The EMBO Journal vol.9 no.13 pp.4535-4541, 1990
UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation Wolfgang Seufert, John P.McGrathl and Stefan Jentsch Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, D-7400 Tubingen, FRG and 'Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Commuicated by G.Gerisch
The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitinconjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubel mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubclubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle. Key words: protein turnover/quiescence/ubiquitinconjugating enzymes/UBC gene family/ubiquitin
Introduction Modification of proteins by the covalent attachment of ubiquitin is universal to all eukaryotes. Ubiquitin is a small, abundant, very highly conserved intracellular protein. Ubiquitin conjugation has been implicated in a number of different cellular functions. Biochemical and genetic evidence suggests that ubiquitin -protein conjugates are necessary intermediates in a major ATP-dependent cytosolic proteolytic pathway (reviewed by Finley and Varshavsky, 1985 and Hershko, 1988). Aside from its participation in selective protein degradation, a role for ubiquitin in the direct modification of the structure and function of proteins has been suggested. Support for this idea comes from the discovery of metabolically stable ubiquitin-protein conjugates. In the nucleus of higher eukaryotes, a large fraction of the chromosomal histones H2A and H2B exist in mono-ubiquitinated form (Wu et al., 1981). Furthermore, Oxford University Press
ubiquitin has been found to be attached to certain integral membrane proteins (Siegelman et al., 1986; Yarden et al., 1986; Leung et al., 1987), Drosophila actin (Ball et al., 1987), and viral coat proteins (Dunigan et al., 1988). Enzymes have also been described which precisely cleave ubiquitin from conjugates (ubiquitin carboxy-terminal hydrolases; for a review see Wilkinson et al., 1989), suggesting that some of these conjugation events are reversible. Ubiquitin conjugation is a multistep process and requires the activities of a ubiquitin-activating enzyme, El, and a family of ubiquitin-conjugating enzymes, E2s (reviewed by Jentsch et al., 1990). Some conjugation reactions require additional factors, known as ubiquitin-protein ligases or E3s, to mediate substrate recognition and subsequent ubiquitination. Studies of the mouse cell line ts85, which has a thermolabile ubiquitin-activating enzyme, indicate that ubiquitin conjugation is essential for cell viability (Ciechanover et al., 1984; Finley et al., 1984). Moreover, deletion of the gene encoding ubiquitin-activating enzyme (UBAI) in yeast results in cell death (McGrath,J.P., Jentsch,S. and Varshavsky,A., manuscript submitted). To further our understanding of the ubiquitin system, we began a systematic genetic analysis aimed at determining the functions of individual ubiquitin-conjugating enzymes. Several of the enzymatic components of the ubiquitin-protein ligase system were purified from the yeast Saccharomyces cerevisiae, and the corresponding genes were isolated. These studies led to the discovery that the previously characterized yeast DNA repair gene RAD6 and the cell cycle gene CDC34 both encode ubiquitin-conjugating enzymes (Jentsch et al., 1987; Goebl et al., 1988). More recently, we have isolated the yeast genes UBC4 and UBC5 which code for nearly identical 16 kd ubiquitinconjugating enzymes. These enzymes were shown to be central components of the ubiquitin-dependent proteolytic pathway (Seufert and Jentsch, 1990). Ubiquitin-dependent protein turnover involves the processive coupling of a branched, multi-ubiquitin chain to proteolytic substrates and their subsequent degradation by an ATP-dependent protease complex (Matthews et al., 1989). The ubc4ubc5 double mutants exhibit severe deficiencies in the formation of high molecular weight ubiquitin conjugates and in the degradation of short-lived and abnormal proteins, thereby defining UBC4/UBC5-mediated protein turnover as a major proteolytic pathway in yeast cells. Moreover, this pathway of protein degradation was shown to serve essential functions of the eukaryotic stress response. This report describes the gene cloning and functional characterization of a novel ubiquitin-conjugating enzyme, UBC 1. The UBCI-encoded enzyme is also involved in selective protein degradation, however, this enzyme is specifically required in the early stages of growth after germination of spores. Our observation that UBC 1, UBC4 and UBC5 constitute a subfamily of ubiquitin-conjugating 4535
W.Seufert, J.P.McGrath and S.Jentsch
enzymes required for growth and viability suggests a vital function for ubiquitin-dependent protein degradation in eukaryotic cells. Furthermore, this report provides evidence to suggest that the observed enzymatic diversity at the level of ubiquitin conjugation may contribute to the precise regulation and selectivity of intracellular protein turnover.
A E1
Results
P
_-UBC1
B
D
C El-ub
uBclUBC1-ub
Cloning of the UBC1 gene Using ubiquitin-Sepharose affinity chromatography, we have purified several enzymes of the yeast ubiquitin-protein ligase system (Jentsch et al., 1987). This purification scheme is based on ATP-dependent thiolester formation of these enzymes with immobilized ubiquitin, thereby exploiting their enzymatic activities. By this method, we isolated ubiquitinactivating enzyme, E1, and at least five distinct ubiquitinconjugating enzymes, E2s (Figure IA). Antibodies were raised against the mixture of purified El and E2s. In yeast whole cell extracts, these antibodies reacted strongly with El and with the largest of the purified E2 enzymes, E230K (Figure iB). When used to screen a yeast genomic library in the expression vector Xgtl 1, immunoreactive phage clones were identified which carried the genes for ubiquitinactivating enzyme, UBA] (McGrath,J.P., Jentsch,S. and Varshavsky,A., manuscript submitted), and for the E230K ubiquitin-conjugating enzyme. The gene encoding E230K was designated UBCJ. Analysis of induced Escherichia coli lysogens harboring the UBCI-containing phage showed that a full-length UBCJ gene product was expressed, rather than the expected lacZ fusion product (data not shown). One phage clone bearing a 4.7 kb yeast DNA insert was selected and used for rapid mapping of the UBCI coding region by transposon tagging. Transposition events in which the engineered mini TnJO transposon (mTnJO/URA3/supF; Snyder et al., 1986) disrupted the UBCJ coding sequence were identified by the loss of immunoreactivity of these clones. Subcloning of DNA fragments corresponding to the tagged region into an E. coli expression vector established that a 1.7 kb EcoRl-PstI fragment directs the synthesis of a correctly sized, immunoreactive protein (Figures IC and 2). To verify that we have isolated the gene encoding the UBC 1 ubiquitin-conjugating enzyme we assayed the cloned gene product for enzymatic activity. The formation of a ubiquitin-enzyme thiolester, a metastable intermediate of ubiquitin -protein conjugation reactions was assayed. Only extracts of E.coli cells expressing the gene product were capable of forming a thiolester-bonded protein complex with 1251-labeled ubiquitin in a reaction dependent upon ATP and purified yeast ubiquitin-activating enzyme. The mobility of the observed radioactive protein complex corresponded to the predicted size of the ubiquitin-E230K thiolester adduct (Figure 1D). Nucleotide sequence of UBC1 Having localized the UBCJ coding region by transposon tagging and verified the identity of the protein encoded by this gene, we next determined its nucleotide sequence (Figure 3). The molecular weight of the UBC 1 protein calculated from the deduced amino acid sequence is 24.2 kd. Apparently, UBC 1 displays a slower mobility in SDS -polyacrylamide gels. The predicted amino acid 4536
E2s
-- C1ub_
ub
Fig. 1. Purification, immunodetection, and assay of enzymatic activity of UBCl protein. (A) Purified components of the yeast ubiquitin-protein ligase system include ubiquitin-activating enzyme, El, and a family of ubiquitin-conjugating enzymes, E2s (Coomassie stained 18% polyacrylamide-SDS gel). (B) Immunodetection of El and UBC1 protein in a total yeast extract. (C) Immunodetection of UBC1 protein expressed from the cloned gene in an E.coli extract (pKUBCl, Figure 2). (D) In vitro thiolester formation of El (El ub) and UBC1 (UBCl ub) with 12'1-labeled ubiquitin (ub). sequence shows that UBC 1 is highly similar in sequence to all other characterized members of the ubiquitin-conjugating enzyme family (Figure 7 and our unpublished results). In particular, sequences surrounding the single cysteine residue of the protein which is required for ubiquitin-enzyme thiolester formation are highly conserved.
UBC1
gene expression
The expression of a number of the genes encoding components of the ubiquitin system appears to be highly regulated. The polyubiquitin gene, UBI4, and the genes for at least two of the E2 enzymes, UBC4 and UBCS, have been shown to be transcriptionally responsive to heat shock (Finley et al., 1987; Seufert and Jentsch, 1990). Similarly, transcription from UBI4 and UBC5 increases when yeast cells enter stationary phase. We therefore examined the expression of the UBCJ gene under different growth conditions and in response to environmental stress. On Northern blots with total yeast RNA, a single UBCJ transcript of 1 kb was detected (Figure 4A). The UBCJ transcript was present in exponentially growing cells. Its level increased substantially when cells entered stationary phase. Western analysis using antibodies raised against a trpE UBC 1 fusion protein revealed a similar increase in UBC1 protein levels upon entry into stationary phase (Figure 4B) indicating that UBCJ expression is largely controlled at the level of transcription. Analysis of RNA isolated from cells shifted from 23 to 38°C for 30 min indicated that, unlike some other UBC genes, transcription of UBCJ is not induced by this heat shock treatment (Figure 4A). -
UBC1 gene disruption causes slow growth To assess the in vivo function of UBC 1 protein, ubcl mutants were generated by gene disruption. The mTnJO transposon used for gene tagging carries a selectable yeast marker, the URA3 gene. One mutant allele was selected where the UBCJ gene was disrupted by the insertion of a transposon at a position amino-terminal of the active-site cysteine of the enzyme (Tn 18, Figure 5A). A second ubcl mutant allele was constructed by replacing a segment of the coding region
Ubiquitin-mediated protein degradation I kb
E
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EXPRESSION pta.
E
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Fig. 2. Identification of the UBCJ coding region. A map of the yeast DNA insert in a Xgtl 1 clone expressing UBCI is shown with its relative orientation to the lac promoter (plac) of the vector. Restriction sites for enzymes EcoRI (E) and PstI (P) are given. Mini TnJO insertions disrupting the UBCJ coding region are indicated by open triangles. Subfragments were cloned in a tac promoter (ptac) plasmid and UBCJ expression in E.coli was analyzed on Western blots. Plasmid pKUBCI carries the complete UBC1 coding region (open box; the arrow indicates the direction of UBCI gene transcription). This plasmid directs expression of UBCJ in Ecoli (Figure IC).
GAATTCGGAGGACGTGGAACTAAATTAAAATATACGAACTCTAAG
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CATAGTCCGATACGAAGCATTAATATGGTTTTT
-201
TGTTACATTTTCTTCCATCTGAAMTATTTGGAGGGGCAAAAACAATGAGATGACAGTTGMATGCATGAACTTCCGATCGCGGGCGTATAGGTAAAGTCAA
-101
GAAAAAACGTAGTCTATATAACGCATATTTAACTAACGAGAAAAACAATATCGATGACAGTTG
TTGAAGCAAGTGTGACGACATAAGTATCGTAATTTAGTGGTTGGATACATTAAAAAAMCAAGTGGTATATATATAAGTAGTAGTAGTAAGAAGTAAGCG
-1
ATG TCT AGG GCT MG AGA ATT ATG AAA GM ATC CM GCT GTG MG GAT GAT CCT GCA GCT CAC ATT ACT CTT GM Met Ser Arg Ala Lys Arg Ile Met Lys Glu Ile Gln Ala Val Lys Asp Asp Pro Ala Ala His Ile Thr Leu Glu
75
TTT GTG AGT GM TCT GAT ATC CAC CAT TTA MA GGC ACA TTT TTG GGC CCA CCT GGA ACA CCT TAC GAG GGT GGC Phe Val Ser Glu Ser Asp Ile His His Leu Lys Gly Thr Phe Leu Gly Pro Pro Gly Thr Pro Tyr Glu Gly Gly
150
AM TTT GTC GTG GAT ATC GM GTA CCT ATG GAG TAT CCA TTC MA CCA CCA MG ATG CAG TTC GAC ACA MA GTA Lys Phe Val Val Asp Ile Glu Val Pro Met Glu Tyr Pro Phe Lys Pro Pro Lys Met Gln Ph. Asp Thr Lys Val
225
TAC CAT CCA MT ATA TCA TCA GTG ACA GGT GCC ATT TGT TTA GAT ATT CTT MG MT GCA TGG TCG CCA GTG ATA Tyr His Pro Asn Ile Sor Ser Val Thr Gly Ala Ile C Leu Asp Ile Leu Lys Asn Ala Trp Ser Pro Val Ile
300
ACA CTA MG TCT GCA TTG ATT TCG CTA CAG GCG CTA CTA CM TCT CCA GAG CCT MC GAT CCT CM GAT GCA GM Thr Leu Lys Ser Ala Leu Ile Ser Lou Gln Ala Leu Lou GLn Ser Pro Glu Pro Asn Asp Pro Gln Asp Ala Glu
375
GTA GCA CM CAC TAT TTA CGG GAT AGA GM TCT TTT MT MG ACT GCT GCA CTA TGG ACG AGG TTA TAC GCC AGT Val Ala Gln His Tyr Lou Arg Asp Arg Glu Ser Phe Asn Lys Thr Ala Ala Leu Trp Thr Arg Leu Tyr Ala Sor
450
GM ACT TCC MT GGT CM AAA GGC MT GTA GAG GAG TCT GAT TTA TAT GGG ATT GAC CAC GAT CTG ATT GAC GAG Glu Thr Ser Asn Gly Gln Lys Gly Asn Val Glu Glu Ser Asp Lou Tyr Gly Il. Asp His Asp Leu Ile Asp Glu
525
TTT GM TCT CM GGT TTT GM MG GAC MG ATT GTG GM GTG TTG AGA CGA TTA GGC GTC AAG TCC TTA GAC CCC Phe Glu Ser Gln Gly Phe Glu Lys Asp Lys Ile Val Glu Val Lou Arg Arg Lou Gly Val Lys Ser Leu Asp Pro
600
MT GAC AC MC ACA GCC AAC CGT ATC ATC GAG GM TTG TTG MG TGMTAGATMAAAAAMCGCACCMGTMGTMGTAA Asn Asp Aan Asn Thr Ala Asn Arg Ile Ile Glu Glu Lou Leu Lys
685
TAAAGMTAAATMACTATATGAGTAAAACACCAAGCGAGGATGTTTCATTGTGCATCCGTGTTCTTGATGATCA
760
Fig. 3. Nucleotide sequence of the UBCJ gene and predicted amino acid sequence of the encoded protein. The unique cysteine residue of UBCl required for thiolester formation with ubiquitin is underlined. Nucleotide numbers starting at the first nucleotide of the coding region are given on the right.
by the yeast HIS3 marker (Figure 5A). Both ubcl mutant alleles were used to transform the diploid strain DF5 by homologous recombination to marker prototrophy. These ubcl/UBCJ heterozygotes were then sporulated and subjected to tetrad analysis. The genotypes of the meiotic segregants were determined by Southern hybridization analysis using UBCJ specific probes. Both of the ubcl insertional mutations gave rise to viable cells which were obtained at the expected 2:2 ratio indicating that UBCJ is not an essential gene. Western analysis of the mutant strains indicated that the UBC1 protein was absent in these cells (Figure SB). The loss of UBC1 function lead to a slowgrowth phenotype (Table I) and an aberrant cell morphology: mutant cells were abnormally large and tended to form aggregates.
UBC 1 is specifically required during eardy stages of growth after germination The most striking phenotype of ubcl mutants was observed after germination of mutant spores. Tetrad analysis following sporulation of ubcl/UBCJ heterozygotes showed that unlike the wild-type spores, the ubcl mutant spores gave rise to tiny, amorphously shaped colonies indicative of slow growth and poor cell viability (Figure 6A). However, cells from these colonies recovered after several divisions and formed colonies of normal appearance. Such colonies were only slightly smaller than wild-type colonies (Figure 6B), reflecting the 1.5-fold increase in doubling time displayed by ubel cultures as compared with wild-type (Table I). Mating, sporulation, and germination of ubcl mutants appeared to be essentially unaffected. The specific 4537
W.Seufert, J.P.McGrath and S.Jentsch
requirement for UBC 1 function upon resumption of growth following a resting state also manifested itself after prolonged nutrient deprivation or extended storage on plates (data not shown). Together these data suggest that in addition to a role for exponentially growing cells, UBC 1-mediated functions are critical for growth and viability at a specific point in the yeast life cycle: during the transition period after a state of quiescence. UBC1 is involved in protein turnover One strategy employed by cells for protection against the toxic effects of abnormal proteins is selectively to eliminate these proteins by conjugation to ubiquitin and subsequent degradation. To evaluate the involvement of UBC 1 in ubiquitin-mediated protein turnover, we examined the sensitivity of ubc] mutants to the amino acid analog
A
B x
_
_
U x.0
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Fig. 4. UBCJ gene expression. (A) Northern analysis of UBCI RNA levels in exponentially growing cells (exp; 6 h at 30°C, OD6 = 1), in cells entering stationary phase (sta; 18 h at 300C, OD6w=25), in exponentially growing cells prior to heat shock (-hs; 6 h at 25°C; OD6W = 1), and following heat shock (+hs; shift-up to 38°C for 30 min). Identical amounts of total RNA were loaded in each lane. (B) Western analysis of UBC1 protein levels in exponentially growing cells (exp) and in cells entering stationary phase (sta). Identical amounts of total protein were loaded.
A
canavanine. When incorporated into proteins, canavanine results in structurally altered proteins which are usually rapidly degraded. When compared to congenic wild-type cells, ubcl mutants were more sensitive to canavanine (Table I), suggesting that UBC1 functions as an enzymatic component of the ubiquitin-mediated pathway for selective protein degradation. This conclusion is further supported by direct measurements of the turnover of canavanyl-proteins which showed a slight but significant reduction of selective protein degradation in ubcl mutants (Table I). UBC1, UBC4, and UBC5 constitute a UBC subfamily We have previously demonstrated that the E2 enzymes UBC4 and UBC5 mediate most of the ubiquitin-dependent protein degradation in yeast (Seufert and Jentsch, 1990). The moderate defects in protein degradation observed in ubcl mutants might indicate a functional overlap of UBC 1 with the UBC4 and UBC5 enzymes. To address this possibility, double and triple mutants in these UBC genes were constructed, either by direct disruption of genes in existing haploid ubc mutants, or by crossing different ubc mutant strains followed by meiotic segregation. Disruption of UBCJ in a ubc4 mutant background resulted in double mutants exhibiting more severe phenotypes than those of the single mutants combined. In particular, ubclubc4 double mutants showed significantly prolonged doubling times (Table II), suggesting important overlapping functions for these two genes during exponential growth. The critical role of UBC 1 after sporulation and germination was again evident when ubclubc4 haploids were constructed by mating and meiotic segregation: ubclubc4 spores failed to form visible colonies even after extended incubation. Microscopic inspection revealed that most of the ubclubc4 spores had germinated and formed microcolonies of a few abnormally shaped cells. Since no viable cells could be recovered from these microcolonies, we conclude that UBC1/UBC4-mediated functions are essential for cell viability after germination of ascospores. In contrast, ubclubcS double mutants showed no apparent phenotypic difference from ubcl single mutants. This is consistent with the previous observation that UBC5 is
B § or _
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B
3
i
i
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Tn18
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Fig. 5. Outline and analysis of UBCI gene disruption. (A) Open boxes indicate coding sequences and arrows give the direction of transcription. Single letters denote restriction sites for enzymes BamHI (B), EcoRI (E), EcoRV (V), and PstI (P). The mTnJO transposon (upper part; Snyder et al., 1986) carries the terminal repeats of TnJO (closed boxes) and in addition to the yeast marker gene URA3 the supF marker for selection in E.coli. In clone Tnl8 the transposon had integrated at codon 62 in the UBCJ gene. The unique cysteine residue of UBC1 at position 88 is indicated by an asterisk. Replacement of an internal EcoRV fragment of UBC1 (codon 31-55) by the HIS3 marker (1.7 kb BamHI fragment of YEp6; Struhl et al., 1979) is illustrated in the lower part. (B) Western analysis of UBC1 protein in wild-type and ubcl mutant strains. Proteins were isolated from cells entering stationary phase and identical amounts were loaded in each lane. The position of UBC1 protein and the origin of the gel (orn) is indicated.
4538
Ubiquitin-mediated protein degradation
dispensible as long as UBC4 is present (Seufert and Jentsch, 1990). Neither by crossing and meiotic segregation, nor by direct gene disruption, could ubclubc4ubc5 triple mutants be recovered, indicating that the combined loss-of-function of these three genes was incompatible with cell viability. To confirm this result, UBCJ, UBC4 and UBC5 genes were disrupted in a strain carrying a 2,1 plasmid with a functional UBC4 gene and a URA3 marker. Spontaneous plasmid loss during growth in non-selective medium should give rise to cells that are ura3 and therefore resistant to the toxic effects of 5-fluoro-orotic acid (5-FOA) (Boeke et al., 1984). Mitotic loss of the UBC4 plasmid was readily observed in wild-type cells and various combinations of ubc mutants, however, growth of the chromosomal triple ubclubc4ubc5 mutant was found to be plasmid-dependent. These cells failed to give rise to 5-FOA resistant colonies and no uracil auxotrophs were observed after growth in non-selective medium. Therefore, UBCJ, UBC4, and UBCS constitute a gene family essential for cell growth. The similarity of UBCJ gene function to UBC4 and UBC5 was substantiated by the observation that overexpression of
B
A
Fig. 6. Colony formation of germinated wild-type and ubcl mutant spores compared with growing cells. (A) Colony formation of germinated spores obtained from tetrad dissection after sporulation of UBCJ/ubcl diploid cells. The four meiotic segregants of a single diploid cell were placed in a vertical line. Wild-type spores formed large colonies, ubcl mutant spores formed tiny colonies. (B) Colony formation of cells from the exponential growth phase. Using a micromanipulator single wild-type cells were placed in the first and third horizontal line, single ubcl mutant cells in the second and fourth horizontal line.
I Q AV K D
S R AK RRIM
UBCI
D]PA
UBCJ partially complemented ubc4ubc5 mutant phenotypes (Table III). An -10-fold overproduction of UBC 1 protein was achieved by placing the gene on a high-copy number 2, plasmid (data not shown). This overexpression of UBCJ caused no detectable phenotypic effects in wild-type cells, and, as expected, the plasmid functionally complemented a chromosomal ubc] null mutation. In ubc4ubc5 mutants, the overexpression of UBCJ improved growth (Table III). In particular, high-level expression of UBCI restored growth of ubc4ubc5 mutants at elevated temperatures and increased resistance of these mutants to canavanine at least 500-fold (Table III). Apparently, the defects of ubc4ubcS mutants in the ubiquitin-mediated proteolysis pathway can be complemented by overexpression of UBCI.
Discussion The covalent attachment of ubiquitin to cellular proteins is catalyzed by a family of ubiquitin-conjugating (E2) enzymes. This paper describes the cloning and functional characterization of the gene UBCJ from the yeast S. cerevisiae coding for a novel member of this enzyme family. The UBC1 protein is related in sequence to other ubiquitin-conjugating enzymes and was found to be an enzymatic component of the complex ubiquitin-mediated pathway for intracellular protein turnover. Proteins have strikingly different half-lives from a few minutes to several hours. In several cases protein degradation has a regulatory role e.g. in reducing the levels of key enzymes of metabolic pathways and in the down-regulation of cellular regulators. In particular, some transcription factors, certain products of cellular and viral oncogenes and crucial cell cycle regulators such as the cyclins are shortlived in vivo. Turnover rates for individual proteins can vary considerably depending on the cell type, nutritional and other influences and the position within the cell cycle. Another function of intracellular protein degradation is the elimination of misfolded, misassembled, mislocalized, damaged, or other abnormal proteins. Protein degradation by a ubiquitin-dependent pathway involves covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by specific ATPdependent protease complexes (Matthews et al., 1989).
H IT L E F V S E S D I H H L K G T F L G P P G T P Y E G G M L Y H W QASIIGPADSPYAIGG
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N D N N T A N R I I E E L L K
A
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D K I V E V L R RL G V K S L D P
Fig. 7. Amino acid sequence similarity of UBCI to UBC4 and UBC5 proteins. Amino acids of UBCI identical to UBC4 and UBC5 proteins are boxed. Only those residues of UBC5 which differ from UBC4 are shown. The active-site cysteine of UBC1 required for thiolester formation with ubiquitin is marked by an asterisk.
4539
W.Seufert, J.P.McGrath and S.Jentsch Table I. Phenotypes of ubcl mutants
wild-type ubcl
Doubling time
Resistance to canavanine
(h)a
(%
1.5 2.3
82 12
Degradation of canavanyl-peptides MC
b
100 76
aCells were grown in YPD liquid medium at 30°C. OD6W was followed for determination of doubling times. bResistance to canavanine was determined with cells grown in SD medium containing required nutrients. Appropriate aliquots were spread on supplemented SD plates with or without canavanine at 1.5 fg/ml. Resistance is given as the fraction of colonies formed in the presence of the amino acid analog. cCells were pre-treated for 90 min with canavanine at 20 tg/ml and labeled for 5 min with [35S]methionine. Protein degradation was measured as the fraction of total incorporated radioactivity released from cells during the chase period (Seufert and Jentsch, 1990). The wild-type value at a 3 h chase was defined as 100%.
Table II. Growth phenotypes of ubc mutants
wild-type ubel ubc4 ubc5 ubclubc4 ubclubc5 ubc4ubc5 ubclubc4ubcS
Vegetative growth (doubling time in h)a
Viability after germination of sporesb
1.5 2.3 2.0 1.5 5.5 2.3 6.2
+ + + +
not viable
-
+ +
aCells were grown in YPD liquid medium at 30°C. OD6W was followed for determination of doubling times. bSpores obtained after tetrad dissection were transferred on YPD plates for germination and growth.
Table III. Complementation of ubc4ubc5 mutant phenotypes by overexpression of UBCI
ubc4ubc5 ubc4ubcS UBClhC
Doubling time (h)a
Growth at 37OCb
Resistance to canavanine (%)c
8.8 7.2
+
UBC1 encodes a novel member of an essential subfamily of yeast ubiquitin-conjugating enzymes involved in protein degradation Wolfgang Seufert, John P.McGrathl and Stefan Jentsch Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, D-7400 Tubingen, FRG and 'Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA Commuicated by G.Gerisch
The covalent attachment of ubiquitin to cellular proteins is catalyzed by members of a family of ubiquitinconjugating enzymes. These enzymes participate in a variety of cellular processes, including selective protein degradation, DNA repair, cell cycle control, and sporulation. In the yeast Saccharomyces cerevisiae, two closely related ubiquitin-conjugating enzymes, UBC4 and UBC5, have recently been shown to mediate the selective degradation of short-lived and abnormal proteins. We have now identified a third distinct member of this class of ubiquitin-conjugating enzymes, UBC1. UBC1, UBC4 and UBC5 are functionally overlapping and constitute an enzyme family essential for cell growth and viability. All three mediate selective protein degradation, however, UBC1 appears to function primarily in the early stages of growth after germination of spores. ubel mutants generated by gene disruption display only a moderate slow growth phenotype, but are markedly impaired in growth following germination. Moreover, yeast carrying the ubclubc4 double mutation are viable as mitotic cells, however, these cells fail to survive after undergoing sporulation and germination. This specific requirement for UBC1 after a state of quiescence suggests that degradation of certain proteins may be crucial at this transition point in the yeast life cycle. Key words: protein turnover/quiescence/ubiquitinconjugating enzymes/UBC gene family/ubiquitin
Introduction Modification of proteins by the covalent attachment of ubiquitin is universal to all eukaryotes. Ubiquitin is a small, abundant, very highly conserved intracellular protein. Ubiquitin conjugation has been implicated in a number of different cellular functions. Biochemical and genetic evidence suggests that ubiquitin -protein conjugates are necessary intermediates in a major ATP-dependent cytosolic proteolytic pathway (reviewed by Finley and Varshavsky, 1985 and Hershko, 1988). Aside from its participation in selective protein degradation, a role for ubiquitin in the direct modification of the structure and function of proteins has been suggested. Support for this idea comes from the discovery of metabolically stable ubiquitin-protein conjugates. In the nucleus of higher eukaryotes, a large fraction of the chromosomal histones H2A and H2B exist in mono-ubiquitinated form (Wu et al., 1981). Furthermore, Oxford University Press
ubiquitin has been found to be attached to certain integral membrane proteins (Siegelman et al., 1986; Yarden et al., 1986; Leung et al., 1987), Drosophila actin (Ball et al., 1987), and viral coat proteins (Dunigan et al., 1988). Enzymes have also been described which precisely cleave ubiquitin from conjugates (ubiquitin carboxy-terminal hydrolases; for a review see Wilkinson et al., 1989), suggesting that some of these conjugation events are reversible. Ubiquitin conjugation is a multistep process and requires the activities of a ubiquitin-activating enzyme, El, and a family of ubiquitin-conjugating enzymes, E2s (reviewed by Jentsch et al., 1990). Some conjugation reactions require additional factors, known as ubiquitin-protein ligases or E3s, to mediate substrate recognition and subsequent ubiquitination. Studies of the mouse cell line ts85, which has a thermolabile ubiquitin-activating enzyme, indicate that ubiquitin conjugation is essential for cell viability (Ciechanover et al., 1984; Finley et al., 1984). Moreover, deletion of the gene encoding ubiquitin-activating enzyme (UBAI) in yeast results in cell death (McGrath,J.P., Jentsch,S. and Varshavsky,A., manuscript submitted). To further our understanding of the ubiquitin system, we began a systematic genetic analysis aimed at determining the functions of individual ubiquitin-conjugating enzymes. Several of the enzymatic components of the ubiquitin-protein ligase system were purified from the yeast Saccharomyces cerevisiae, and the corresponding genes were isolated. These studies led to the discovery that the previously characterized yeast DNA repair gene RAD6 and the cell cycle gene CDC34 both encode ubiquitin-conjugating enzymes (Jentsch et al., 1987; Goebl et al., 1988). More recently, we have isolated the yeast genes UBC4 and UBC5 which code for nearly identical 16 kd ubiquitinconjugating enzymes. These enzymes were shown to be central components of the ubiquitin-dependent proteolytic pathway (Seufert and Jentsch, 1990). Ubiquitin-dependent protein turnover involves the processive coupling of a branched, multi-ubiquitin chain to proteolytic substrates and their subsequent degradation by an ATP-dependent protease complex (Matthews et al., 1989). The ubc4ubc5 double mutants exhibit severe deficiencies in the formation of high molecular weight ubiquitin conjugates and in the degradation of short-lived and abnormal proteins, thereby defining UBC4/UBC5-mediated protein turnover as a major proteolytic pathway in yeast cells. Moreover, this pathway of protein degradation was shown to serve essential functions of the eukaryotic stress response. This report describes the gene cloning and functional characterization of a novel ubiquitin-conjugating enzyme, UBC 1. The UBCI-encoded enzyme is also involved in selective protein degradation, however, this enzyme is specifically required in the early stages of growth after germination of spores. Our observation that UBC 1, UBC4 and UBC5 constitute a subfamily of ubiquitin-conjugating 4535
W.Seufert, J.P.McGrath and S.Jentsch
enzymes required for growth and viability suggests a vital function for ubiquitin-dependent protein degradation in eukaryotic cells. Furthermore, this report provides evidence to suggest that the observed enzymatic diversity at the level of ubiquitin conjugation may contribute to the precise regulation and selectivity of intracellular protein turnover.
A E1
Results
P
_-UBC1
B
D
C El-ub
uBclUBC1-ub
Cloning of the UBC1 gene Using ubiquitin-Sepharose affinity chromatography, we have purified several enzymes of the yeast ubiquitin-protein ligase system (Jentsch et al., 1987). This purification scheme is based on ATP-dependent thiolester formation of these enzymes with immobilized ubiquitin, thereby exploiting their enzymatic activities. By this method, we isolated ubiquitinactivating enzyme, E1, and at least five distinct ubiquitinconjugating enzymes, E2s (Figure IA). Antibodies were raised against the mixture of purified El and E2s. In yeast whole cell extracts, these antibodies reacted strongly with El and with the largest of the purified E2 enzymes, E230K (Figure iB). When used to screen a yeast genomic library in the expression vector Xgtl 1, immunoreactive phage clones were identified which carried the genes for ubiquitinactivating enzyme, UBA] (McGrath,J.P., Jentsch,S. and Varshavsky,A., manuscript submitted), and for the E230K ubiquitin-conjugating enzyme. The gene encoding E230K was designated UBCJ. Analysis of induced Escherichia coli lysogens harboring the UBCI-containing phage showed that a full-length UBCJ gene product was expressed, rather than the expected lacZ fusion product (data not shown). One phage clone bearing a 4.7 kb yeast DNA insert was selected and used for rapid mapping of the UBCI coding region by transposon tagging. Transposition events in which the engineered mini TnJO transposon (mTnJO/URA3/supF; Snyder et al., 1986) disrupted the UBCJ coding sequence were identified by the loss of immunoreactivity of these clones. Subcloning of DNA fragments corresponding to the tagged region into an E. coli expression vector established that a 1.7 kb EcoRl-PstI fragment directs the synthesis of a correctly sized, immunoreactive protein (Figures IC and 2). To verify that we have isolated the gene encoding the UBC 1 ubiquitin-conjugating enzyme we assayed the cloned gene product for enzymatic activity. The formation of a ubiquitin-enzyme thiolester, a metastable intermediate of ubiquitin -protein conjugation reactions was assayed. Only extracts of E.coli cells expressing the gene product were capable of forming a thiolester-bonded protein complex with 1251-labeled ubiquitin in a reaction dependent upon ATP and purified yeast ubiquitin-activating enzyme. The mobility of the observed radioactive protein complex corresponded to the predicted size of the ubiquitin-E230K thiolester adduct (Figure 1D). Nucleotide sequence of UBC1 Having localized the UBCJ coding region by transposon tagging and verified the identity of the protein encoded by this gene, we next determined its nucleotide sequence (Figure 3). The molecular weight of the UBC 1 protein calculated from the deduced amino acid sequence is 24.2 kd. Apparently, UBC 1 displays a slower mobility in SDS -polyacrylamide gels. The predicted amino acid 4536
E2s
-- C1ub_
ub
Fig. 1. Purification, immunodetection, and assay of enzymatic activity of UBCl protein. (A) Purified components of the yeast ubiquitin-protein ligase system include ubiquitin-activating enzyme, El, and a family of ubiquitin-conjugating enzymes, E2s (Coomassie stained 18% polyacrylamide-SDS gel). (B) Immunodetection of El and UBC1 protein in a total yeast extract. (C) Immunodetection of UBC1 protein expressed from the cloned gene in an E.coli extract (pKUBCl, Figure 2). (D) In vitro thiolester formation of El (El ub) and UBC1 (UBCl ub) with 12'1-labeled ubiquitin (ub). sequence shows that UBC 1 is highly similar in sequence to all other characterized members of the ubiquitin-conjugating enzyme family (Figure 7 and our unpublished results). In particular, sequences surrounding the single cysteine residue of the protein which is required for ubiquitin-enzyme thiolester formation are highly conserved.
UBC1
gene expression
The expression of a number of the genes encoding components of the ubiquitin system appears to be highly regulated. The polyubiquitin gene, UBI4, and the genes for at least two of the E2 enzymes, UBC4 and UBCS, have been shown to be transcriptionally responsive to heat shock (Finley et al., 1987; Seufert and Jentsch, 1990). Similarly, transcription from UBI4 and UBC5 increases when yeast cells enter stationary phase. We therefore examined the expression of the UBCJ gene under different growth conditions and in response to environmental stress. On Northern blots with total yeast RNA, a single UBCJ transcript of 1 kb was detected (Figure 4A). The UBCJ transcript was present in exponentially growing cells. Its level increased substantially when cells entered stationary phase. Western analysis using antibodies raised against a trpE UBC 1 fusion protein revealed a similar increase in UBC1 protein levels upon entry into stationary phase (Figure 4B) indicating that UBCJ expression is largely controlled at the level of transcription. Analysis of RNA isolated from cells shifted from 23 to 38°C for 30 min indicated that, unlike some other UBC genes, transcription of UBCJ is not induced by this heat shock treatment (Figure 4A). -
UBC1 gene disruption causes slow growth To assess the in vivo function of UBC 1 protein, ubcl mutants were generated by gene disruption. The mTnJO transposon used for gene tagging carries a selectable yeast marker, the URA3 gene. One mutant allele was selected where the UBCJ gene was disrupted by the insertion of a transposon at a position amino-terminal of the active-site cysteine of the enzyme (Tn 18, Figure 5A). A second ubcl mutant allele was constructed by replacing a segment of the coding region
Ubiquitin-mediated protein degradation I kb
E
E
p
P
P P E m1mmlim
PIS
:
EXPRESSION pta.
E
P
[ p
Pta
E
C::>_
p
P
pKUBCI
+
Fig. 2. Identification of the UBCJ coding region. A map of the yeast DNA insert in a Xgtl 1 clone expressing UBCI is shown with its relative orientation to the lac promoter (plac) of the vector. Restriction sites for enzymes EcoRI (E) and PstI (P) are given. Mini TnJO insertions disrupting the UBCJ coding region are indicated by open triangles. Subfragments were cloned in a tac promoter (ptac) plasmid and UBCJ expression in E.coli was analyzed on Western blots. Plasmid pKUBCI carries the complete UBC1 coding region (open box; the arrow indicates the direction of UBCI gene transcription). This plasmid directs expression of UBCJ in Ecoli (Figure IC).
GAATTCGGAGGACGTGGAACTAAATTAAAATATACGAACTCTAAG
-301
CATAGTCCGATACGAAGCATTAATATGGTTTTT
-201
TGTTACATTTTCTTCCATCTGAAMTATTTGGAGGGGCAAAAACAATGAGATGACAGTTGMATGCATGAACTTCCGATCGCGGGCGTATAGGTAAAGTCAA
-101
GAAAAAACGTAGTCTATATAACGCATATTTAACTAACGAGAAAAACAATATCGATGACAGTTG
TTGAAGCAAGTGTGACGACATAAGTATCGTAATTTAGTGGTTGGATACATTAAAAAAMCAAGTGGTATATATATAAGTAGTAGTAGTAAGAAGTAAGCG
-1
ATG TCT AGG GCT MG AGA ATT ATG AAA GM ATC CM GCT GTG MG GAT GAT CCT GCA GCT CAC ATT ACT CTT GM Met Ser Arg Ala Lys Arg Ile Met Lys Glu Ile Gln Ala Val Lys Asp Asp Pro Ala Ala His Ile Thr Leu Glu
75
TTT GTG AGT GM TCT GAT ATC CAC CAT TTA MA GGC ACA TTT TTG GGC CCA CCT GGA ACA CCT TAC GAG GGT GGC Phe Val Ser Glu Ser Asp Ile His His Leu Lys Gly Thr Phe Leu Gly Pro Pro Gly Thr Pro Tyr Glu Gly Gly
150
AM TTT GTC GTG GAT ATC GM GTA CCT ATG GAG TAT CCA TTC MA CCA CCA MG ATG CAG TTC GAC ACA MA GTA Lys Phe Val Val Asp Ile Glu Val Pro Met Glu Tyr Pro Phe Lys Pro Pro Lys Met Gln Ph. Asp Thr Lys Val
225
TAC CAT CCA MT ATA TCA TCA GTG ACA GGT GCC ATT TGT TTA GAT ATT CTT MG MT GCA TGG TCG CCA GTG ATA Tyr His Pro Asn Ile Sor Ser Val Thr Gly Ala Ile C Leu Asp Ile Leu Lys Asn Ala Trp Ser Pro Val Ile
300
ACA CTA MG TCT GCA TTG ATT TCG CTA CAG GCG CTA CTA CM TCT CCA GAG CCT MC GAT CCT CM GAT GCA GM Thr Leu Lys Ser Ala Leu Ile Ser Lou Gln Ala Leu Lou GLn Ser Pro Glu Pro Asn Asp Pro Gln Asp Ala Glu
375
GTA GCA CM CAC TAT TTA CGG GAT AGA GM TCT TTT MT MG ACT GCT GCA CTA TGG ACG AGG TTA TAC GCC AGT Val Ala Gln His Tyr Lou Arg Asp Arg Glu Ser Phe Asn Lys Thr Ala Ala Leu Trp Thr Arg Leu Tyr Ala Sor
450
GM ACT TCC MT GGT CM AAA GGC MT GTA GAG GAG TCT GAT TTA TAT GGG ATT GAC CAC GAT CTG ATT GAC GAG Glu Thr Ser Asn Gly Gln Lys Gly Asn Val Glu Glu Ser Asp Lou Tyr Gly Il. Asp His Asp Leu Ile Asp Glu
525
TTT GM TCT CM GGT TTT GM MG GAC MG ATT GTG GM GTG TTG AGA CGA TTA GGC GTC AAG TCC TTA GAC CCC Phe Glu Ser Gln Gly Phe Glu Lys Asp Lys Ile Val Glu Val Lou Arg Arg Lou Gly Val Lys Ser Leu Asp Pro
600
MT GAC AC MC ACA GCC AAC CGT ATC ATC GAG GM TTG TTG MG TGMTAGATMAAAAAMCGCACCMGTMGTMGTAA Asn Asp Aan Asn Thr Ala Asn Arg Ile Ile Glu Glu Lou Leu Lys
685
TAAAGMTAAATMACTATATGAGTAAAACACCAAGCGAGGATGTTTCATTGTGCATCCGTGTTCTTGATGATCA
760
Fig. 3. Nucleotide sequence of the UBCJ gene and predicted amino acid sequence of the encoded protein. The unique cysteine residue of UBCl required for thiolester formation with ubiquitin is underlined. Nucleotide numbers starting at the first nucleotide of the coding region are given on the right.
by the yeast HIS3 marker (Figure 5A). Both ubcl mutant alleles were used to transform the diploid strain DF5 by homologous recombination to marker prototrophy. These ubcl/UBCJ heterozygotes were then sporulated and subjected to tetrad analysis. The genotypes of the meiotic segregants were determined by Southern hybridization analysis using UBCJ specific probes. Both of the ubcl insertional mutations gave rise to viable cells which were obtained at the expected 2:2 ratio indicating that UBCJ is not an essential gene. Western analysis of the mutant strains indicated that the UBC1 protein was absent in these cells (Figure SB). The loss of UBC1 function lead to a slowgrowth phenotype (Table I) and an aberrant cell morphology: mutant cells were abnormally large and tended to form aggregates.
UBC 1 is specifically required during eardy stages of growth after germination The most striking phenotype of ubcl mutants was observed after germination of mutant spores. Tetrad analysis following sporulation of ubcl/UBCJ heterozygotes showed that unlike the wild-type spores, the ubcl mutant spores gave rise to tiny, amorphously shaped colonies indicative of slow growth and poor cell viability (Figure 6A). However, cells from these colonies recovered after several divisions and formed colonies of normal appearance. Such colonies were only slightly smaller than wild-type colonies (Figure 6B), reflecting the 1.5-fold increase in doubling time displayed by ubel cultures as compared with wild-type (Table I). Mating, sporulation, and germination of ubcl mutants appeared to be essentially unaffected. The specific 4537
W.Seufert, J.P.McGrath and S.Jentsch
requirement for UBC 1 function upon resumption of growth following a resting state also manifested itself after prolonged nutrient deprivation or extended storage on plates (data not shown). Together these data suggest that in addition to a role for exponentially growing cells, UBC 1-mediated functions are critical for growth and viability at a specific point in the yeast life cycle: during the transition period after a state of quiescence. UBC1 is involved in protein turnover One strategy employed by cells for protection against the toxic effects of abnormal proteins is selectively to eliminate these proteins by conjugation to ubiquitin and subsequent degradation. To evaluate the involvement of UBC 1 in ubiquitin-mediated protein turnover, we examined the sensitivity of ubc] mutants to the amino acid analog
A
B x
_
_
U x.0
_ -.- ~~~~~~~~4
_
0M
Fig. 4. UBCJ gene expression. (A) Northern analysis of UBCI RNA levels in exponentially growing cells (exp; 6 h at 30°C, OD6 = 1), in cells entering stationary phase (sta; 18 h at 300C, OD6w=25), in exponentially growing cells prior to heat shock (-hs; 6 h at 25°C; OD6W = 1), and following heat shock (+hs; shift-up to 38°C for 30 min). Identical amounts of total RNA were loaded in each lane. (B) Western analysis of UBC1 protein levels in exponentially growing cells (exp) and in cells entering stationary phase (sta). Identical amounts of total protein were loaded.
A
canavanine. When incorporated into proteins, canavanine results in structurally altered proteins which are usually rapidly degraded. When compared to congenic wild-type cells, ubcl mutants were more sensitive to canavanine (Table I), suggesting that UBC1 functions as an enzymatic component of the ubiquitin-mediated pathway for selective protein degradation. This conclusion is further supported by direct measurements of the turnover of canavanyl-proteins which showed a slight but significant reduction of selective protein degradation in ubcl mutants (Table I). UBC1, UBC4, and UBC5 constitute a UBC subfamily We have previously demonstrated that the E2 enzymes UBC4 and UBC5 mediate most of the ubiquitin-dependent protein degradation in yeast (Seufert and Jentsch, 1990). The moderate defects in protein degradation observed in ubcl mutants might indicate a functional overlap of UBC 1 with the UBC4 and UBC5 enzymes. To address this possibility, double and triple mutants in these UBC genes were constructed, either by direct disruption of genes in existing haploid ubc mutants, or by crossing different ubc mutant strains followed by meiotic segregation. Disruption of UBCJ in a ubc4 mutant background resulted in double mutants exhibiting more severe phenotypes than those of the single mutants combined. In particular, ubclubc4 double mutants showed significantly prolonged doubling times (Table II), suggesting important overlapping functions for these two genes during exponential growth. The critical role of UBC 1 after sporulation and germination was again evident when ubclubc4 haploids were constructed by mating and meiotic segregation: ubclubc4 spores failed to form visible colonies even after extended incubation. Microscopic inspection revealed that most of the ubclubc4 spores had germinated and formed microcolonies of a few abnormally shaped cells. Since no viable cells could be recovered from these microcolonies, we conclude that UBC1/UBC4-mediated functions are essential for cell viability after germination of ascospores. In contrast, ubclubcS double mutants showed no apparent phenotypic difference from ubcl single mutants. This is consistent with the previous observation that UBC5 is
B § or _
_supF I
B
3
i
i
I~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ Tn18
uBC1I
_
Fig. 5. Outline and analysis of UBCI gene disruption. (A) Open boxes indicate coding sequences and arrows give the direction of transcription. Single letters denote restriction sites for enzymes BamHI (B), EcoRI (E), EcoRV (V), and PstI (P). The mTnJO transposon (upper part; Snyder et al., 1986) carries the terminal repeats of TnJO (closed boxes) and in addition to the yeast marker gene URA3 the supF marker for selection in E.coli. In clone Tnl8 the transposon had integrated at codon 62 in the UBCJ gene. The unique cysteine residue of UBC1 at position 88 is indicated by an asterisk. Replacement of an internal EcoRV fragment of UBC1 (codon 31-55) by the HIS3 marker (1.7 kb BamHI fragment of YEp6; Struhl et al., 1979) is illustrated in the lower part. (B) Western analysis of UBC1 protein in wild-type and ubcl mutant strains. Proteins were isolated from cells entering stationary phase and identical amounts were loaded in each lane. The position of UBC1 protein and the origin of the gel (orn) is indicated.
4538
Ubiquitin-mediated protein degradation
dispensible as long as UBC4 is present (Seufert and Jentsch, 1990). Neither by crossing and meiotic segregation, nor by direct gene disruption, could ubclubc4ubc5 triple mutants be recovered, indicating that the combined loss-of-function of these three genes was incompatible with cell viability. To confirm this result, UBCJ, UBC4 and UBC5 genes were disrupted in a strain carrying a 2,1 plasmid with a functional UBC4 gene and a URA3 marker. Spontaneous plasmid loss during growth in non-selective medium should give rise to cells that are ura3 and therefore resistant to the toxic effects of 5-fluoro-orotic acid (5-FOA) (Boeke et al., 1984). Mitotic loss of the UBC4 plasmid was readily observed in wild-type cells and various combinations of ubc mutants, however, growth of the chromosomal triple ubclubc4ubc5 mutant was found to be plasmid-dependent. These cells failed to give rise to 5-FOA resistant colonies and no uracil auxotrophs were observed after growth in non-selective medium. Therefore, UBCJ, UBC4, and UBCS constitute a gene family essential for cell growth. The similarity of UBCJ gene function to UBC4 and UBC5 was substantiated by the observation that overexpression of
B
A
Fig. 6. Colony formation of germinated wild-type and ubcl mutant spores compared with growing cells. (A) Colony formation of germinated spores obtained from tetrad dissection after sporulation of UBCJ/ubcl diploid cells. The four meiotic segregants of a single diploid cell were placed in a vertical line. Wild-type spores formed large colonies, ubcl mutant spores formed tiny colonies. (B) Colony formation of cells from the exponential growth phase. Using a micromanipulator single wild-type cells were placed in the first and third horizontal line, single ubcl mutant cells in the second and fourth horizontal line.
I Q AV K D
S R AK RRIM
UBCI
D]PA
UBCJ partially complemented ubc4ubc5 mutant phenotypes (Table III). An -10-fold overproduction of UBC 1 protein was achieved by placing the gene on a high-copy number 2, plasmid (data not shown). This overexpression of UBCJ caused no detectable phenotypic effects in wild-type cells, and, as expected, the plasmid functionally complemented a chromosomal ubc] null mutation. In ubc4ubc5 mutants, the overexpression of UBCJ improved growth (Table III). In particular, high-level expression of UBCI restored growth of ubc4ubc5 mutants at elevated temperatures and increased resistance of these mutants to canavanine at least 500-fold (Table III). Apparently, the defects of ubc4ubcS mutants in the ubiquitin-mediated proteolysis pathway can be complemented by overexpression of UBCI.
Discussion The covalent attachment of ubiquitin to cellular proteins is catalyzed by a family of ubiquitin-conjugating (E2) enzymes. This paper describes the cloning and functional characterization of the gene UBCJ from the yeast S. cerevisiae coding for a novel member of this enzyme family. The UBC1 protein is related in sequence to other ubiquitin-conjugating enzymes and was found to be an enzymatic component of the complex ubiquitin-mediated pathway for intracellular protein turnover. Proteins have strikingly different half-lives from a few minutes to several hours. In several cases protein degradation has a regulatory role e.g. in reducing the levels of key enzymes of metabolic pathways and in the down-regulation of cellular regulators. In particular, some transcription factors, certain products of cellular and viral oncogenes and crucial cell cycle regulators such as the cyclins are shortlived in vivo. Turnover rates for individual proteins can vary considerably depending on the cell type, nutritional and other influences and the position within the cell cycle. Another function of intracellular protein degradation is the elimination of misfolded, misassembled, mislocalized, damaged, or other abnormal proteins. Protein degradation by a ubiquitin-dependent pathway involves covalent attachment of ubiquitin to proteolytic substrates and their subsequent degradation by specific ATPdependent protease complexes (Matthews et al., 1989).
H IT L E F V S E S D I H H L K G T F L G P P G T P Y E G G M L Y H W QASIIGPADSPYAIGG
UBC4
IM S
UBCI UBC4
K
UBC4
TLIS K VILLS I C S IL LT D ANIPIDID PIL V PE IA
UECS
KRIA K
L SD LE RDPIPTSCSAGPVGD
F V V D IE V M E Y P F K P P K M Q V F L SIIH FIPT DYPFKPPKIS i
U
L
LJJ
UBC
QK G
FD KV Y H P N I SV T GAI C L D I L K N A W S P V I TTK I H P N I|N A N NI C L DILKD Q WSPA L L V NWW S LIL-L A [El
L
iW
WL
IYIYK TDRP
N V E E S D L Y G I D H D L I D E F E S Q G F E
UBCI
E T S N G
UBEC
N D N N T A N R I I E E L L K
A
K
Y E A T AIR EIWTIK KYAV
UKU
D K I V E V L R RL G V K S L D P
Fig. 7. Amino acid sequence similarity of UBCI to UBC4 and UBC5 proteins. Amino acids of UBCI identical to UBC4 and UBC5 proteins are boxed. Only those residues of UBC5 which differ from UBC4 are shown. The active-site cysteine of UBC1 required for thiolester formation with ubiquitin is marked by an asterisk.
4539
W.Seufert, J.P.McGrath and S.Jentsch Table I. Phenotypes of ubcl mutants
wild-type ubcl
Doubling time
Resistance to canavanine
(h)a
(%
1.5 2.3
82 12
Degradation of canavanyl-peptides MC
b
100 76
aCells were grown in YPD liquid medium at 30°C. OD6W was followed for determination of doubling times. bResistance to canavanine was determined with cells grown in SD medium containing required nutrients. Appropriate aliquots were spread on supplemented SD plates with or without canavanine at 1.5 fg/ml. Resistance is given as the fraction of colonies formed in the presence of the amino acid analog. cCells were pre-treated for 90 min with canavanine at 20 tg/ml and labeled for 5 min with [35S]methionine. Protein degradation was measured as the fraction of total incorporated radioactivity released from cells during the chase period (Seufert and Jentsch, 1990). The wild-type value at a 3 h chase was defined as 100%.
Table II. Growth phenotypes of ubc mutants
wild-type ubel ubc4 ubc5 ubclubc4 ubclubc5 ubc4ubc5 ubclubc4ubcS
Vegetative growth (doubling time in h)a
Viability after germination of sporesb
1.5 2.3 2.0 1.5 5.5 2.3 6.2
+ + + +
not viable
-
+ +
aCells were grown in YPD liquid medium at 30°C. OD6W was followed for determination of doubling times. bSpores obtained after tetrad dissection were transferred on YPD plates for germination and growth.
Table III. Complementation of ubc4ubc5 mutant phenotypes by overexpression of UBCI
ubc4ubc5 ubc4ubcS UBClhC
Doubling time (h)a
Growth at 37OCb
Resistance to canavanine (%)c
8.8 7.2
+
