©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cloning and Expression of a Yeast Gene Encoding a Protein with ATPase Activity and High Identity to the Subunit 4 of the Human 26 S Protease (*)

Héctor A. Lucero (1)(§), Eric W. T. Chojnicki (2)(¶), Sreekala Mandiyan (3), Hannah Nelson (3), Nathan Nelson (3)

From the (1) Department of Physiology, Boston University School of Medicine, Boston, Massachusetts 02118, (2) Bristol Myers Squibb Company, Pharmaceutical Research Institute, East Syracuse, New York 13221, and (3) Roche Institute of Molecular Biology, Nutley, New Jersey 07110

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The cloning, expression, and biochemical characterization of an essential gene of Saccharomyces cerevisiae that encodes for a new member of the TBP1-like subfamily of putative ATPases are described. The protein is 72% identical at the amino acid level to subunit four (S4) of the human 26 S protease and 73% identical to Schizosaccharomyces pombe MTS2 gene product. The purified, recombinant protein, designated Yhs4p, has an estimated molecular mass of 49 kDa and exhibits a Mg-dependent ATPase activity with nucleotide specificity and Kfor ATP similar to those exhibited by the human 26 S protease. The observed ATPase activity was reduced by 73% upon the introduction of point mutation K229Q in the ``P-loop'' domain of the ATP-binding site relative to the nonmutated form of the protein. This is the first direct biochemical evidence supporting the putative ATPase activity of a member of the TBP1-like subfamily. Furthermore, the experimental results demonstrate a regulatory function for the amino-terminal region of the molecule. The amino-terminal truncated form of Yhs4p lacking two clusters of positively charged amino acids exhibits a greater ATPase activity. The ATPase activity of both the truncated and complete forms of Yhs4p is stimulated by polyanions. Polylysine partially inhibits the ATPase activity of the amino-terminal truncated form having no observable effect on the complete protein. N-Ethylmaleimide inhibits the ATPase activity of both forms of Yhs4p. We propose that Yhs4p ATPase may play an essential role in the regulatory function of the proteolytic activity of the yeast 26 S protease.


INTRODUCTION

The 20 S proteasome is a widely occurring intracellular, multicatalytic protease of 700 kDa (for review, see Ref. 1) composed of 24-28 subunits of 20-30 kDa encoded by the ``proteasome gene family'' (for review, see Ref. 2). Several hetero-oligomeric protein complexes of about 10 subunits ranging from 40 to 110 kDa (700 kDa) have been described as activators of the 20 S proteasome. Among the activator complexes are CF-1 (3) and ``ball'' (4) from rabbit reticulocyte, the µ particle (5) from Drosophila, and PA-700 (6) from bovine blood cells. These complexes are called ``19 S cap complex'' (7) and assemble with the 20 S proteasome to generate the 26 S protease. The similarities and differences among these complexes are still under investigation (4, 8) . However, some functional aspects of their interaction with the 20 S proteasome have been established (for a review see Ref. 7). (i) The assembly between the 20 S proteasome and each of the activator complexes is coupled to ATP hydrolysis, and only CTP substitutes for ATP (3) . (ii) The 26 S protease exhibits an ATP-dependent proteolytic activity on ubiquitin-conjugated proteins, which is not observed in the 20 S proteasome. All four nucleotides stimulate proteolysis of ubiquitin conjugates by the assembled 26 S protease (9) , and are hydrolyzed by it (10) . Therefore, the subunits of the activator complexes confer ATP dependence and ubiquitin recognition to the proteolytic core provided by the 20 S proteasome subunits (9, 11) .

The discovery of the human immunodeficiency virus Tat-binding protein TBP1 (12) unveiled a new subfamily of putative ATPases of approximately 45-50 kDa containing a single ATP binding site called ``P-loop'' domain (13) . This ``TBP1-like'' subfamily initially included the transcription activator Tat-binding protein TBP7 (14) , a modulator of human immunodeficiency Tat-mediated transactivation MSS1 (15) and the yeast protein SUG1 (16) . More recently, this subfamily has been expanded to include components of the regulatory subunits of the 26 S protease. Subunit four (S4) of the 26 S protease is a putative ATPase that shares identity with TBP1, TBP7, SUG1, and MSS1 (17) . Partial sequence analysis proved that MSS1 is subunit 7 (18) and TBP7 is subunit 6 (19) of the 26 S protease. Therefore, the putative ATPases among the larger subunits of the 26 S protease may have a dual role in the regulation of transcription and proteolysis. Alternatively, they may only function as 26 S protease subunits, and the regulation of transcription may be a consequence of the selective proteolysis catalyzed by the complex (19) .

Three other members of this TBP1-like subfamily MTS2 from Schizosaccharomyces pombe (20) and CIM5 and CIM3 from Saccharomyces cerevisiae (21) are involved in the regulation of the yeast proteasome in vivo. MTS2 is 75% identical to S4 of the human 26 S protease. CIM3 is identical to SUG1, and CIM5 is 33% identical to S4. The yeast 20 S proteasome, known as yscE proteinase, has been extensively characterized (for a review, see Ref. 22). The yeast 26 S protease has not yet been purified. Therefore, no information is available on its subunit composition.

We have cloned and expressed an S. cerevisiae essential gene encoding the 49-kDa protein Yhs4p that is 72% identical to S4 of the human 26 S protease and is a member of the TBP1-like subfamily of putative ATPases. We present the strategy of expression and purification of Yhs4p that allowed a characterization of its ATPase activity. Yhs4p is the first member of the TBP1-like subfamily on which an ATPase activity is demonstrated in vitro. The amino terminus of Yhs4p is involved in negative modulation of the ATPase activity.


EXPERIMENTAL PROCEDURES

Yeast Strains and Analysis of Mutants, Saccharomyces cerevisiae

Diploid strain W303 ( leu2, his3, ade2, trp1, ura3) was used for the disruption of the gene YHS4. Wild-type and mutants were grown in 1% yeast extract, 2% peptone, 2% glucose medium (23) . Cells were transformed using the lithium acetate method (24) and then grown on minimal media supplemented with the appropriate nutritional requirements (25) . The YHS4 gene, deleted in the 5` end of the open reading frame, was cloned by serendipity while screening a yeast cDNA gt11 library. The cDNA was used as a probe for screening a yeast genomic library in YEp13 kindly donated by Dr. Michael Douglas. A 2.3 kb() genomic fragment containing a complete open reading frame was isolated. To interrupt the YHS4 gene, a 1.3-kb EcoRI fragment of the gt11 clone was subcloned into the EcoRI site of pBluescript. The resulting plasmid was linearized with HpaI, which cuts after 0.45 kb from the 5` end of the insert, and LEU2 gene was cloned into the site. The construct was excised by HindIII leaving 0.43 and 0.75 kb at the 5` and 3` ends, respectively. The above HindIII fragment was used to interrupt the YHS4 gene in diploid W303 cells as described previously (23) . Colonies that grew on minimal medium lacking leucine were analyzed by polymerase chain reaction, sporulated on acetate medium, and subjected to tetrad dissection.

Mutation of the P-loop Motif and Subcloning

Two primers were synthesized to mutate the lysine residue (Lys) in the P-loop motif to either histidine or glutamine. The sense primer was 5`-CCC GGT ACA GGT CAT ACA TTG CTA GCA AAG-3`, and the antisense primer was 5`-TAG CAA TGT CTG ACC TGT ACC GGG TG-3`. The mutation was generated as described previously (26) by polymerase chain reaction overlap extension using the 2.3-kb genomic fragment as template and the 5` primer ATA GAA AAT GGA TCC GGA CAA GGT GTA TCA TCT G and the 3` primer GCA TTA CTT GGA TCC GTA AAT CTA TTA TAG AAA AAT GTA. Each primer carries a BamHI site ( underlined) inframe with the corresponding BamHI site of the pGEX-2T plasmid. The mutant used in the expression and ATPase activity assays presented in this report is AAG to CAG in codon 229 of the open reading frame, corresponding to K229Q in the protein sequence. The reading frame of the wild-type YHS4 gene was adapted by polymerase chain reaction amplification with the same 5` and 3` primers. Wild-type and mutated YHS4 gene were subcloned into the BamHI site of pGEX-2T following standard procedures (27) . A 1.3-kb EcoRI fragment of the gt11 clone was subcloned inframe into the EcoRI site of the pGEX-2T. Escherichia coli cells BL21(DE3)pLysS, transformed with the wild-type, mutated, and truncated YHS4 genes were grown at 37 °C in 100 ml of LB/ampicillin medium in a 500-ml flask with shaking at 300 rpm. At a cell density of 0.4 OD units at 600 nm, isopropyl-1-thio-- D-galactopyranoside (1 m M final concentration) was added and growth was continued for 2 h at 30 °C. Cells were sedimented at 3,000 g at 4 °C for 15 min, resuspended in 5 ml of 137 m M NaCl, 2.7 m M KCl, 4.3 m M NaHPO7HO, 1.4 m M KHPO, pH 7.3 (PBS), and disrupted by three passages through a French press. The homogenates were then centrifuged at 30,000 g for 20 min at 4 °C, pellets were resuspended in 5 ml of PBS, and supernatants were absorbed for 5 min at 4 °C with 0.5 ml of GSH-agarose (50% (v/v) slurry in PBS). GSH-agarose beads were collected by microcentrifugation for 5 s and washed 3 times with 1 ml of PBS. Washed beads were resuspended for 5 min at 4 °C with 0.5 ml of 50 m M Tris-HCl, pH 7.3, containing 5 m M GSH. Beads were spun down as above, and supernatants containing the fusion proteins were concentrated 5-fold by ultrafiltration in a Centricon-10 (Amicon) cartridge. ATPase activity was assayed immediately after this step in a basic medium (50 µl) containing 50 m M Tris-HCl, pH 7.6, 5 m M dithiothreitol, 5 m M MgCl, and 50 µ M ATP/[-P]ATP (1,000-2,000 cpm/pmol) at 37 °C. Nucleoside triphosphate, EDTA, polycations (polylysine and polyarginine), polyanions (M13 single-stranded DNA, yeast total RNA, poly-Glu-Ala-Tyr (6:3:1), and pBluescript double-stranded DNA), and N-ethylmaleimide were added to this basic medium where indicated. The reaction was initiated by the addition of 20 ng of fusion protein and ended by the addition of 500 µl of 10% trichloroacetic acid. POreleased from [-P]ATP was determined as described (28) . Protein was determined by the method of Lowry et al. (29) . SDS-PAGE gels (30) were stained with Coomassie Blue R-250.


RESULTS

Cloning and Characterization of Yhs4p cDNA

Initial cloning efforts resulted in the isolation of a yeast cDNA containing a 1.3-kb EcoRI fragment encoding a amino-terminal truncated protein of 387 residues (43 kDa) with homology to the TBP1-like subfamily. The cDNA truncated in the 5` region was used to probe a yeast genomic library resulting in the isolation of a 2.33-kb fragment from a yeast genomic library that encodes a protein of 437 residues (49 kDa). The 1.3-kb cDNA fragment lacks the 5`-untranslated region and 150 base pairs of the open reading frame encoding the first 50 amino acid residues from the amino-terminal. The deduced protein sequence designated Yhs4p is shown in Fig. 1. Underlined are the 50 amino acid residues that are absent in the amino-terminal truncated molecule. Two clusters of positively charged amino acids, KKKKKand RKKRK, are present at the amino terminus. The sequence also contains a P-loop domain GAPGTGK, found in ATP/GTP binding proteins (13) , two additional domains, DEIDAIGand MATNKIE, characteristic of GTP binding proteins (31) , and the LIRPGRIDRdomain homologous to the domain VI of a subfamily of ATP-dependent DNA/RNA helicases (32, 33) . These four domains, characteristic of ATP/GTP-binding proteins and ATP-dependent DNA/RNA helicases, and the cysteine residue (Cys) near the carboxyl terminus of the sequence are conserved in all members of the TBP1-like subfamily. Fig. 2 depicts the sequence of these sites, designated with the roman numerals from I to IV, and their position in the primary structure of the members of this subfamily. Interestingly these sites cluster at approximately 250 amino acid residues from the amino termini and span 100 amino acid residues. Yhs4p sequence is 73% identical to MTS2 and 72% identical to S4 of 26 S protease in the whole span of their primary sequence comprising around 450 amino acid residues. The identity is lower (40-50%) in the first 25 amino acid residues at the amino-terminal region. These structural features suggest that MTS2 and Yhs4p are yeast homolog to human S4. The identity of Yhs4p with other members of the TBP1-like family is lower (50-60%), and it is restricted to the 200-300 amino acid residues containing domains I-IV, characteristic of several NTP-binding proteins. The amino-terminal region of these proteins has only a 6-7% identity with Yhs4p. The consensus sequences of domains I-VI in the TBP1-like subfamily and the proposed function of similar sites in members of other NTP binding families are summarized in the lower part of Fig. 2.


Figure 1: Predicted amino acid sequence encoded by YHS4. The protein sequence of Yhs4p is presented using the single letter amino acid code. Underlined in italics are the 50 amino acid residues that are missing in the amino-terminal truncated molecule. The cysteine residues are underlined in boldface letters. Shaded are the two clusters of positively charged amino acid residues. Double underlined in boldface letters are the domains present in ATP/GTP-binding proteins and in ATP-dependent DNA/RNA helicases . The mutation K229Q is shown by the arrow.




Figure 2: Primary sequence identity and conserved domains in members of the TBP1-like subfamily of ATPases. The sequences of the four domains ( I-IV) conserved in members of the TBP1-like subfamily of ATPases are shown in the upper portion of the diagram. In the central portion the bars represent the primary structure of the proteins. Empty boxes indicate the localization of the four domains in the primary structure. Circle near the carboxyl termini indicates the position of the conserved cysteine residue. Numbers on top of the bars indicate percent of sequence identity of the region with the corresponding region in Yhs4p. The lower part shows the consensus sequence for domains I-IV in this subfamily, and the interactions proposed for similar domains in other NTP-binding molecules. In brackets are all possible amino acids found in that particular position in all members of the subfamily.



Gene Disruption and Tetrad Analysis

YHS4 gene was disrupted with the S. cerevisiae LEU2 gene. Polymerase chain reaction analysis of colonies that were prototrophic for leucine was used to confirm the presence of the yhs4::LEU2 disruption allele (data not shown). Eight Leuisolates were sporulated, and tetrads were dissected. In every case but two, the four spores segregated in a ratio of two viable to two nonviable (Fig. 3). The viable spores were auxotrophic for leucine and did not carry the marker for the disruption.

Expression of Yhs4p

Fragments encoding the complete Yhs4p, the amino-terminal truncated protein, and a protein with a K229Q mutation in the P-loop domain were subcloned in the vector pGEX-2T and expressed to render fusion proteins with glutathione S-transferase. Wild-type and P-loop-mutated Yhs4p proteins are well expressed in E. coli cells (Fig. 4, Whole cell after induction). However, most of the expressed proteins remain in the pellet fraction obtained after disruption and subsequent sedimentation (Fig. 4, Pellet). Purification of the fusion proteins that remain soluble after sedimentation (Fig. 4, Supernatant) was undertaken using glutathione-agarose affinity chromatography. The affinity-purified supernatants (Fig. 3, GSH- agarose purified supernatant) showed no protein in the supernatant from control cells ( lane 1) and a single 77 kDa band in supernatants from cells expressing the wild-type ( lane 2) and P-loop-mutated ( lane 3) fusion proteins. 77 kDa is the expected molecular mass for a fusion product between Yhs4p of 49 kDa and glutathione S-transferase of 28 kDa. Amino-terminal truncated protein was also expressed and purified as above (data not shown). As expected, amino-terminal truncated Yhs4p travels slightly faster than the wild-type and mutated proteins (Fig. 5). The yield of affinity-purified fusion proteins varied between 0.5 and 3 µg/300 ml liquid culture.


Figure 4: Expression of wild-type and K229Q mutated Yhs4p proteins in E. coli. Complete and P-loop-mutated YHS4 DNAs were subcloned in pGEX-2T plasmid inframe with the glutathione S-transferase gene and expressed as described under ``Experimental Procedures.'' E. coli cells that were not transformed, called control cells, ( lane 1) cells that were transformed with the complete ( lane 2) and with the mutated ( lane 3) Yhs4p gene and fractions of these cells were resolved in 15% SDS-PAGE gel. Whole cells (15 µg of protein) were analyzed before and after induction with isopropyl-1-thio-- D-galactopyranoside. Equal volumes of Pellet fraction containing 15 µg of protein and Supernatant fraction containing 5 µg of protein and 30 µl of the GSH- agarose purified supernatants (10 ng protein for lanes 2 and 3) from cells after induction with isopropyl-1-thio-- D-galactopyranoside were loaded in the gel. The arrow on the right indicates the positions of the expressed fusion proteins.




Figure 3: Tetrad analysis of yhs4::LEU2 disruption allele. The disruption allele was constructed and spores were analyzed as described under ``Experimental Procedures.'' All viable spores were phenotypically Leuconfirming that the yhs4::LEU2 disruption allele is lethal.



Kinetic Parameters and Effectors of Yhs4p ATPase Activity

The affinity-purified Yhs4p is a Mg-ATPase with a Kfor ATP of 5 µ M and a Vof 7 pmol of ATP/min/µg of protein (Fig. 6). The amino-terminal truncated protein exhibited the same Kvalue but a Vof 20 pmol of ATP/min/µg of protein. Mutational analysis of the P-loop domain G XXXXGKT (where X can be any amino acid) of several ATP/GTP-binding proteins revealed that the lysine residue is essential for optimal activity. It was recently reported that the K155Q mutation in the P-loop domain of the subunit of E. coli F-ATPase impairs its catalytic activities (34) . The mutation K229Q in P-loop of Yhs4p inhibited 73% of the wild-type ATPase activity, suggesting that the lysine residue is required for optimal function of the ATPase. No ATPase activity was observed in GSH-agarose purified supernatants from control cells. The ATPase activity of complete and N-truncated Yhs4p was 95% inhibited by EDTA and by 50 µ M N-ethylmaleimide (not shown).

The observation that the amino-terminal truncated Yhs4p is a more active ATPase than the complete molecule (Fig. 6) suggests that the amino-terminal region of Yhs4p has a negative regulatory function. A distinctive feature of the amino-terminal region is the presence of two clusters of positively charged amino acid residues (See Fig. 1). To learn if these clusters were involved in the observed negative modulation, we studied the effect of several polyanions and two polycations on the ATPase activity of complete and amino-terminal truncated Yhs4p. Both Mg-ATPase activities were stimulated by polyanions. Single-stranded DNA, double-stranded DNA, RNA, and poly(Glu-Ala-Tyr) produced a 4-6-fold stimulation of the ATPase activity in the complete protein, and a 1.5-2-fold stimulation in the amino-terminal truncated protein (Fig. 7). The polyanions produced maximal effect at a concentration of 10 µg/ml under the experimental conditions used in the ATPase activity assay. The polycation polylysine had only a small inhibitory effect on the ATPase activity of the complete Yhs4p and inhibited 48% the activity of the amino-terminal truncated molecule (Fig. 7). Polyarginine elicited a similar pattern of inhibition on both forms of Yhs4p (not shown). These data suggest that the positively charged clusters at the amino terminus of the protein are involved in a negative modulation of the ATPase activity. The absence of these clusters in the amino-terminal truncated protein or their neutralization by polyanions prevents the negative modulation. Polyanions also stimulate the ATPase activity of the amino-terminal truncated protein, suggesting a regulatory functionality for other domains of the molecule.


Figure 6: Kinetic parameters of complete and amino-terminal truncated Yhs4p ATPase. ATPase activity was measured for 10 min as a function of four ATP concentrations in the range from 5 to 100 µ M as described under ``Experimental Procedures.'' Vand K values were calculated by Lineweaver-Burk plot. Each point represents the mean of three determinations assayed in duplicate. Number in parentheses is percent of inhibition. K229Q point mutation in Yhs4p and was obtained as described under ``Experimental Procedures.'' n.d. indicates ``not determined.''




Figure 7: Effect of several polyanions and polylysine on the ATPase activity of complete and amino-terminal truncated Yhs4p. ATPase activity was assayed as described under ``Experimental Procedures.'' Each point represents the average of three determinations. Polyanions and polylysine were added to a final concentration of 10 µg/ml. The ATPase activity was assayed without additions ( closed circles) and after the addition of polylysine ( circles) or single-stranded DNA ( inverted triangles), double-stranded DNA ( triangles), RNA ( diamonds), or poly(Glu-Ala-Tyr) ( squares).



The nucleotide specificity of the Yhs4p, examined by testing the effect of unlabeled nucleotides on the hydrolysis of [-P]ATP, showed that GTP, UTP, and CTP compete with [-P]ATP, resulting in a significant inhibition of the ATPase activity (Fig. 8). Therefore, Yhs4p interacts with all four nucleotides as predicted by the domains found in its sequence (Fig. 1).


Figure 8: Nucleoside triphosphate specificity of complete and amino-terminal truncated Yhs4p ATPase. ATPase activity was assayed for 15 min as described under ``Experimental Procedures.'' Cold nucleoside triphosphate was added to the ATPase assay medium at a final concentration of 500 µ M. Each bar represents the average of three determinations.




DISCUSSION

We have cloned and expressed a yeast cDNA encoding a new member of the TBP1-like subfamily of putative ATPases, designated Yhs4p. Disruption of YHS4 gene (Fig. 3) shows that the production of Yhs4p is essential for cell viability. In previous studies, using strategies of gene disruption/complementation in yeast (12, 14, 15) and co-transfection/over expression in human cells (16, 20, 21) , some members of this subfamily have been identified as transcriptional regulators (12, 14, 15, 16) , while others were proposed to be essential for the function of the 26 S protease involved in ATP/ubiquitin-dependent proteolytic pathway (20, 21) . Initially the TBP1-like subfamily of putative ATPases was composed of proteins identified as transcriptional regulators (TBP1, TBP7, MSS1, and SUG1). Recent findings contributed to expand our understanding on the biological function of members of this subfamily. (i) Subunit 4 of 26 S protease is a member of this subfamily (17) , and (ii) MSS1 and TBP7 are identical to subunits of the 26 S protease (18, 19) . These findings suggested that alterations in the activity of the 26 S protease may alter the pattern of transcriptional regulation (19) . It is also possible that some members of this subfamily may play a dual role in transcriptional regulation and in proteolysis catalyzed by the 26 S protease. It is essential to characterize their biochemical activities to gain further insight into their biological role. No member of this subfamily has been biochemically characterized yet. Their primary protein structure contain four domains that are characteristically found in ATP/GTP binding proteins (13, 31) and in DNA/RNA helicases (32, 33) . These domains are highly conserved and cluster at a constant distance of approximately 250 residues from the amino-terminal end. Yhs4p, MTS2, and S4 share the highest identity (73%) in the whole span of their primary sequence with a lower identity (50%) in the first 40 amino acid residues of their amino termini. The divergence observed between Yhs4p and other members of this subfamily is greatest at their amino-terminal region. For instance Yhs4p and CIM5, a S. cerevisiae protein proposed to be involved in 26 S protease activity in vivo (21) , share only 4% identity in the first 180 residues from their amino-terminal region (Fig. 2, central diagram). A distinct feature of the Yhs4p amino-terminal is the presence of two clusters of positively charged amino acid residues (Fig. 1). The amino-terminal truncated Yhs4p has a V3 times higher than the complete molecule (Fig. 6). To prove the hypothesis that the clusters of positive amino acid residues confer a negative modulation on the ATPase activity of Yhs4p we studied the effect of several polyanions and two polycations on the activity of amino-terminal truncated and complete Yhs4p. The stimulation by polyanions was overall 3-fold higher in the complete molecule than in the amino-terminal truncated molecule, while polylysine inhibited only the activity of amino-terminal truncated Yhs4p by 48% (Fig. 7). These results support the idea that the positive residues in the amino-terminal region result in it functioning as an internal modulator of Yhs4p ATPase activity. The activation of Yhs4p by polyanions appears to be unspecific, suggesting a putative mechanism for in vivo modulation of the ATPase through the interaction with negatively charged epitopes of neighboring proteins. Yhs4p as part of a macromolecular complex may become activated upon interaction with the proper subunit during the assembly of the 26 S protease complex. Regarding this possibility, it is interesting to note that some subunits in the 20 S proteasome and in the activator PA-700 contain a cluster of three or more acidic residues at their carboxyl termini as for instance in the 20 S proteasome subunits HC8 (EEDESDDD) and HC9 (EEEE) in human (35) ; Y13 (EDEEADED) (36) , YC1 (DDEEDEDD) (37) , and PRE2 (DDD) (38) in yeast; XC8 (EEEDDSDDD) and XC9-1 (EEEE) (39) in Xenopus; and p58 (EDDDD) (8) in the PA700 activator.

Yhs4p contains four cysteine residues (Fig. 1). The cysteine residue closest to the carboxyl terminus is conserved in all members of TBP1-like subfamily (Fig. 2, central diagram). The ATPase activity of the regulatory subunit of the 26 S protease PA-700 is inhibited by low concentrations of the sulfydryl reagent N-ethylmaleimide (8) . Similarly, ATPase activities of complete and amino-terminal truncated Yhs4p were 90% inhibited by 50 µ M N-ethylmaleimide (not shown), suggesting that one or more of its four cysteine residues are essential for the activity.

Yhs4p has substrate specificity (Fig. 8) and kinetic parameters (Fig. 5) resembling those described for the ATPase activity of the 26 S protease (10) . The high identity of Yhs4p with human S4 of the 26 S protease (17) and S. pombe MTS2 involved in regulation of the yeast proteasome in vivo (20) suggests that this protein may have a role in the regulation of the proteasome in the yeast S. cerevisiae. The similarity between the ATPase activity of Yhs4p reported here and 26 S protease suggests that the role may be as a catalytic subunit for the hydrolysis of ATP coupled to the hydrolysis of ubiquitin conjugates by the 26 S protease. Other putative activities that may be attributed to Yhs4p based on observed sequence homology are the function of a protein kinase, DNA/RNA helicase, and GTPase. These putative activities may require oligomerization of Yhs4p or interaction with the appropriate regulatory protein(s).

While this work was in progress, the S. cerevisiae gene designated YTA5 was cloned and sequenced (40) and was shown to encode a protein identical to Yhs4p. This work, however, shows a direct biochemical activity for the protein product of the gene as an ATPase and proves that the gene is essential. The production of Yhs4p as a fusion protein enabled its purification and the partial characterization of its ATPase activity. To reiterate, no other member of the TBP1-like subfamily of putative ATPases has been characterized yet in terms of its ATPase activity or any other putative activity predicted by its primary sequence.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked `` advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank/EMBL Data Bank with accession number(s) L17040.

§
To whom correspondence should be addressed: Tel.: 617-638-4394; Fax: 617-638-5047; E-mail: hlucero@acs.bu.edu.

Present address: Athena Neurosciences, Inc., Technical Operations Group, 800F Gateway Bvd., South San Francisco, CA 94080.

The abbreviations used are: kb, kilobases(s); PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

H. A. L. thanks Dr. Benjamin Kaminer for continued support.


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