An Essential Yeast Gene Encoding a Homolog of Ubiquitin-activating Enzyme*

Ubiquitin (Ub) activation by the Uh-activating (El) enzyme is the initial and essential step common to all of the known processes that involve post-translational conjugation of Ub to itself or other proteins. The "acti­ vated" Ub, linked via a thioester bond to a specific cys­ teine residue of El enzyme, can be transferred to a cys­ teine residue in one of several Uh-conjugating (E2) enzymes, which catalyze the formation of isopeptide bonds between the C-terminal glycine of Ub and lysine residues of acceptor proteins. In the yeast Saccharomy­ ces


encoded yeast El and El enzymes of other organisms.
The regions of similarities between Ubalp and Uba2p encompass a putative ATP-binding site as well as a se quence that is highly conserved between the known El enzymes and contains the active-site cysteine of El. This cysteine is shown to be required for an essential func tion of Uba2p, suggesting that Uba2p-catalyzed reac tions involve a transient thioester bond between Uba2p and either Ub or another protein. Uba2p is located largely in the nucleus. The putative nuclear localization signal of Uba2p is near its C terminus. The Ubalp (El enzyme) and Uba2p cannot complement each others es sential functions even if their subcellular localization is altered by mutagenesis. Uba2p appears to interact with itself and several other S. cerevisiae proteins with ap parent molecular masses of 52, 63, 87, and 120 kDa. Uba2p is multiubiquitinated in vivo, suggesting that at least a fraction of Uba2p is metabolically unstable. Uba2p is likely to be a component of the Ub system that functions as either an E2 or El/E2 enzyme.
* This work was supported by a grant from the Bundesministerium fur Bildung und Forschung (Fiirderkennzeichen 0316711) (to R. J. D.) and by Grant GM31530 from the National Institutes of Health (to A. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Z48725. § To whom correspondence should be addressed: lnstitut fiir Mikro biologie, Heinrich-Heine-Universitat Diisseldorf, Universitiitsstra/3e 1, Geb. 26.12,Diisseldorf, Ubiquitin (Ub) 1 is a highly conserved 76-residue protein whose covalent conjugation to other proteins (often in the form of a multi-Uh chain) plays a role in a number of processes, primarily through routes that involve protein degradation (reviewed by Rechsteiner (1991), Finley and Chau (1991), Hershko and Ciechanover (1992), Jentsch (1992), Varshavsky (1992), Goldberg (1992), Hochstrasser (1992), Vierstra (1993), Parsell and Lindquist (1993), and Ciechanover (1994)). Ub activation by the Uh-activating (El) enzyme is the initial and essential step common to all of the known processes that in volve post-translational conjugation of Ub to itself or other proteins. The El enzyme activates Ub in an ATP-dependent reaction by first adenylating the C-terminal Gly-76 of Ub and thereafter linking this residue to the side chain of a Cys residue in the same El molecule, yielding an El� Ub thioester and free AMP (Ciechanover et al., 1982;Haas et al., 1983;Pickart, 1988;Pickart et al., 1994). In the presence of a Uh-conjugating (E2) enzyme (one of several such enzymes in a cell), the El-linked Ub is transferred to a Cys residue in E2. The E2 enzymes conjugate their cysteine-linked Ub moiety to its ultimate pro tein acceptors, yielding isopeptide bond-mediated Uh-protein conjugates (reviewed by Jentsch (1992)). The targeting of pro teins for ubiquitination by at least some E2 enzymes involves their association with distinct proteins called recognins, E2s or Uh-protein ligases, which mediate the initial recognition of a substrate by a recognin-E2 complex (Dohmen et al., 1991a;Sung et al., 1991;Scheffner et al., 1995). At least some recognins also form a thioester with Ub in the presence of El, appropriate E2, and a "downstream" protein substrate 2 (Scheffner et al., 1995), suggesting the existence of a Ub trans esterification cascade (El -E2 -recognin) that mediates the formation of isopeptide bond-linked Uh-protein conjugates.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM I EMBL Data Bank with accession number(s)
Genes encoding El enzymes have been cloned from a variety of organisms, including the yeast Saccharomyces cerevisiae, plants, and mammals such as mouse and man (Hatfield et al., 1990;McGrath et al., 1991;Handley et al., 1991;Imai et al., 1992;Kok et al., 1993;Hatfield and Vierstra, 1992). All of these genes encode 110-120-kDa proteins highly similar in se quence. UBAl, which encodes the 114-kDa El enzyme of S. cerevisiae, is essential for cell viability (McGrath et al., 1991). Biochemical and genetic analyses of ts85 and analogous tem perature-sensitive (ts) mammalian cell lines demonstrated that these cells carry ts mutations in their genes for El enzyme, and that the activity of El is required for ATP-dependent proteolysis, as well as for other (possibly proteolysis-mediated) cellular functions Ciechanover et al., 1984;Kulka et al., 1988;Aysawa et al., 1992;Nishitani et al., 1992). A gene required for spermatogenesis was identified in the Sxr (sex-reversed) region of the mouse Y chromosome and shown to encode an El enzyme whose expression is testis-specific (Mitchel et al., 1991;Kay et al., 1991).
We report the isolation and analysis of a novel essential S. cerevisiae gene, termed UBA2, that encodes a 636-residue protein whose amino acid sequence is highly similar to those of El enzymes, but whose activity appears to be distinct from that of the known El enzymes. MATERIALS AND METHODS Media, Genetic Techniques, and Construction of Strains-S. cerevisiae cultures were grown at 30 °C in rich (YP) or synthetic (S) media (Sherman et al., 1986) containing either 2% dextrose (YPD or SD media) or 2% galactose (YPG or SG media). Transformation of S. cerevisiae was carried out as described by Dohmen et al. (1991b). The strain SUB312 (a gift from Dr. D. .2::LEV2 (pUBlO0, expressing a non-Uh portion of Ubilp) (pUB23-X, expressing Ub-/3gal from PGALJ) lacks chromosomal sequences encoding Uh and expresses Uh (an essential protein) from the PGALJ promoter in the plasmid pUB23 (Finley et al., 1994). In the present work, pUb23 ofSUB312 were replaced with plasmids YEp96 or YEp105, which expressed Uh or mycUb, respectively, from the P cuPJ promoter (Ellison and Hochstrasser, 1991). All other S. cerevisiae strains were derivatives of   (Madura et al., 1993). JD51, a diploid derivative of JD47-13C, was constructed through a transient expression of HO endonuclease from the plasmid YCp50-HO (Herskowitz and Jensen, 1986).
The diploid JD90 (uba2/:,.::HIS3/UBA2) was produced from JD51 using one-step gene transplacement (Rothstein, 1991). The Pstl DNA fragment containing the uba2/:,.::HIS3 allele used in the transplacement was isolated from pJD346. The latter plasmid was constructed using oligonucleotide-directed deletion (Finley et al., 1987) of the UBA2 gene and simultaneous introduction of a BamHI site, which was used to insert the H/S3 marker as an 1.8-kilobase pair BamHI fragment (H/S3 was isolated from YEp6; Struhl et al. (1979)). The strain JD62 (PGALr UBA2 / UBA2) was produced by integrating the plasmid pJDA9-5 (which had been linearized by a cut with BstXI in the UBA2 part of the plasmid) into the UBA2 locus of the strain JD51. pJDA9-5 was constructed in the background of the vector pRS306 (Sikorski and Hieter, 1989) and contained 470 bp of UBA2, from the EcoRI site (which was introduced immediately in front of the ATG start codon using polymerase chain reaction (PCR)) to the Smal site. This 4 70-bp fragment was ligated to a 500-bp fragment containing the P GALI promoter. The latter fragment was generated by first introducing, using oligonucleotidedirected mutagenesis, a Smal site between the promoters P GALI and P GAL JO in a fragment originally isolated from of pBM272 (Hovland et al., 1989), and thereafter cloning the resulting Smal-BamHI fragment into the polylinker of pRS306. Integration of the resulting (linearized) pJDA9-5 at the UBA2 locus of the diploid strain JD51 yielded, in one of the two UBA2-containing chromosomes of JD51, a nonfunctional (truncated) copy of UBA2, and the intact UBA2 open reading frame (ORF) expressed from P GALJ• JD62-6A is a haploid MATa segregant of the strain JD62 bearing the altered UBA2 locus.
Cloning of UBA2 and Construction of Plasmids-Standard methods were used (Ausubel et al., 1992). The UBA2 gene was initially identified in a library of Agtll phages (Young and Davis, 1983) carrying inserts of S. cerevisiae genomic DNA Specifically, UBA2 was isolated as a result of a cross-reaction of a UBA2-expressing phage plaque with a polyclonal antiserum raised against a yeast plasma membrane ATPase kinase. 3 An oligonucleotide constructed on the basis of initial sequence analysis was then used to isolate UBA2 clones through plaque hybridization with the EMBL3A phage library of S. cerevisiae genomic DNA (a gift from Dr. R. Young). For mapping and sequencing of UBA2, yeast DNA fragments derived from two overlapping EMBL3A phage inserts were subcloned into YCplac22 (Gietz and Sugino, 1988), and a restriction map of UBA2 was produced using standard methods. The sequence of UBA2 was determined on both strands using a variety of UBA2 subclones, synthetic oligonucleotides as primers, the chain termination method (Ausubel et al., 1989), and a Sequenase Kit (U. S. Biochemical Corp.).
A -3-kilobase pair Pstl fragment of the YCplac22-based UBA2-bearing plasmid pJDA2-A contained the UBA2 ORF, -an 0.6 kilobase pair of the 5'-flanking sequence, and a 3'-flanking region of the UBA2 locus that included part of the adjacent SAC7 gene (Dunn and Shortle, 1990). The Pstl fragment was subcloned into the phagemid pRS315 (Sikorski and Hieter, 1989), yielding pJDA315. (One Pstl site of this fragment was present in the polylinker ofYCplac22; the other Pstl site was present 3' to the insert/EMBL3A junction.) In the construction of the uba2!:,., uba2-C177A, and uba2-C177S alleles of UBA2 (see the main text), singlestranded pJDA315 DNA and synthetic oligonucleotides were used as described by Kunkel (1985). To express UBA2 from P cuPi (derived from YEp46; see below) or P GALI (see above), a Bglll site was placed in front of the ATG start codon of UBA2 using PCR and synthetic primers.
For C-terminal tagging of Uba2p, a Kpnl recognition sequence was linked to the 3' end of the UBA2 ORF using PCR. This Kpnl site was then used to link, in-frame, the UBA2 ORF and fragments encoding specific sequence tags. A restriction fragment encoding 23 residues and containing two repeats of the 9-residue ha epitope (Field et al., 1988) was a gift from Dr. N. Schnell. The FLAG tag (Brizzard et al., 1994) and the His6 tag (Hoffmann and Roeder, 1991) were produced using PCR and synthetic primers. The GFP gene (Chalfie et al., 1994) was amplified from the plasmid TU#65 (a gift from Dr. N. Johnsson) using PCR and primers that added appropriate restriction sites for in-frame fusions with UBA2-derived genes (see above). In the constructs containing untagged UBA2, this gene was flanked by its original 3' sequences, whereas the tagged UBA2 derivatives bore Teyc 1 , the transcription termination sequence of the CYCl gene that was transplanted from the plasmid YEp46 (Ecker et al., 1987) (a gift from Dr. D. Finley). The tagged derivatives of UBAl and the chimeric genes produced by swapping sequences of UBAl and UBA2 were constructed using PCR-generated fragments of these genes.
The final constructs were inserted into the 2µ-based plasmids YE-plac181 (LEU2) or YEplac195 (URA3) (Gietz and Sugino, 1988), using multistep protocols (details available on request). For the expression of His6-Ub (Uh bearing a Met-Arg-Gly-Ser-His 6 -Gly-Ser N-terminal extension) in Escherichia coli, a DNA fragment encoding the Uh ORF was amplified from plasmid YEp46 using PCR and synthetic primers, and cloned as a BamHI-Kpnl fragment into pQE-40 (Qiagen), yielding pJD359. To express a His6-tagged Uh (Beers and Callis, 1993) in S. cerevisiae, an EcoRI-Kpnl fragment from pJD359 encoding His6-Ub was inserted between PcuPJ and Teyci (both from YEp46) in the background of YEplacl81 (see above), yielding pJD421. Most of the junctures and tags generated by PCR and synthetic oligonucleotides were verified by sequencing. In the cases where sequence testing was incomplete, biological functions of the constructs were verified using independently produced clones.
Immunoprecipitation of Radiolabeled Proteins-The S. cerevisiae strain JD62-6A was transformed with either YEplac181 (a vector), pJD389, pJD302, pJD306, or pJD396 that expressed, respectively, no Uba2p, Uba2p-ha, Uba2p-C177A-ha, Uba2p-C177S-ha, or Uba2p-GFPha. The cultures were grown in SG medium to A 600 of -1, then diluted 5-fold into SD or SG media containing 50 µM CuSO 4 , and incubated for 8 h. The cells from 15-ml cultures were collected by centrifugation, resuspended in 0.6 ml of SD or SG media containing 50 µM CuSO 4 and 0.1 mCi ofTran 35 S-label (ICN), and incubated for 10 min at 30 °C. The cells were collected by centrifugation for 10 s at 14,000 X g and resuspended in 600 µI of the lysis buffer (0.15 M NaCl, 1% Triton X-100, 1 mM EDTA, 50 mM Na-HEPES, pH 7.5) containing also 10 mM N-ethylmaleimide, 1 mM phenylmethylsulfonyl fluoride, as well as leupeptin, pepstatin A, antipain, chymostatin, and aprotinin (each at 20 µg/ml). 0.5 ml of 0.5-mm glass beads was then added, and the cells were lysed by vortexing in the lysis buffer. The extracts were centrifuged at 14,000 x g for 15 min. Immunoprecipitations were carried out with the supernatants (containing approximately equal amounts of CHCl 3 COOH-pre-cipitable 35 S) by adding ascitic fluid (1.5 µ1/0.6 ml of supernatant) containing the monoclonal anti-ha antibody 12CA5 (Field et al., 1988) (Babco Inc., Richmond, CA). The samples were incubated on ice for 2 h; 25 µl of Protein A-Sepharose (0.1 g/ml) (Pharmacia Biotech Inc.) was then added, and the suspensions were incubated, with end-over-end rotation, for 1 h at 4 °C, followed by a 5-s centrifugation in a microcentrifuge. Pellets were washed four times with 1 ml of the lysis buffer, resuspended in the thioester-preserving electrophoretic sample buffer (10% glycerol, 2% SDS, 0.1 % bromphenol blue, 50 mM Tris-HCl, pH 6.8), and centrifuged at 14,000 X g for -20 s. One-half of each supernatant was left untreated; another half was made 0.25 Min /3-mercaptoethanol, followed by incubation at 100 °C for 3 min. Both samples were subjected to SDS-8% PAGE and fluorography. 14 C-Labeled protein size standards were from Amersham.
Immunoblot Analysis of Immunoprecipitated Proteins-The strain SUB312 (Finley et al., 1994), which expressed either wild-type (wt) Ub or mycUb from plasmids YEp96 or YEp105, respectively (see above), was transformed with either the vector YEplac195 (Gietz and Sugino, 1988), its derivative pJD426, or its derivative pJD428, which expressed, respectively, Ubalp-FLAG or Uba2p-FLAG. Cells from exponential cultures (A 600 of -1) of the transformants growing in SD media containing 50 µM CuSO 4 were collected by centrifugation, and extracts were prepared as described above. Total protein concentration in an extract was determined using the Bradford assay (Bio-Rad). An extract containing 50 µg of total protein was added to 40 µl of an agarose affinity gel containing the monoclonal anti-FLAG (M2) antibody (Eastman Kodak), followed by incubation for 1 h at 4 °C, with end-over-end rotation. The anti-FLAG resin was then washed four times with 1 ml of the lysis buffer, and the retained proteins were fractionated by SDS-8% PAGE, followed by electroblotting onto nitrocellulose membranes (Schleicher & Schuell). Sequential detection of immunoreactive proteins with the anti-FLAG or anti-myc antibodies was carried out using the Amersham ECL detection system.
Detection of Ub Thioesters---S. cerevisiae SUB312 cells carrying YEp96 (which expressed wt Ub; see above) and either pJD426 or pJD428 (which expressed, respectively, Ubalp-FLAG or Uba2p-FLAG from PcuPI) were grown in SD media. The expression of FLAG-tagged proteins was induced with 50 µM CuSO 4 for 6 h before making the extracts, which were prepared as described above except that N-ethylmaleimide was replaced with 0.5 mM dithiothreitol. The FLAG-tagged proteins were immunoprecipitated as described above from extracts containing 50 µg of total protein. The anti-FLAG resin was washed twice with 1 ml of the lysis buffer and once with 1 ml of 10 mM MgC1 2 , 25 mM Tris-HCl (pH 8.0) (Tris-Mg buffer). The washed resin was resuspended in 50 µl of the thioester assay buffer (10 mM MgC1 2 , 1 mM ATP, 10 mM creatine phosphate, 25 mM Tris-HCl (pH 8.0)) containing also, 100 µ,g/ml creatine kinase and -30 pmol of[ 35 S]methionine-labeled His6-Ub (10 6 cpm) (see below), and incubated at 37 °C for 10 min. The resin was then washed twice with Tris-Mg buffer, and resuspended in electrophoretic sample buffer (see above) containing or lacking 0.25 M /3-mercaptoethanol. The former (but not the latter) sample was incubated at 100 °C for 3 min before SDS-8% PAGE. The gel was stained with Coomassie (to verify the presence of approximately equal amounts of FLAG-tagged test proteins), then dried and subjected to autoradiography.
Ub Affinity Chromatography-The strain SUB312, lacking pUB23 (see above) and expressing His6-Ub from pJD421, was transformed with plasmids pJD426 or pJD428, which expressed, respectively, Ubalp-FLAG or Uba2p-FLAG. Cells grown in 1 liter of SD, 50 µM CuSO 4 medium to A 600 of -1 were lysed with glass beads in 20 ml of 1 % Triton X-100, 0.3 M NaCl, 0.33 mM /3-mercaptoethanol, 50 mM sodium phosphate (pH 8.0). The extracts (diluted 4-fold) were adjusted to 0.25% Triton X-100, 1 M NaCl and loaded onto Ni-NTA-Sepharose columns (Qiagen) that bind the His6 tag. The columns were washed with 0.3 M NaCl, 5 mM MgC1 2 , 0.33 mM /3-mercaptoethanol, 10 mM imidazole, 2 mM ATP, 50 mM sodium phosphate (pH 8.0). The Ubalp-FLAG protein was eluted with the same buffer containing 2 mM AMP and 40 µM sodium pyrophosphate instead of ATP. The His6-Ub bound to the resin was subsequently eluted with a low pH buffer (Beers and Callis, 1993). Fractions were assayed for the presence of FLAG-tagged proteins by immunoblotting with the anti-FLAG antibody.
The supernatant was adjusted to a final concentration of 1 M NaCl, 1 mM imidazole, 0.5 mM /3-mercaptoethanol in a final volume of30 ml, and loaded onto a Ni-NTA-Sepharose column (at a flow rate of0.2 ml/min). The column was washed with 30 ml of pH 8 buffer (5% glycerol, 5 mM MgC1 2 , 1 mM imidazole, 1 M NaCl, 0.5 mM /3-mercaptoethanol, 50 mM sodium phosphate (pH 8.0)), and thereafter with 45 ml of pH 6 buffer (the same as pH 8 buffer but containing 10 mM imidazole and adjusted to pH 6.0 with 1 M NaOH). Uba2p-FLAG-His6 was eluted with pH 3 buffer (the same as pH 8 buffer but adjusted to pH 3 with 1 M HCl). The eluted fractions (12 ml) were adjusted (in a final volume of 25 ml) to 8 M urea, 0.1 M sodium phosphate, 10 mM Tris (pH 8.0), and loaded onto a second Ni-NTA-Sepharose column that had been equilibrated with the same pH 8-urea buffer. The column was washed with 25 ml of pH-urea buffer followed by elution of Uba2p-FLAG-His6 with pH 6-urea buffer (the same as pH 8-urea buffer but adjusted to pH 6 with 1 M HCl). Proteins in the eluted fractions were assayed for the presence of Uba2p-FLAG-His6 and mycUb by immunoblotting, using the M2 anti-FLAG and 9E10 anti-myc antibodies, respectively.
Preparation ofin Vivo Labeled His6-Ub from E. coli-E. coli JM105 (Ausubel et al., 1989), transformed with the plasmid pJD395 that expressed His6-Ub (see above), were grown in 20 ml of Luria broth containing 50 µg/ml ampicilin to A 600 of -0.6. The cells were centrifuged at 5,000 X g for 10 min, washed in M9 medium (Ausubel et al., 1989), and resuspended in M9 medium supplemented with glucose (0.2%), thiamine (2 µg/ml), methionine assay medium (0.06%) (Difeo), and isopropylthiogalactoside (1 mM). The cells were incubated in this medium at 37 °C for 1 h. 0.3 mCi of[ 35 S]Prolabel (Amersham) was then added, followed by incubation at 37 °C for 30 min. The cells were collected by centrifugation at 5,000 X g for 10 min, washed once in 0.3 M NaCl, 50 mM sodium phosphate (pH 8.0), and resuspended in 0.4 ml of the same buffer containing 5 mM /3-mercaptoethanol and protease inhibitors (see above). The cells were lysed by vortexing with 0.1-mm glass beads for 1 min three times. The extracts were centrifuged at 14,000 X g for 15 min. [ 35 S]Methionine-labeled His6-Ub was purified from the supematants using Ni-NTA-Sepharose (Qiagen) and the manufacturer's recommendations.

RESULTS AND DISCUSSION
The UBA2 Gene-A DNA fragment containing a S. cerevisiae gene that was later termed UBA2 was isolated from a library of Agtll phages (Young and Davis, 1983) carrying inserts of S. cerevisiae genomic DNA. The finding of UBA2 resulted from a cross-reaction of a UBA2-expressing phage plaque with a polyclonal antiserum raised against a yeast plasma membrane ATPase kinase, a presumably unrelated protein. 3 Initial analyses of a partial ORF in a cloned DNA fragment revealed extensive similarities of the deduced amino acid sequence to the sequences of Uh-activating (El) enzymes (see below). AS. cerevisiae DNA clone containing the complete gene (termed UBA2) and its flanking sequences was then isolated (see "Materials and Methods"). Mapping and sequence analyses showed that UBA2 is located on the right arm of Chromosome IV, flanked by SAC7 and SUF3 (Fig. IA). SUF3 encodes a glycine tRNAccc• The 3'-end of this gene is only 161 bp away from the presumed UBA2 start codon, inferred so as to yield the largest ORF. The next in-frame ATG is located 126 bp downstream of the inferred start codon. A protein initiated from this downstream ATG would lack a putative ATP-binding motif (see below).
The 1908-bp UBA2 encodes an acidic (calculated pl of 4.8), 636-residue (71 kDa) protein (Fig. lC). The Codon Adaptation Index (calculated according to Sharp and Li (1987)) of UBA2 is 0.151, characteristic of weakly expressed yeast genes. The sequence of the first 561 residues ofUba2p is highly similar (28% identical, 49% similar) to the sequence between residues 414 and 1020 of the 114-kDa Ubalp, the yeast El enzyme (Fig. 2). The sequence ofUba2p is also highly similar to those of human, mouse, and wheat El enzymes (Fig. 2). By contrast, the 636residue Uba2p is only 12% identical (and lacks regions of contiguous identity longer than 3 residues) to the 540-residue Axrlp protein of Arabidopsis thaliana that is required for auxin response in this plant; the sequence of Axrlp has signif-

SUF3 :
:  icant similarities to sequences of the known El enzymes (Fig.  2). Although both El and E2 enzymes interact with Ub and form enzyme -Ub thioesters, there are no statistically significant sequence similarities between the known Els and E2s (Jentsch, 1992). Sequence comparisons failed to detect significant similarities between Uba2p and the known E2 enzymes. The sequence similarities between Uba2p and El enzymes include the consensus sequence for a nucleotide binding site, GXGXXG (positions 28-33 in Uba2p) (Wierenga and Hol, 1983), and also the consensus sequence KXXPZCTXXXXP (Z is a non-polar residue) (positions 172-183 in Uba2p) (Fig. 2) around the essential cysteine (C) in El enzymes that becomes linked to Ub in an El-Ub thioester (Hatfield and Vierstra, 1992). These motifs are present in all of the known El enzymes, but are absent from the Axrlp protein (Fig. 2). The known El enzymes contain an N-terminal domain of -400 residues that are absent from Uba2p (Fig. 2). Conversely, the 82-residue C-terminal region of Uba2p is absent from the known El enzymes. This region contains the sequence KRTK (positions 619-622) that matches one consensus sequence for nuclear localization signals (NLSs).
The known El enzymes contain two -150-residue regions of similar sequence (Fig. 2). Only one such region is present in Uba2p (positions 12-156); it contains stretches of similarity to the N-terminal region of the E. coli ClnNp protein, which functions in the biosynthesis of the organic component of molybdopterin, a molybdenum-containing prosthetic cofactor (McGrath et al., 1991;Johnson and Rajagopalan, 1987; and data not shown). (The -150-residue duplication in the El enzymes are the reason for a difference between their alignment with the A. thaliana Axrlp in Fig. 2 and the alignment described by Leyser et al. (1993).) UBA2 Is Essential for Cell Viability-A null allele of UBA2 was produced by replacing the entire UBA2 ORF with the H/S3 gene (Fig. lA). The resulting construct was used to replace, by homologous recombination (Rothstein, 1991), one of the two copies of wt UBA2 in the diploid JD51. The resulting uba2t:./ UBA2 strain was sporulated and subjected to tetrad analysis. In virtually all of the 20 tetrads examined, no more than 2 of the 4 spores gave rise to growing colonies (Fig. lB). All of the viable spores were His-, indicating that the absence of UBA2 is incompatible with spore viability. Microscopic examinations of germinated uba2!1 spores showed growth-arrested microcolonies of 50-100 enlarged cells. Microdissection of such colonies yielded -60% cells with large buds, -10% with two buds, and -30% without buds (data not shown).
To determine whether UBA2 is also required for vegetative growth, the wt UBA2 was replaced by an otherwise wt UBA2 that was marked with the URA3 gene and expressed from the galactose-inducible, glucose-repressible P GALJ promoter. The resulting heterozygous (P aALrUBA2 I UBA2) diploid was sporulated, and tetrads were dissected onto galactose-containing medium. The Ura+ spores (in which UBA2 was linked to P GALJ) gave rise to smaller colonies than the Ura -(wt UBA2) spores). However, no significant difference in colony sizes could be observed upon restreaking of the colonies (data not shown), suggesting that smaller sizes of the initial Ura+ colonies were due to a phenotypic lag in the galactose-induced expression of UBA2. When PaALrUBA2 cells (strain JD62-6A) were streaked onto glucose-containing media, no single colonies were observed (Fig. 3), except for an occasional appearance of spontaneous suppressors (data not shown), indicating that UBA2 is essential for vegetative growth as well. Microscopic examination of PaALrUBA2 cells that were grown in glucose for 15 h showed them to be arrested at more than one stage of the cell cycle. Many (not all) of these cells were larger than wt cells, and some of them had two or three buds (data not shown). An Essential Cysteine in Uba2p-Uba2p contains the consensus sequence of El enzymes, KXXPZCTXXXXP (Z is a non-polar residue), which encompasses the essential Cys residue (C) that becomes linked to Ub in an El-Ub thioester (see above and Fig. 2). To determine whether the corresponding Cys-177 ofUba2p (Fig. 2) is also essential for the function of Uba2p, we converted Cys-177 into Ala or Ser (see "Materials and Methods"). Low copy (CEN-based) plasmids expressing no Uba2p (vector alone), wt Uba2p, or its mutant variants Uba2p-C 177 A and Uba2p-C 177S were introduced into strain JD62-6A, in which wt UBA2 was expressed from P GALJ, and the transformants were selected on galactosecontaining (SG) plates. Upon restreaking, the transformants of all four classes yielded colonies of similar sizes on SG plates. However, only the transformants that carried the plasmid-borne wt UBA2 grew on glucose-containing (SD) plates, conditions that repressed the expression of the chromosomal UBA2 from PaALJ (Fig. 3A). Thus, Cys-177 is required for an essential function of Uba2p.
We also determined the growth rate effects of overproducing either wt Uba2p or its inactive variants Uba2p-C 177 A and Uba2p-Cl 77S. These proteins were expressed from high copy (2µ-based) plasmids and P GALJ (Fig. 3B). Overproduction of wt Uba2p did not result in a significant growth defect (Fig. 3B). By contrast, the expression of either Uba2p-C 177 A or Uba2p-C 177S in the same Uba2+ cells greatly impaired their growth (Fig. 3B). Thus, Uba2-C177A and Uba2-C177S are dominant negative alleles of UBA2, suggesting that the corresponding inactive proteins (Uba2p-C 177 A and Uba2p-C 177S) compete with wt Uba2p for binding to itself or other relevant proteins. This interpretation is consistent with the finding that Uba2p is a part of a heterooligomeric complex (see below).
3. Cysteine 177 of Uba2p is essential for Uba2p function. Panel A, the S. cereuisiae strain JD62-6A, in which UBA2 was expres ed from P GAL, (see "Mate1·ials and Methods") was transformed with the CEN6/ARSH4/LEU2 vector pRS315 (Sikorski and Hieter, 1989); or with the same vector expressing wt Uba2p; or with the same vector expressing either Uba2p-Cl 77 A or Uba2p-Cl 77S. Transformants were elected on SG plates lacking uracil and leucine (SG/-Ura/ -Leu), and were then restreaked onto the same medium (denoted as "galacto e") or onto SD/-Ura/-Leu plates (denoted as "glucose"). Panel B, dominant negative effects of mutants at po ition 177 of Uba2p. The wt strain JD52 (a haploid segregant of JD51; see "Materials and Methods," and Fig. 1) wa transformed with the high copy LEU2 vector YEplacl81 (Gietz and Sugino, 1988); or the same vector expressing (from P GAL.,) either wt Uba2p, or Uba2p-Cl 77 A, or Uba2p-Cl 77S. Transformants were selected on SDI-Leu media, and were then restreaked onto the same (SD/-Leu) or SG/-Leu plates. The plates were photographed after 2 and 3 days of growth at 30 °C on gluco e and galactose, respectively. tional, P cup,-inducing levels of Cu 2 + in the medium (data not shown).
Immunoblot analysis of extracts (prepared using a detergent-lacking lysis buffer) from cells expressing Uba2p-ha indicated that the bulk ofUba2p-ha was present in the 14,000 X g pellet, from which it could be recovered after resuspension of the pellet in a lysis buffer containing 1 % Triton X-100 (data not shown), suggesting that Uba2p-ha was located in a membraneenclosed compartment. Fluorescence microscopy was then used to examine S. cereuisiae transformants that expressed the Uba2p-GFP fusion protein. The bulk of fluorescent GFP was located in an organelle identified as the nucleus since it could also be stained with DAPI, a DNA-binding fluorescent marker (Fig. 5, B and C). In a control transformant that expressed GFP alone the bulk of fluorescent GFP was located in the cytoplasm and probably also in the nucleus, because no "negative" staining of the nucleus was observed (Fig. 51). Fractionation experiments with extracts from S. cereuisiae that expressed Uba2-FLAG confirmed the nuclear localization of Uba2p (data not shown).
Ubalp and Uba2p Cannot Substitute for Each Other-We asked whether increased expression of Ubalp (the yeast El enzyme) could compensate for the absence of Uba2p, or vice versa. Ubalp was overproduced by expressing it from a high copy plasmid and the induced PcuP, (see "Materials and Methods"). Overproduction of Ubalp in the PaALrUBA2 strain JD62-6A did not rescue these cells from growth arrest on glucose-containing media (conditions that repressed the expression ofUba2p). In a reciprocal test, Uba2p was expressed from  Fig. 2, and variants of these proteins that have been constructed and tested in the present work. The corresponding genes were expressed from Pcu,,, in the high copy plasmid YEplacl81 (see Fig. 3). The constructs were transformed into JD62-6A (P 0 A,_,. UBA2; see the legend to Fig. 3) or JD77-1A (ubal :i::HIS3) carrying the low copy plasmid pJD320 that expressed wt Ubalp from P 0 AL,· Transformants were selected on galactose-containing plates and then assayed for growth on glucose-containing plate as hown in Fig. 3. "+" on the right indicates colony growth on glucose-containing plates;"-" denotes the absence of a significant difference in the kinetics of growth arrest in comparison to that observed with the same strain carrying vector alone. Subcellular localization of these proteins was analyzed by fluorescent microscopy (Fig. 5), using fusions between the GFP and C termini of the above protein constructs (see "Materials and Methods"). These GFP fusions (whose properties in complementation as ays were indistinguishable from those of their counterparts lacking the GFP moiety) were expres ed in wt S. cereuisiae a described above for the GFPlacking fusions. Subcellular localization (denoted as SL) of the GFP fusions is abbreviated in the diagram as "C" for largely cytoplasmic (apparently cytosolic) and "N' for la1·gely nuclear (see Fig. 5). Uba2pspecific portions of these fusions are shaded in blaclz or gray. Ubalp is represented as a white rectangle. Additional amino acid sequences at the C termini of truncated variants or at the junctions within specific fusion proteins are shown in single letter abbreviations. The positions of regions of identical or similar sequences among the e constructs are indicated by dotted lines. a high copy plasmid and PcuP, in a uba16. strain (McGrath et al., 1991) that expressed Ubalp from a low copy plasmid and PaALi (see "Materials and Methods"). Overproduction ofUba2p did not prevent growth arrest of P aAL 1 -UBA1 cells on glucosecontaining media ( Fig. 4 and data not shown). A derivative of Uba2p that lacked its 82-residue C-terminal region (which is absent from the known El enzymes) (Fig. 4111) was also unable to rescue cells depleted for Ubalp. The same results were obtained with a Uba2p derivative bearing the 412-residue Nterminal region of Ubalp (this region is absent from Uba2p) (Fig. 4, V and VI). (Either of these Uba2p-based constructs could rescue cells depleted for Uba2p (data not shown).) Fluorescence patterns of cells expressing Ubalp-GFP (this fusion was functionally active in that it could rescue uba16. cells) were indistinguishable from those expressing GFP alone (see above and Fig. 5D), suggesting that Ubalp was located in both the cytosol and the nucleus. Fluorescence patterns of cells expressing a GFP fusion to the Uba2p derivative bearing the El-specific N-terminal region (Fig. 4Vl) indicated that the presence of this region did not impair the accumulation of Uba2p in the nucleus (Fig. 5H). By contrast, a Uba2p-GFP fusion Jacking the 82-residue C-terminal region of Uba2p (this region contained a putative NLS) (Fig. 4lll) accumulated in the nucleus much less efficiently: the relative level of cytoplasmic fluorescence was much higher with this fusion than with GFP fusions to either wt Uba2p or Uba2p bearing the El-specific N-terminal region (Fig. 5E). Thus, the C-terminal region of Uba2p is important for its transport to the nucleus but may be not the sole NLS-containing region of Uba2p. That the Cterminal region of Uba2p actually bears a portable NLS was confirmed by fusing this region to the C terminus of Ubalp (Fig. 41V). The resulting fusion was unable to rescue cells depleted for Uba2p (see above), but did rescue cells depleted for Ubalp (Fig. 41V). Examination of cells expressing the corresponding GFP fusion (Fig. 41V) showed that the bulk of the Ubalp derivative bearing the 86-residue C-terminal region of Uba2p was located in the nucleus (Fig. 5F). We conclude that the S. cerevisiae Ubalp (El enzyme) and Uba2p have distinct, apparently nonoverlapping, essential functions.
Does Uba2p Activate Ubiquitin?-The sequence similarities between Uba2p and the known El enzymes, and the functional essentiality of the conserved Cys-177 ofUba2p were consistent with the possibility that Uba2p might be a nuclear ubiquitinactivating (El) enzyme. We therefore tested whether Uba2p could activate Ub in vitro under conditions permissive for Ub activation by Ubalp, the known S. cerevisiae El enzyme. Ubalp-FLAG and Uba2p-FLAG were precipitated with a monoclonal anti-FLAG antibody linked to an agarose-based affinity resin. The antibody-immobilized Ubalp-FLAG and Uba2p-FLAG were incubated in the presence of ATP and [ 35 S]methionine-labeled His6-Ub (see "Materials and Methods"). The incubation products were fractionated by SDS-PAGE with or without prior treatment with mercaptoethanol at 100 °C (this treatment cleaves Ub thioesters but not isopeptide bond-mediated Ub conjugates). While Ubalp-FLAG formed a Ub adduct (Ubalp-Ub thioester) sensitive to mercaptoethanol, no such adduct could be detected with Uba2p-FLAG (data not shown).
In a different test, we prepared an extract from a yeast strain that expressed both Ubalp-FLAG and His6-Ub, and used chromatography on Ni-NTA-Sepharose to retain His6-Ub and its covalent or noncovalent complexes with other proteins. The preparation of extract and its loading onto the column were carried out in the presence of ATP. Under these conditions, a fraction of Ubalp-FLAG in the extract bound to the column, and could be specifically eluted with a buffer containing AMP and pyrophosphate (see "Materials and Methods"). The binding of Ubalp-FLAG to Ni-NTA-Sepharose under these conditions resulted from the formation of a thioester between Ubalp-FLAG (an El enzyme) and the column-bound His6-Ub. Since AMP and pyrophosphate are the other products of this ATPdependent reaction (Haas et al., 1982), high concentrations of AMP and pyrophosphate can partially reverse it (Ciechanover et al., 1982), yielding ATP and free El (Ubalp-FLAG).
In an otherwise identical test with cells expressing His6-Ub and Uba2p-FLAG (instead of Ubalp-FLAG), no binding of Uba2p-FLAG to the column containing His6-Ub was observed (data not shown). We conclude that Uba2p does not form a thioester with Ub under conditions that allow the formation of Ub thioesters by a known El enzyme such as Ubalp.
Uba2p Is Associated with Itself and Other Proteins-To examine the interactions of Uba2p with other S. cerevisiae proteins, we expressed various Uba2p derivatives tagged with the ha epitope in the strain JD62-6A (which expressed wt Uba2p from P cAL 1 ). Cells expressing Uba2p-ha, Uba2p-Cl 77 A-ha, Uba2p-Cl 77S-ha, or Uba2p-GFP-ha were grown in SG medium; a portion of this culture was shifted to the glucosecontaining SD medium for 8 h before labeling with [ 35 S]methionine (these conditions extinguished the expression of wt Uba2p from PcAL 1 ). SDS-PAGE of proteins immunoprecipitated from whole cell extracts with anti-ha antibody yielded not only the expected Uba2p species but several other proteins as well (Fig. 6). Uba2p-ha (and its Cys-177 derivatives) ran as doublets with apparent molecular masses of-88 and -90 kDa, significantly higher than the calculated molecular mass of Uba2p-ha (73 kDa). The same anomalous mobility was observed with Uba2p expressed in E. coli (data not shown). The -90-kDa form was also detected with anti-FLAG antibody in immunoblot analyses of cells expressing Uba2p-FLAG (Fig. 7). The -90-kDa Uba2p appears to be a phosphorylated derivative of the -88-kDa Uba2p. 4 Several non-Uba2p species (termed p52, p63, p87, and p120), with apparent molecular masses of-52, -63, -87, and -120 kDa, were specifica1ly coimmunoprecipitated with Uba2p-ha (Fig. 6). The same proteins were coimmunoprecipitated with either the functiona1ly active Uba2p-ha (Fig. 6, lanes 2 , 5, and 6) or the inactive (mutant at position 177) derivatives of Uba2p-ha (lanes 3 and 4), indicating that Uba2p interactions with these proteins do not require its putative active-site Cys-177. (Proteins of the same molecular masses were also copurified together with Uba2p-FLAG-His6 fusion on either an anti-FLAG antibody (agarose-based) affinity resin or on Ni-NTA-Sepharose; these results (data not shown) provided independent evidence for a direct interaction between Uba2p and at least one polypeptide among p52, p63, p87, and p120.) The electrophoretic mobilities of Uba2p-interacting proteins were the same irrespective of whether the samples were treated with mercaptoethanol before SDS-PAGE (data not shown).
Immunoprecipitation with anti-ha antibody was also carried -- Fie. 6. Uba2p interacts with itself and other proteins. 35 8-Labeled proteins from the strain JD62-6A (PcM.,-UBA2; see the legend to Fig. 3) transformed with either a vector (control) or vector-based plasmids expressing ha-tagged Uba2p derivative were immunoprecipitated with a monoclonal anti-ha antibody, and analyzed by SDS-PAGE and fluorography. Each of the samples wa split in two and either heat-treated in the presence of mercaptoethanol before SDS-8% PAGE ( ee "Materials and Methods") or left untreated. The data shown here were obtained with untreated amples (mercaptoethanol-treated samples yielded the ame re ults). The cells carried either a control vector  5 and 6). The cells were grown in galacto e and shifted to a glucose-containing medium 8 h before ' 15 8 labeling (lanes 1-5). Alternatively, the cells were labeled in a galactose-containing medium (lane 6). The sizes of marker proteins (M) are in kDa, on the left. Positions of different Uba2p derivatives and coimmunoprecipitated protein (denoted a p52, p63, p87, and pl20) are indicated on the right. Uba2p-ha* denotes a post-translationally modified Uba2p-ha (see also Fig. 7 and main text). A dot mark the position of a S. cereuisiae protein that cross-reacted with anti-ha antibody (Dohmen et al., 1991a). out with an otherwise identical extract prepared from JD62-6A cell grown in the presence of galactose; these cells, unlike those grown on glucose-containing media, contained the untagged wt Uba2p as well. This immunoprecipitation yielded, in addition to the non-Uba2p species described above, a protein with an apparent molecular mass (-86 kDa) expected for the untagged, wt Uba2p (Fig. 6, lane 6). (The expected apparent mass of the untagged Uba2p was calculated by subtracting 2 kDa (the mass ofha tag) from 88 kDa, the apparent mass of the 73-kDa Uba2p-ha.) In addition, extracts from cells lacking wt Uba2p and expressing both Uba2p-FLAG and Uba2p-GFP-ha yielded both Uba2p-FLAG and Uba2p-GFP-ha upon immunoprecipitation with either anti-FLAG or anti-ha antibody (data not shown). Taken together, these findings ( Fig. 6 and data not shown) strongly suggested that Uba2p interacts with itself and is a part of a complex or complexes that contain proteins p52, p63, p87, and pl20.
Uba2p Is Ubiquitinated-In the course of testing for the putative Uh-activating function of Uba2p, we found that Uba2p-FLAG is multiubiquitinated in vivo (Fig. 7). In these experiments, the anti-FLAG antibody was used to immunoprecipitate proteins from extracts of the S. cereuisiae strain SUB312, which lacked the chromosomal Ub-coding sequences, expressed either wt Ub or epitope-tagged mycUb from a plasmid (Finley et al., 1994;Ellison and Hochstrasser, 1991), and in addition carried either a plasmid expressing Uba2p-FLAG (from the induced Pcup,) or a corresponding control vector (see "Materials and Methods"). Immunoblot analysis, using a monoclonal anti-myc antibody, of proteins that had been immune- Fie. 7. Post-translational modifications of Uba2p. Immunoblot analyses of proteins immunoprecipitated with the monoclonal anti-FLAG antibody from the S. cereuisiae strain SUB312 (Finley et al., 1994) that lacked the chromosomal Ub-coding sequences, expressed either wt Ub or mycUb from a plasmid, and in addition carried either a pla mid expres ing Uba2p-FLAG or a corresponding (control) vector (see "Materials and Methods," and the main text). Panel A, immunoblotting with anti-FLAG antibody after SDS-8% PAGE of proteins immunoprecipitated with the same antibody. The presence (or absence) of mycUb and Uba2p-FLAG in each of the initial extracts is indicated above the lanes. An extract used to produce the sample in the third lane was identical to an extract in the fourth lane except that the latter was heated at 100 °C in the presence of 0.6% SDS before immunoprecipitation with anti-FLAG antibody (see main text). Panel B, the immunoblot in A was reprobed with a monoclonal anti-myc antibody. The position of a heavy chain of anti-FLAG antibody that reacted with the secondary antibody is indicated by a dot on the left. precipitated with anti-FLAG antibody from cells expressing both Uba2p-FLAG and mycUb showed an extensive smear of mycUb-containing species extending from below the position of Uba2p-FLAG to the top of the gel (Fig. 7B). This pattern was observed exclusively with cells expressing both Uba2p-FLAG and mycUb, and was the same irrespective of whether the samples were heat-treated in the presence of mercaptoethanol or left untreated before SDS-PAGE; the latter result indicated that most species in the mycUb-containing smear lacked mycUb thioesters ( Fig. 7 and data not shown).
No smear of mycUb-containing species was observed in otherwise identical experiments with cells that expressed mycUb and Ubalp-FLAG (but not Uba2p-FLAG). Instead, a band corresponding to the expected size of a Ubalp -mycUb thioester was detected by the anti-myc antibody; this band was not observed with mercaptoethanol-pretreated samples (data not shown). Since no smear ofUba2p-FLAG-containing species was detected in the samples immunoprecipitated with anti-FLAG antibody and then imrnunoblotted with the same antibody, we asked whether mycUb moieties were linked directly to Uba2p-FLAG or to a protein(s) coimrnunoprecipitated with it by anti-FLAG antibody. The lysis buffer (see "Materials and Methods") was made 0.6% in SDS; the extract was heated at 100 °C for 3 min, and then diluted with SDS-lacking lysis buffer to the final SDS concentration of 0.03%, followed by immunoprecipitation with anti-FLAG antibody. The results were indistinguishable from those obtained in the absence of SDS pretreatment (Fig. 7), strongly suggesting that the mycUb-containing species of the smear were largely those of multiubiquitinated Uba2p-FLAG. This interpretation was supported by the results of experiments in which Uba2p-FLAG-His6 was expressed in cells that also expressed mycUb. Uba2p-FLAG-His6 was purified from an extract of these cells on Ni-NTA-Sepharose in the presence of 8 M urea, conditions expected to disrupt noncovalent proteinprotein interactions but not the isopeptide bonds of Ub conjugates. Immunoblotting with anti-myc antibody still detected mycUb in fractions from the column that contained Uba2p-FLAG-His6 (data not shown).
Taken together, these findings ( Fig. 7 and data not shown) suggest that a relatively small proportion of Uba2p-FLAG is ubiquitinated in vivo, because otherwise these multiubiquitinated Uba2p-FLAG species should have also been detectable with anti-FLAG antibody. (The sensitivity of detection of mycUb-containing multiubiquitinated Uba2p-FLAG species would be expected to be higher with anti-myc than with anti-FLAG antibody, given the increasing molar ratio of mycUb to Uba2p-FLAG in higher molecular mass species of the smear.) The observation that a minor fraction of the smear of mycUbcontaining species extends below the position of Uba2p-FLAG (Fig. 7B) suggests, among other possibilities, that multiply ubiquitinated Uba2p-FLAG is proteolyzed in vivo.
Concluding Remarks-We describe the isolation and analysis of an essential S. cerevisiae gene, termed UBA2, that encodes a 71-kDa nuclear protein Uba2p with extensive sequence similarities to Uh-activating (El) enzymes, an evolutionarily conserved family of 110-120-kDa proteins. Until the finding of Uba2p, the previously identified S. cerevisiae Ubalp El enzyme (McGrath et al., 1991) has been the only known member of the El sequence family in this organism.
A variety of tests for El activity ofUba2p, the ability to form a thioester with Ub, yielded negative results (see above), suggesting that Uba2p is not an El enzyme. We also showed that Uba2p, which is located largely in the nucleus, cannot complement the essential function of the largely cytosolic Ubalp El enzyme even if Uba2p is rendered partially cytosolic through a deletion of its NLS-containing C-terminal region. Conversely, Ubalp cannot complement the (unknown) essential function of Uba2p even ifUbalp is made partially nuclear by fusing it to the NLS-containing C-terminal region of Uba2p.
One constraint on the range of possible functions of Uba2p was provided by the finding that Cys-177 of Uba2p is required for an essential (cell viability-sustaining) function of Uba2p. Cys-177 of Uba2p is located within a region conserved among El enzymes and at a position where the active-site cysteine is present in the known Els. (This cysteine forms a thioester bond to the C terminus of Ub in an El -Ub thioester (see Introduction).) Thus it is likely that an enzymatic reaction catalyzed by Uba2p involves the formation of a Cys-177-mediated thioester between Uba2p and either Ub or another protein. One possibility is that Uba2p acts as a Uh-conjugating (E2) enzyme (see Introduction). If so, Uba2p would be the first example of an E2 whose sequence is El-like and dissimilar to sequences of the currently known E2 enzymes.
Another clue that should help understand the function of Uba2p was provided by the finding that Uba2p interacts with itself and several other, presently unknown proteins, termed p52, p63, p87, and p120 (Fig. 6). Uba2p was also found to be multiubiquitinated in vivo (Fig. 7), suggesting that at least a fraction of Uba2p is metabolically unstable.
We recently learned that C. Moore and her colleagues had also identified the UBA2 gene, which they termed PIP2 (for polymerase-interacting protein 2). 5 Pip2p (Uba2p) was isolated in a two-hybrid screen (Chien et al., 1991) for proteins that interact with Paplp, the poly(A) polymerase of S. cerevisiae. PAPI is essential for cell viability and encodes a 64.5-kDa protein (Lingner et al., 1991). Uba2p (Pip2p) is apparently not required for mRNA processing but plays a role in its regulation. 5 We note that p63, a protein of the size ( -63 kDa) similar to that of Paplp, coimmunoprecipitated with the Uba2p protein in our experiments (Fig. 6). Paplp is also known to interact with CFl, a cleavage and polyadenylation specificity factor whose components include Rna14p and Rna15p (Chen and 5 M. del Olmo, S. Gross, and C. Moore, manuscript in preparation. Minvielle-Sebastia, 1994). The presence of active Uba2p in these or other heterooligomeric complexes appears to be essential for cell viability, because overexpression of inactive Uba2p derivatives that lack the putative active-site Cys-177 residue has a dominant negative effect on cell growth (Fig. 3).
It is likely that Uba2p (Pip2p) regulates the rate or specificity ofmRNA polyadenylation by mediating ubiquitination and possibly also degradation of at least one component of a machinery involved. Further work, currently under way, is aimed at identifying this component and other aspects of the Uba2p function.