference by gel mobility (refer to Figs. 1 and 3). The lack of methylation and metabolic instability of P0* distinguish it from P1, which is fully methylated and metabolically stable. These methylation differences between P1 and P0* stem from the absence or presence of the AAX tripeptide, respectively, which must be absent for the methyltransferase to act. The P0* species of a-factor is transient, and thus is not readily apparent in a wild-type strain. Presumably this is because each step of CAAX processing occurs in rapid succession, resulting in the fast conversion of P0* to P1; we can detect fully processed a-factor (M) even after a short 5 min of pulse labeling. The accumulation of the unmethylated (and unAAXed) P0* in the double mutant (ste24 rce1) provides the basis for our conclusion that Ste24p and Rce1p have redundant roles for the COOH-terminal AAXing of a-factor.
A role for Ste24p in NH2-terminal processing of a-factor
was implicated by a block in P1 P2 processing that results in P1 accumulation (neither P2 nor M is formed) in
our initial study of the ste24 mutant (Fujimura-Kamada et al.,
1997
). In the present study, we provide independent confirmation that Ste24p is in fact necessary for this step by
use of Ubi-a-factor fusions. The observation that production of mature a-factor from a construct that lacks the
P1
P2 cut site (Ubi-P2) can bypass the Ste24p requirement for NH2-terminal processing is consistent with the
role of Ste24p in the first NH2-terminal processing step of
a-factor maturation (Fig. 6). Interestingly, although Ste24p
and Rce1p function redundantly for CAAX processing of
a-factor, Rce1p is unlikely to contribute to NH2-terminal
processing, based on the finding that P1 fails to be converted to P2 in the ste24 mutant where Rce1p is present
(refer to Fig. 3 A).
As described above, Ste24p has dual roles in a-factor processing and functionally overlaps with Rce1p in one of these steps. This predicts that Rce1p should be dispensable for a-factor processing. Indeed, we observe that in the rce1 single mutant, the a-factor biogenesis profile is similar to wild-type; P2 and M are generated at similar rates in the rce1 mutant as in a wild-type strain and export of M is also indistinguishable in these two strains (refer to Fig. 3 A and Fig. 2).
Having established here that Ste24p participates in two
distinct proteolytic processing steps for a-factor, we can explain the previous discrepant findings for the role of
Ste24p (Boyartchuk et al., 1997; Fujimura-Kamada et al.,
1997
). These were due to (a) the use of two different assays and (b) the complexity arising from the situation that
one protein, Ste24p, carries out two functions and that two
proteins, Rce1p and Ste24p, mediate a common step. Our
combined approach of examining the NH2- and COOH-terminal processing steps of a-factor in strains deleted for
STE24 and RCE1 has now resolved this issue.
a-Factor Production and Mating in ste24 and rce1 Single and Double Mutants
In light of our current view of dual functions for Ste24p
(COOH-terminal AAXing and NH2-terminal cleavage)
and a single function for Rce1p (COOH-terminal AAXing), we can reevaluate the mature a-factor production
and mating phenotypes of the ste24 and rce1 single and
double mutants (Fig. 2). The double mutant (ste24 rce1) is
completely sterile because it can carry out neither COOH-
nor NH2-terminal processing, and thus does not produce
any mature a-factor. As expected, the rce1 mutant has a
wild-type mating efficiency because Ste24p mediates both
NH2- and COOH-terminal processing of a-factor in this
strain. In contrast, the ste24 mutant has a dramatically decreased mating efficiency (5% of wild-type) (Fujimura-Kamada et al., 1997), that is due solely to a block in NH2-terminal processing, since COOH-terminal AAXing can
be carried out by Rce1p in the ste24 mutant.
The residual mating exhibited by the ste24 mutant is significant since it reflects a low level of mature a-factor production. What gene product is responsible for the residual
a-factor processing in the ste24 mutant? An unknown enzyme, or possibly even Rce1p, could mediate a very low
level of P1 P2 cleavage. Alternatively, the P1
P2 processing step might be bypassed at a low level by a one-step
removal of the entire NH2-terminal extension from P1. A
candidate for this latter possibility is Axl1p, whose normal
role is to mediate the second NH2-terminal (P2
M) cleavage step (between N21 and Y22) in a-factor processing.
Axl1p may be able to generate mature a-factor directly from
P1 somewhat inefficiently (P1
M cleavage). We favor the
second hypothesis because overexpression of AXL1 can
significantly suppress the ste24 mating defect (Fujimura-
Kamada, K., and S. Michaelis, unpublished results).
Overlapping Roles for Ste24p and Rce1p
As discussed above, the SDS-PAGE and phenotypic analyses of the rce1 and ste24 single and double mutants indicate that either Ste24p or Rce1p is sufficient for the
COOH-terminal AAXing of a-factor. Using the Ubi-P2
construct to circumvent the Ste24p requirement for NH2-terminal processing, we could investigate the individual
contributions of Ste24p and Rce1p solely to the COOH-terminal AAXing of a-factor. Based upon a-factor spot dilution assays and SDS-PAGE analysis (refer to Fig. 7), we
concluded that Ste24p and Rce1p each can carry out a substantial amount of the AAXing of a-factor, since the single
mutants produced approximately equivalent amounts of
the bioactive mature species. Although the levels of P2
methylation in the single mutants expressing Ubi-P2 are
indistinguishable from one another at a later time point, at
an early time point their methylation levels differ by
~50% (i.e., a twofold lower level of methylation in the
rce1 versus the ste24 strains for P2 [refer to Fig. 8]). This is
consistent with findings for the somewhat asymmetric
roles of Ste24p and Rce1p in a-factor AAXing (35 versus
60%, respectively) using an in vitro AAXing assay with a
synthetic peptide substrate and membrane extracts from
mutant strains (Boyartchuk et al., 1997).
It is rather surprising that Ste24p and Rce1p function redundantly in the CAAX processing of a-factor, since they
are neither homologues nor do they share common consensus sequences. Whereas Ste24p possesses the HEXXH zinc
metalloprotease motif, Rce1p lacks any known protease
motifs. The sole resemblance between Ste24p and Rce1p is
that both are extremely hydrophobic with several predicted
membrane spans. This situation contrasts with another redundant pair of a-factor processing components, Axl1p and
Ste23p, that are structurally similar to each other and contain the same protease motif (Adames et al., 1995). Both
Axl1p and Ste23p can mediate the final cleavage step
(P2
M) in a-factor maturation, accounting for >90% and
<10% of processing, respectively (Adames et al., 1995
).
Given the dissimilarity of Ste24p and Rce1p and that their roles do not overlap completely, it is possible that one (or both) of these proteins acts indirectly in a-factor maturation. Ste24p and/or Rce1p may regulate another protease(s) that cleaves a-factor. Because Rce1p lacks any currently known protease motifs, it may either be a cofactor involved in AAXing or it may represent a novel protease. The development of an in vitro assay with purified Ste24p (or Rce1p) and its substrate will resolve this issue. Since both candidate proteases, Ste24p and Rce1p, as well as the potential substrate, prenylated a-factor, are quite hydrophobic and likely membrane bound, the purification and assay development will be challenging.
STE24 likely encodes a protease, however, because the
conserved protease motif of Ste24p is necessary to complement the mating and a-factor production defects of a ste24
mutant (Boyartchuk et al., 1997, Fujimura-Kamada et al.,
1997
). Furthermore, mutations in the HEXXH motif are
sufficient to abolish AAXing as well as NH2-terminal processing (data not shown). Aside from the Ste24p subfamily
of unusually hydrophobic zinc metalloproteases (Fujimura-Kamada et al., 1997
), only one other subclass of HEXXH
multispanning membrane proteases is known, the unrelated S2P protease involved in cholesterol homeostasis
(Rawson et al., 1997
). Both the S2P protease and its substrate are very hydrophobic (Rawson et al., 1997
). S2P releases the sterol regulatory element binding protein transcription factor by cleaving within a transmembrane span
(Hua et al., 1996
; Sakai et al., 1996
). It will be interesting to
see how such a hydrophobic protease recognizes and processes its similarly hydrophobic substrate and whether
Ste24p and S2P use analogous processing mechanisms.
If Ste24p directly cleaves a-factor, then how does Ste24p
cut its substrate at two distinct sites? The COOH-terminal AAXing site (between residues C32, which is prenylated,
and V33) bears no resemblance to the NH2-terminal
P1 P2 processing site (between residues T7 and A8).
One explanation is that the prenyl cysteine serves as a
landmark or recognition site for recruitment of Ste24p and
that cleavage occurs at certain nearby sites. Alternatively, Ste24p could have broad substrate specificity, like the signal peptidases that recognize hydrophobic residues (for
review see Dalbey et al., 1997
). Ferreting out the rules for
Ste24p cleavage will require assaying a large number of
defined substrates.
Since Ste24p and Rce1p are not homologues but do
function redundantly in a-factor processing, it is reasonable to postulate that each enzyme recognizes subsets of
overlapping substrates. Various CAAX-containing proteins possess different CAAX sequences. Specific CAAX
sequences can direct farnesylation, geranylgeranylation, or even both (Moores et al., 1991; Trueblood et al., 1993
,
1997
; Caplin et al., 1994
, 1998
), which could also provide
another level of substrate discrimination for Ste24p and
Rce1p. Although Ste24p and Rce1p mediate processing of
the a-factor CAAX box, each is also likely to promote
AAXing of other yeast CAAX proteins, such as Ras or
the
subunit (Ste18p) of the heterotrimeric G protein that
transduces the pheromone response. For example, it has
been suggested that Rce1p is involved in AAXing of
Ras2p, whereas Ste24p is not (Boyartchuk et al., 1997
;
Schmidt et al., 1998
). The function of another CAAX protein, Ste18p, does not require Ste24p and Rce1p since the
MAT
ste24 rce1 mutant does not have a mating defect
(Fig. 2 B). This suggests that (a) Ste18p does not require
AAXing by Ste24p and Rce1p for its function, (b) AAXing is not necessary for Ste18p function, or (c) another unidentified CAAX protease processes Ste18p. The substrate specificities of Ste24p and Rce1p remain to be
elucidated.
Insight into the Role of the NH2-terminal Extension of a-Factor by Ubi-a-Factor Fusions
In addition to reaffirming the role of Ste24p in NH2-terminal processing, the Ubi-a-factor fusions provided a
method to verify the order of a-factor processing events
and also to examine the role of the NH2-terminal extension in the production and export of mature a-factor. First,
the NH2-terminal extension must be removed in two successive steps for production of mature a-factor. This sequence of events is based on the finding that the second
NH2-terminal processing step by Axl1p can proceed efficiently only after completion of the first NH2-terminal cleavage, either via Ste24p or as encoded by Ubi-P2. The
P1 P2 cut could expose the substrate recognition site for
Axl1p. Second, since the NH2-terminal extension is ultimately removed from a-factor before its export, a reasonable hypothesis is that the NH2-terminal extension could
play a transient role, such as targeting a-factor to its transporter, Ste6p. However, our results with Ubi-M suggest that this is not the case because Ubi-M, which lacks the
NH2-terminal extension, can still produce mature a-factor
that is properly exported (refer to Fig. 5 B). Instead, the
major difference between mature a-factor generated conventionally from MFA1 or unconventionally from Ubi-M
is that the latter is highly metabolically unstable. An interesting possibility is that the NH2-terminal extension plays
a role in protecting a-factor from degradation. A candidate for a degradation pathway is the N-end rule pathway since the first residue (tyrosine) of mature a-factor is a destabilizing residue. Another possibility is that the NH2-terminal extension acts as a chaperone to stabilize mature
a-factor. Alternatively, the mature species derived from
Ubi-M may not be methylated as efficiently as wild-type
a-factor, and thus could be degraded by the same machinery that degrades P0*.
Model for Intracellular a-Factor Processing and Trafficking
Most secreted molecules, such as the other S. cerevisiae
mating pheromone a-factor, undergo posttranslational processing in the lumenal compartments of the secretory
pathway. In contrast, a-factor and other CAAX proteins
are thought to be COOH terminally processed in the cytosol or on the cytosolic face of membranes. For example, it
has been suggested that most of unprocessed Ras2p accumulates on intracellular membranes in yeast strains deleted for the CAAX proteases (ste24 rce1) (Boyartchuk et al., 1997). We have sought elsewhere to define the intracellular site where a-factor CAAX processing and NH2-terminal processing take place (Schmidt et al., manuscript submitted for publication). In those studies, we demonstrated
by subcellular fractionation and indirect immunofluorescence that the CAAX proteases (Ste24p and Rce1p) and
the methyltransferase (Ste14p) are localized to the membrane of ER, presumably with their active sites facing the
cytosol (Romano et al., 1998
, Schmidt et al., manuscript
submitted for publication). Those findings, together with
the results presented here that show dual roles for Ste24p,
lead to our current view of the intracellular trafficking and
processing of a-factor (refer to Fig. 1). The newly synthesized a-factor precursor (P0) is prenylated by the cytosolic
Ram1p/Ram2p complex, permitting its association with the
ER membrane. At the cytosolic face of the ER membrane,
prenylated a-factor (P0*) completes COOH-terminal CAAX processing, including AAXing by Rce1p or Ste24p,
followed by carboxyl methylation by Ste14p to form P1.
Next, the first NH2-terminal cleavage step is mediated by
Ste24p to yield P2. P2 is subsequently shuttled to another
compartment where Axl1p (or Ste23p) (Schmidt, W.K., and
S. Michaelis, unpublished data) performs the final cleavage
step to generate M, which is then exported by Ste6p.
This model raises two intriguing issues. First, since
Ste24p, Rce1p, and Ste14p are localized to the same intracellular membrane and act sequentially in CAAX processing, these three components could form a complex, a notion
that we are presently investigating. Second, how does a prenylated protein, and a-factor in particular, traffic from the
ER to its final destination (the plasma membrane)? We
propose that a-factor could diffuse through the cytosol, use
a carrier, or hitchhike on the outside of vesicles using the
classical secretory pathway (Schmidt et al. 1998).
Human Ste24p Functions in Yeast
The COOH-terminal CAAX protein motif and its processing enzymes are conserved evolutionarily among eukaryotes. Here we report the cloning of the first mammalian CAAX prenyl protease, human (Hs) STE24. We have
shown elsewhere that S. cerevisiae (Sc) STE24 defines a
novel subfamily of zinc metalloproteases containing homologues from bacteria (E. coli, H. influenza) and other
fungi (S. pombe) (Fujimura-Kamada et al., 1997). Additionally, ESTs that are similar to STE24 exist in many multicellular organisms (A. thaliana, C. elegans, D. melanogaster, M. musculus, H. sapiens). Members of the Ste24p
subfamily are characterized by multiple membrane spans,
a zinc metalloprotease motif (HEXXH), several highly
conserved regions designated I, II, and III, and a COOH-terminal dilysine motif potentially involved in ER retrieval.
Hs Ste24p and Sc Ste24p are 36% identical and 51% similar; Hs Ste24p shares the common features of this subfamily. However, instead of the canonical dilysine motif for ER
retrieval (lysines at positions
3 and
4, or
3 and
5
from the COOH terminus), Hs Ste24p possesses what may
be a degenerate dilysine motif (lysines at positions
3 and
6). It will be interesting to determine if human Ste24p, like its S. cerevisiae counterpart, is localized to the ER
membrane. We expect that this will be the case, since another CAAX processing component (Ste14p methyltransferase) localizes to the ER membrane in yeast and mammalian cells (Dai et al., 1998
; Romano et al., 1998
).
We show by complementation that Hs Ste24p can function
in yeast (Fig. 9 C). Notably, Hs Ste24p complements the mating defect of the double mutant (ste24 rce1), suggesting that it
is capable of mediating not only COOH-terminal AAXing,
but also NH2-terminal processing of a-factor (Fig. 9 C). What
is the physiological substrate for Hs Ste24p; is there a human
homologue for a-factor that undergoes similar processing
steps? Clearly, no such human substrate has been discovered
to date. A potential substrate is the prelamin A precursor,
which undergoes a series of maturation steps that includes
COOH-terminal CAAX processing, followed by a subsequent proteolytic cleavage located 14 residues NH2-terminal to the prenyl cysteine (Weber et al., 1989). This latter cleavage may be analogous to the NH2-terminal processing of
a-factor, which occurs at sites that are 26 and 12 residues
away from the prenyl cysteine (Chen et al., 1997b
). The
endoprotease that processes the prelamin A precursor has
yet to be cloned, although an enzymatic activity has been
detected in nuclear extracts (Kilic et al., 1997
). In addition to
a possible role in NH2-terminal proteolytic cleavage similar
to the P1
P2 cut of the a-factor precursor, Hs Ste24p is also
likely to be involved in the processing of multiple CAAX
proteins found in diverse human cell types. ESTs of Hs
STE24 have been identified in cDNA libraries derived from
a variety of tissues (fetal brain, bone, prostate, fetal lung, pancreas tumor, human tonsillar cells enriched for germinal center B cells, retina, and heart) indicating a wide expression
pattern. We anticipate that future studies will reveal the
mammalian substrates of Ste24p, some of which may use a
processing pathway similar to that of yeast a-factor.
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Footnotes |
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Received for publication 21 May 1998 and in revised form 10 July 1998.
Address all correspondence to Susan Michaelis, Department of Cell Biology and Anatomy, The Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205. Tel.: (410) 955-8286. Fax: (410) 955-4129. E-mail: susan_michaelis{at}qmail.bs.jhu.eduWe thank L. Roman, R. Jensen, and members of the Michaelis laboratory for critical reading of the manuscript (all from Johns Hopkins University School of Medicine, Baltimore, MD). We are grateful to the members of the Michaelis laboratory for valuable discussions. We thank A. Toh-e and A. Varshavsky (California Institute of Technology, Pasadena, CA) for plasmids; S. Elledge for the human B cell cDNA library; L. Roman, P. Bhanot, and D. Cabin (all three from Johns Hopkins University School of Medicine) for technical advice.
This study was supported by a grant from the National Institutes of Health (GM41223) to S. Michaelis.
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Abbreviations used in this paper |
---|
E, extracellular; EST, expressed sequence tag; Hs, Homo sapiens; I, intracellular; M, mature; ORF, open reading frame; Sc, Saccharomyces cerevisiae; SD, synthetic minimal medium; Sp, Schizosaccharomyces pombe; Ubi, ubiquitin; Ubp, ubiquitin-specific protease; YPD, yeast extract/peptone/dextrose medium.
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References |
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---|
1. | Adames, N., K. Blundell, M.N. Ashby, and C. Boone. 1995. Role of yeast insulin-degrading enzyme homologs in propheromone processing and bud site selection. Science 270: 464-467 [Abstract]. |
2. | Altschul, S.F., W. Gish, W. Miller, E.W. Myers, and D.J. Lipman. 1990. Basic local alignment search tool. J. Mol. Biol. 215: 403-410 |
3. | Andres, D.A., A. Milatovich, T. Ozcelik, J.M. Wenzlau, M.S. Brown, J.L. Goldstein, and U. Francke. 1993. cDNA cloning of the two subunits of human CAAX farnesyltransferase and chromosomal mapping of FNTA and FNTB loci and related sequences. Genomics 18: 105-112 |
4. | Ashby, M.N., and J. Rine. 1995. Ras and a-factor converting enzyme. Methods Enzymol. 250: 235-250 |
5. | Ausubel, F.M., R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman, J.A. Smith, and K. Struhl. 1987. Current Protocols in Molecular Biology. Vol. 1. Greene Publishing Associates, New York. |
6. | Bachmair, A., D. Finley, and A. Varshavsky. 1986. In vivo half-life of a protein is a function of its amino-terminal residue. Science 234: 179-186 |
7. |
Backlund, P.S..
1997.
Post-translational Processing of RhoA.
J. Biol. Chem
272:
33175-33180
|
8. |
Baker, R.T.,
J.W. Tobias, and
A. Varshavsky.
1992.
Ubiquitin-specific proteases of Saccharomyces cerevisiae. Cloning of UBP2 and UBP3, and functional analysis of the UBP gene family.
J. Biol. Chem
267:
23364-23375
|
9. |
Boyartchuk, V.,
M. Ashby, and
J. Rine.
1997.
Modulation of Ras and a-factor
function by carboxyl-terminal proteolysis.
Science
275:
1796-1800
|
10. | Caplin, B.E., L.A. Hettich, and M.S. Marshall. 1994. Substrate characterization of the Saccharomyces cerevisiae protein farnesyltransferase and type-I protein geranylgeranyltransferase. Biochim. Biophys. Acta 1205: 39-48 |
11. |
Caplin, B.E.,
Y. Ohya, and
M.S. Marshall.
1998.
Amino acid residues that define both the isoprenoid and CAAX preferences of the Saccharomyces cerevisiae protein farnesyltransferase. Creating the perfect farnesyltransferase.
J.
Biol. Chem
273:
9472-9479
|
12. | Casey, P.J., P.A. Solski, C.J. Der, and J.E. Buss. 1989. p21ras is modified by a farnesyl isoprenoid. Proc. Natl. Acad. Sci. USA 86: 8323-8327 [Abstract]. |
13. | Chan, R.K., and C.A. Otte. 1982. Physiological characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by a-factor and alpha factor pheromones. Mol. Cell. Biol. 2: 21-29 |
14. | Chen, P., J.D. Choi, R. Wang, R.J. Cotter, and S. Michaelis. 1997a. A novel a-factor-related peptide of Saccharomyces cerevisiae that exits the cell by a Ste6p-independent mechanism. Mol. Biol. Cell 8: 1273-1291 [Abstract]. |
15. |
Chen, P.,
S.K. Sapperstein,
J.C. Choi, and
S. Michaelis.
1997b.
Biogenesis of the
Saccharomyces cerevisiae mating pheromone a-factor.
J. Cell Biol
136:
251-269
|
16. | Clarke, S.. 1992. Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu. Rev. Biochem. 61: 355-386 |
17. |
Dai, Q.,
E. Choy,
V. Chiu,
J. Romano,
S.R. Slivka,
S.A. Steitz,
S. Michaelis, and
M.R. Philips.
1998.
Human prenylcysteine carboxyl methyltransferase is in
the endoplasmic reticulum.
J. Biol. Chem.
273:
15030-15034
|
18. |
Dalbey, R.E.,
M.O. Lively,
S. Bron, and
J.M. van Dijl.
1997.
The chemistry and
enzymology of the type I signal peptidases.
Protein Sci
6:
1129-1138
|
19. | Elble, R.. 1992. A simple and efficient procedure for transformation of yeasts. Biotechniques 13: 18-20 |
20. | Fleischman, R.D., M.D. Adams, O. White, R.A. Clayton, E.F. Kirkness, A.R. Kerlavage, C.J. Bult, J.-F. Tomb, B.A. Dougherty, J.M. Merrick, et al . 1995. Whole-genome random sequencing and assembly of Haemophilus influenzae. Science 269: 496-512 |
21. |
Fujimura-Kamada, K.,
F.J. Nouvet, and
S. Michaelis.
1997.
A novel membrane-associated metalloprotease, Ste24p, is required for the first step of NH2-terminal processing of the yeast a-factor precursor.
J. Cell Biol.
136:
271-285
|
22. | He, B., P. Chen, S.Y. Chen, K.L. Vancura, S. Michaelis, and S. Powers. 1991. RAM2, an essential gene of yeast, and RAM1 encode the two polypeptide components of the farnesyltransferase that prenylates a-factor and Ras proteins. Proc. Natl. Acad. Sci. USA 88: 11373-11377 [Abstract]. |
23. | Herskowitz, I., and R.E. Jensen. 1991. Putting the HO gene to work: Practical uses for mating-type switching. Methods Enzymol. 194: 132-146 |
24. | Hrycyna, C.A., and S. Clarke. 1990. Farnesyl cysteine C-terminal methyltransferase activity is dependent upon the STE14 gene product in Saccharomyces cerevisiae. Mol. Cell Biol 10: 5071-5076 |
25. |
Hrycyna, C.A., and
S. Clarke.
1992.
Maturation of isoprenylated proteins in
Saccharomyces cerevisiae.
J. Biol. Chem.
267:
10457-10464
|
26. | Hrycyna, C.A., S.K. Sapperstein, S. Clarke, and S. Michaelis. 1991. The Saccharomyces cerevisiae STE14 gene encodes a methyltransferase that mediates C-terminal methylation of a-factor and RAS proteins. EMBO (Eur. Mol. Biol. Organ.) J. 10: 1699-1709 [Abstract]. |
27. | Hrycyna, C.A., S.J. Wait, P.S. Backlund, and S. Michaelis. 1995. Use of the yeast STE14 methyltransferase, expressed as a TrpE-STE14 fusion protein in Escherichia coli, for in vitro carboxylmethylation of isoprenylated polypeptides. Methods Enzymol. 250: 251-256 |
28. |
Hua, X.,
J. Sakai,
M.S. Brown, and
J.L. Goldstein.
1996.
Regulated cleavage of
sterol regulatory element binding proteins requires sequences on both sides
of the endoplasmic reticulum membrane.
J. Biol. Chem.
271:
10379-10384
|
29. | Johnsson, N., and A. Varshavsky. 1994. Ubiquitin-assisted dissection of protein transport across membranes. EMBO (Eur. Mol. Biol. Organ.) J. 13: 2686-2698 [Abstract]. |
30. | Kaiser, C., S. Michaelis, and A. Mitchell. 1994. Methods in Yeast Genetics: A Cold Spring Harbor Course Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 234 pp. |
31. | Kato, K., A. Cox, M. Hisaka, S. Graham, J. Buss, and C. Der. 1992. Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc. Natl. Acad. Sci. USA 89: 6403-6407 [Abstract]. |
32. |
Kilic, F.,
M.B. Dalton,
S.K. Burrell,
J.P. Mayer,
S.D. Patterson, and
M. Sinensky.
1997.
In vitro assay and characterization of the farnesylation-dependent
prelamin A endoprotease.
J. Biol. Chem
272:
5298-5304
|
33. |
Kohl, N.E.,
R.E. Diehl,
M.D. Schaber,
E. Rands,
D.D. Soderman,
B. He,
S.L. Moores,
D.L. Pompliano,
N.S. Ferro,
S. Powers,
K. Thomas, and
J. Gibbs.
1991.
Structural homology among mammalian and Saccharomyces cerevisiae
isoprenyl-protein transferases.
J. Biol. Chem
266:
18884-18888
|
34. | Kornitzer, D., D. Teff, S. Altuvi, and A.B. Oppenheim. 1991. Isolation, characterization, and sequence of an Escherichia coli heat shock gene, htpX. J. Bacteriol 173: 2944-2953 |
35. | Kuchler, K., R.E. Sterne, and J. Thorner. 1989. Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells. EMBO (Eur. Mol. Biol. Organ.) J. 8: 3973-3984 [Abstract]. |
36. | Marcus, S., G.A. Caldwell, D. Miller, C.-B. Xue, F. Naider, and J.M. Becker. 1991. Significance of C-terminal cysteine modifications to the biological activity of the Saccharomyces cerevisiae a-factor mating pheromone. Mol. Cell. Biol. 11: 3603-3612 |
37. | McGrath, J.P., and A. Varshavsky. 1989. The yeast STE6 gene encodes a homologue of the mammalian multidrug resistance P-glycoprotein. Nature 340: 400-404 |
38. | Michaelis, S.. 1993. STE6, the yeast a-factor transporter. Semin. Cell Biol. 4: 17-27 |
39. | Michaelis, S., and I. Herskowitz. 1988. The a-factor pheromone of Saccharomyces cerevisiae is essential for mating. Mol. Cell. Biol. 8: 1309-1318 |
40. |
Moores, S.L.,
M.D. Schaber,
S.D. Mosser,
E. Rands,
M.B. O'Hara,
V.M. Garsky,
M.S. Marshall,
D.L. Pompliano, and
J.B. Gibbs.
1991.
Sequence dependence of protein isoprenylation.
J. Biol. Chem
266:
14603-14610
|
41. | Muhlrad, D., R. Hunter, and R. Parker. 1992. A rapid method for localized mutagenesis of yeast genes. Yeast 8: 79-82 |
42. | Nijbroek, G.L., and S. Michaelis. 1998. Functional assays for analysis of yeast ste6 mutants. Methods Enzymol 292: 193-212 |
43. |
Oldenburg, K.R.,
K.T. Vo,
S. Michaelis, and
C. Paddon.
1997.
Recombination-mediated PCR-directed plasmid construction in vivo in yeast.
Nuc. Acids
Res
25:
451-452
|
44. | Rawson, R.B., N.G. Zelenski, D. Nijhawan, J. Ye, J. Sakai, M.T. Hasan, T.Y. Chang, M.S. Brown, and J.L. Goldstein. 1997. Complementation cloning of S2P, a gene encoding a putative metalloprotease required for intramembrane cleavage of SREBPs. Mol. Cell 1: 47-57 . |
45. | Robzyk, K., and Y. Kassir. 1992. A simple and highly efficient procedure for rescuing autonomous plasmids from yeast. Nuc. Acids Res 20: 3790 |
46. | Romano, J.D., W.K. Schmidt, and S. Michaelis. 1998. The Sacchromyces cerevisiae prenylcysteine carboxyl methyltransferase, Ste14p, is in the endoplasmic reticulum membrane. Mol. Biol. Cell. 9:In press. |
47. | Sakai, J., E.A. Duncan, R.B. Rawson, X. Hua, M.S. Brown, and J.L. Goldstein. 1996. Sterol-regulated release of SREBP-2 from cell membranes requires two sequential cleavages, one within a transmembane segment. Cell 85: 1037-1046 |
48. | Sapperstein, S., C. Berkower, and S. Michaelis. 1994. Nucleotide sequence of the yeast STE14 gene, which encodes farnesylcysteine carboxyl methyltransferase, and demonstration of its essential role in a-factor export. Mol. Cell. Biol. 14: 1438-1449 [Abstract]. |
49. | Schafer, W., and J. Rine. 1992. Protein prenylation: genes, enzymes, targets and functions. Annu.Rev. Genetics 26: 209-237 |
50. | Schafer, W.F., C. Trueblood, C. Yang, M. Mayer, S. Rosenberg, C. Poulter, S.-H. Kim, and J. Rine. 1990. Enzymatic coupling of cholesterol intermediates to a mating pheromone precursor and to the Ras protein. Science 249: 1133-1139 |
51. | Schafer, W.F., R. Kim, R. Sterne, J. Thorner, S.-H. Kim, and J. Rine. 1989. Genetic and pharmacological suppression of oncogenic mutations in ras genes of yeast and humans. Science 245: 379-385 |
52. | Schmidt, W., A. Tam, K. Fujimura-Kamada, and S. Michaelis. 1998. ER membrane localization of CAAX processing components and a protease required for an NH2-terminal proteolytic cleavage of a-factor in yeast. Proc. Natl. Acad. Sci. USA. In press. |
53. |
Sikorski, R.S., and
P. Hieter.
1989.
A system of shuttle vectors and yeast host
strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae.
Genetics
122:
19-27
|
54. |
Tobias, J.W., and
A. Varshavsky.
1991.
Cloning and functional analysis of the
ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae.
J. Biol.
Chem
266:
12021-12028
|
55. | Trueblood, C., Y. Ohya, and J. Rine. 1993. Genetic evidence for in vivo cross-specificity of the CaaX-box protein prenyltransferases farnesyltransferase and geranylgeranyltransferase-I in Saccharomyces cerevisiae. Mol. Cell. Biol 13: 4260-4275 [Abstract]. |
56. |
Trueblood, C.E.,
V.L. Boyartchuk, and
J. Rine.
1997.
Substrate specificity determinants in the farnesyltransferase beta-subunit.
Proc. Natl. Acad. Sci.
USA
94:
10774-10779
|
57. | Weber, K., U. Plessmann, and P. Traub. 1989. Maturation of nuclear lamin A involves a specific carboxy-terminal trimming, which removes the polyisoprenylation site from the precursor; implications for the structure of the nuclear lamina. FEBS (Fed. Eur. Biochem. Soc.) Lett 257: 411-414 . |
58. | Xie, H., H. Yamane, R. Stephenson, O. Ong, B.K.-K. Fung, and S. Clarke. 1990. Analysis of prenylated carboxyl-terminal cysteine methylesters in proteins. Methods (Orlando). 1: 276-282 . |
59. | Yashiroda, H., T. Oguchi, Y. Yasuda, A. Toh-E, and Y. Kikuchi. 1996. Bul1, a new protein that binds to the Rsp5 ubiquitin ligase in Saccharomyces cerevisiae. Mol. Cell. Biol 16: 3255-3263 [Abstract]. |
60. | Zhang, F.L., and P.J. Casey. 1996. Protein prenylation: molecular mechanisms and functional consequences. Annu. Rev. Biochem 65: 241-269 |