PREZ, Highly Homologous to the Human Major Histocompatibility Complex-linked RINGlO Gene, Codes for a Yeast Proteasome Subunit Necessary for Chymotryptic Activity and Degradation of Ubiquitinated Proteins*

We have cloned the yeast PREZ gene by complemen- tation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a 8-type member of the proteasomal gene family, is essential for life and codes for a 287-amino acid proteasomal subunit with a predicted mo- lecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to en- hanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human RinglO protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class I1 region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to RinglO implies the hypothesis that RinglO is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might u r d ) by the two-step gene replacement method (22). The resulting mutant strains were used for generation of isogenic double mutants by mating of a prel-1 strain with a pre2-1 or pre2-2 strain, respectively. The resulting heterozygous diploids were sporu-lated, and co-segregation of the two uncoupled mutations among the haploid progeny was identified within tetrads comprising two wild-type and two mutant spores with respect to chymotryptic activity. Further proof for mutant spore clones indeed carrying the pel-1 allele together with the respective pre2 mutation was obtained by backcrossing them with wild type and following the independent segregation of the pel-1 and pre2 alleles in the spore progeny of the resulting diploid.


PREZ, Highly Homologous to the Human Major Histocompatibility Complex-linked RINGlO Gene, Codes for a Yeast Proteasome Subunit Necessary for Chymotryptic Activity and Degradation of
We have cloned the yeast PREZ gene by complementation of pre2 mutants, which are defective in the chymotrypsin-like activity of the 20 S proteasome (multicatalytic-multifunctional proteinase complex). The PRE2 gene, a 8-type member of the proteasomal gene family, is essential for life and codes for a 287amino acid proteasomal subunit with a predicted molecular mass of 31.6 kDa. Missense mutations in two pre2 mutant alleles were identified. They led to enhanced sensitivity of yeast cells against stress. At the same time, pre2 mutants accumulated ubiquitinated proteins. The Pre2 protein shows striking homology to the human RinglO protein (60% identity excluding the 70 amino-terminal residues), which is encoded in the major histocompatibility complex class I1 region. It represents a component of the low molecular mass polypeptide complex, previously shown to be a special type of the 20 S proteasome. The low molecular mass polypeptide complex is assumed to be involved in antigen presentation, generating peptides from cytosolic protein antigens, which are subsequently presented to cytotoxic T-lymphocytes on the cell surface. The high homology of Pre2 to RinglO implies the hypothesis that RinglO is a subunit of the low molecular mass polypeptide complex central in its chymotryptic activity. One might further suggest that replacement of constitutive proteasomal components by functionally related major histocompatibility complex-linked low molecular mass polypeptides, as is RinglO, adapts mammalian proteasomes for functions in the immune response.
Proteasomes are ubiquitous non-lysosomal proteinase complexes of high molecular mass (about 650 kDa) with a characteristic, hollow cylindrical structure. These 20 S particles, highly conserved from yeast to man (1, 2), are composed of a variable set of nonidentical but structurally related subunits of sizes between 21 and 38 kDa and exhibit multiple proteolytic activities . In vitro analysis of the mammalian 20 S proteasome shows its ability to assemble with additional proteins to a 26 S proteinase complex in an ATP-dependent fashion (6-8), which is able to degrade ubi- quitinated protein substrates (reviewed in Refs. 5 and 9). Recent results from mammals indicate a participation of proteasomes in the pathway of antigen presentation by major histocompatibility complex (MHC)' class I molecules (reviewed in 9-11). Here, proteasomes are thought to generate peptide fragments from foreign proteins in the cytosol, which are delivered, after association with MHC class I glycoproteins and p-microglobulin in the endoplasmic reticulum, to the cell surface and scanned by cytotoxic T-lymphocytes. In both the human and the murine MHC class I1 regions, two y-interferon-inducible genes were identified that encode components of the low molecular mass polypeptide particle, a multiprotein complex shown to be immunologically closely related to the proteasome (12, 13). The low molecular mass polypeptides encoded by the human MHC genes RINGlO (14) and RING12 (15) and the murine RING12 equivalent LMP-2 (16) indeed revealed significant homology to known proteasome components.
Experiments in yeast are gradually uncovering general functions of the enzyme complex in cellular life (17,18). Here, the proteasome is found to function in degradation of shortlived, stress-induced, and abnormal proteins. Among the proteins degraded are those of the ubiquitin-mediated proteolytic pathway (17,18).
These studies were initiated through analysis of mutants defective in proteolytic functions of proteinase yscE, the yeast proteasome. Three isolated mutants define two complementation groups, PREl and PRE2, and show a defect exclusively in the chymotrypsin-like activity of the enzyme particle (17,19). The trypsin-like and the peptidylglutamyl peptide hydrolyzing activities remain unaffected. PREl was cloned and sequenced and was shown to encode an integral proteasomal subunit essential for cell viability (17,19).
Here, we report on the essential yeast proteasomal gene PRE2. The PRE2 gene product shows striking structural similarities to the RinglO protein, which imply related functions of both subunits within yeast and human proteasomes, i.e. a participation in their chymotryptic activity. As in prel mutants, the defect in the chymotrypsin-like activity of pre2 mutant proteasomes leads to hypersensitivity of cells to the amino acid analogue canavanine and to stress-induced accumulation of ubiquitinated proteins.
For immunological detection of ubiquitin-protein conjugates, strains were grown in liquid YPD medium to early stationary phase at 30 "C and subsequently incubated at 37 "C for 3 h. After harvesting and washing, cells were resuspended in distilled water to yield a 50% (v/v) suspension, and washed glass beads, equivalent to the respective cell volume, were added. Cells were heated for 10 min at 95 "C in Eppendorf tubes and subsequently vortex-mixed 6 times for 30 s with intermittent heating for 30 s. SDS-EDTA solution (4.5% SDS, 2.25 mM EDTA), equivalent to the cell volume, was added, and the samples were heated for an additional 10 min at 95 "C. Samples were centrifuged and the supernatants were used for immunoblotting. For SDSpolyacrylamide gel electrophoresis (10% gels), 50 pg of protein were applied onto each lane. Immunoblotting using ubiquitin-protein conjugate antibody was done as described in Ref. 17. Quantification of the dye produced by the peroxidase linked to the rabbit-immunoglobulin antibody was done using the JAVA 1.3 video analysis system (Jandel, Corte Madera, CA).
Gene Cloning and Analysis-Standard molecular biological (20) and yeast genetic/microbiological techniques (20-22) were used. The PRE2 gene was cloned by complementation of the defective chymotrypsin-like activity of a pre2-2 mutant strain using a yeast genomic library in the CEN4-ARS-URA3 shuttle vector YCp50 (23). One recombinant YCp50 plasmid restored the proteolytic activities of both the pre2-1 and the pre2-2 mutant. The complementing portion of its 12-kbp genomic insert was limited to 1.05 kbp after cloning of subfragments into the CENl4-ARS-URA3 shuttle vector pDP83.' For sequence analysis of the PREP locus (see Fig. IA) by the dideoxy chain termination method, pDP83-based plasmids with overlapping inserts from the PRE2 region served as template.
Construction of plasmids carrying a deletion of the PRE2 coding region started from a pDP83-based plasmid harboring a 0.9-kbp fragment from the 5"flanking region of PRE2, which had been generated by exonuclease digestions (left bar in Fig. l.4). A unique polylinker BamHI site was used to introduce a 1.3-kbp BglII/BamHI fragment from the 3"flanking region of PRE2 (right bar in Fig. lA).
In the resulting plasmid pDP83AE2 the 5'-and 3'-non-coding regions are connected, resulting in a 1.0-kbp deletion (see Fig. 1B) of the entire PRE2 coding region. This construct served two purposes. 1) Cloning of the pre2 mutant alleles by "gap repair" (22) was done as follows. Plasmid pDP83AE2 was linearized with BamHI at the 5'-3'junction and introduced into pre2-1 and pre2-2 mutant strains. Plasmids from Ura+ transformants were checked for the correct gap repair event by restriction analysis. The complete nucleotide sequences of the cloned pre2-2 and pre2-2 alleles were determined and a one-base exchange was detected in each allele (see Fig. 1B). Chromosomal introduction by the two-step gene replacement method (22) of each mutant allele into a wild-type strain actually led to loss of wild-type chymotrypsin-like activity. 2) Chromosomal deletion of the PRE2 gene by one-step gene disruption (22) was done as follows. Plasmid pDP83AE2 was cut with BamHI, and a 1.7-kbp BamHI fragment carrying the HIS3 gene was inserted. The diploid strain YS18/18 (MATaIMATa his3-11,15/his3-11,15 leu2-3,112/leu2-3,112 ura3A5/ ura3A5 canRlcanR) was transformed with the pre2AtHI.93 deletion allele excised from the vector. His+ transformants were selected, and replacement of one chromosomal PRE2 copy by the deletion allele was verified by Southern blot analysis. Several of these PRE2/ pre2A:HIS3 heterozygous diploids were sporulated, and asci were subjected to tetrad dissection. Of each tetrad, viability of only two His+ spores was observed.
Construction of Isogenic pre Mutant Strains-Isogenic single-mu-* D. Pridmore, unpublished data. tant strains were generated by introduction of the clonedprel or pre2 alleles into wild-type strains WCG4a or WCG4a (MATa or MATa his3-11,15 leu2-3,112 u r d ) by the two-step gene replacement method (22). The resulting mutant strains were used for generation of isogenic double mutants by mating of a prel-1 strain with a pre2-1 or pre2-2 strain, respectively. The resulting heterozygous diploids were sporulated, and co-segregation of the two uncoupled mutations among the haploid progeny was identified within tetrads comprising two wildtype and two mutant spores with respect to chymotryptic activity. Further proof for mutant spore clones indeed carrying the p e l -1 allele together with the respective pre2 mutation was obtained by backcrossing them with wild type and following the independent segregation of the pel-1 and pre2 alleles in the spore progeny of the resulting diploid.

RESULTS AND DISCUSSION
The PRE2 gene was cloned from a yeast genomic library (23) by complementation of the defective chymotrypsin-like activity in the two pre2 mutants isolated. Sequence analysis of the complementing DNA region revealed an open reading frame that encodes a 287-amino acid protein with a predicted relative molecular mass of 31.6 kDa (Fig. 1).
Data bank search found the nucleotide sequences situated upstream of the PRE2 gene to match almost perfectly those of the RPL16B gene from Saccharomyces carkbergensis (24). RPL16A and RPLI6B are duplicated genes encoding ribosomal proteins and are found in S. cereukiae as well (25). The distance between the translation initiation codons of the divergently transcribed PRES and RPLl6B genes amounts to 600 nucleotides (Fig. 1). Whether these two genes share common regulatory sites is unknown.
Standard genetic procedures (22) revealed the two pre2 mutant genes to be alleles of the isolated PREB gene. The cloned PREB DNA was able to direct integration of a selectable marker gene to the wild-type locus corresponding to the pre2 mutant alleles (not shown). Furthermore, thepre2-1 and pre2-2 mutant alleles were cloned (see "Experimental Procedures") and shown to confer a defective proteasomal chymotryptic activity to wild-type cells. Both mutant genes contain a missense mutation each (Fig. 1B).
PRE2 is essential for cell proliferation. Sporulation of heterozygous diploids carrying a null allele of PRE2 (Fig. 1A) produces asci of which only the two spores harboring a wildtype PRE2 gene grow up to visible colonies. Although the pre2 null mutant spores are capable of germinating, they arrest after two to three cycles of cell division. An identical cell division arrest is seen with spores deleted in the PREl gene (not shown). Seven of eight other hitherto cloned yeast proteasomal genes (26-30)3 are essential for life as well. Our results with PRES confirm the importance of a complete proteasome particle for cell survival.
Biochemical analysis of pre mutants with respect to their defect in the chymotrypsin-like activity revealed a reduction in pre2-2 mutants of 95%, which is similar to the value found in prel-1 mutants (Table I; Ref. 17). Pre2-I mutants exhibit a 3-fold higher residual activity compared with pre2-2 and prel-1 mutants ( Table I). Introduction of thepre2-1 mutation into a prel-1 mutant strain leads to a slight additional decrease in the chymotrypsin-like activity compared with prel-1 single mutants. Combination of the pre2-2 mutation with the prel-1 mutation in a double mutant finally yields a residual activity of only 4% ( Table I).
As shown previously (17), the peptidylglutamyl peptidesplitting activity remains in the prel and pre2 mutants. Interestingly, this activity is even increased in these mutants, most strongly in those carrying the prel-1 allele (Table I)

CCl TCT TTC M C M C CTT A l l CCC T b M T A l W I C E I T 0 3 * U T A T~A T A l -T C T A T A n M T U T A
m C l y S.r ph AM Asn Val 11. Cly ,183 hase without amino acids, 2' ;' glucose, 2"; agar, and sllpplrments R S required (21)) rontaining no ( r ) or ( 1 . 4 pg/ml ~d ) rnnavnnlne s111fate.

T T T A T C C T T C T A C T T C W T A T C C T M T A C T A T T A T C~~T~l T T T T C C T -T M X~~~ l W T A C C C T c G T T C A T A T C C T C C C T C A C C T T C C C C C T T T T~T U T U C C T T~f f i T~-~T C T -
Plates were incuhatcd at 30 "C (n. r. d ) or :18 "(: f h l fnr t 1 p t o 2 ti:lys. Because of the high activity of other proteases against the substrate of the trypsin-like proteasomal activity (C.'hz-AIa-Arg-Arg-4-methoxy-ij-naphthylamide) in cnlde extracts, effects of the prrI and prP2 mutations on this activity could not be determined.
Cells defective in chymotrypsin-like activity caused by t h e prel-I mutation have been reported to exhibit increased sensitivity to stresses, as are elevated temperature (38 " C ) a n d application of t h e amino acid analop.le canavanine (17). Even growth at 30 "C on YPD and, more pronounced, on "\.' medium is affected hv the p r r l -I mutation (Fig. 2 , n a n d r ) .

Yeast PRE2 and Human RING10 Are Homologous Proteasomal Gcncs
The stress conditions mentioned were shown to lead to accumulation of uhiquitinated proteins in p e l -l mutant cells (17). In part, similar phenotypes are induced by mutations residing in the I'IiE2 gene. With its high residual chymotrypsin-like activity, a pre2-I mutant strain does not exhihit heat and canavanine sensitivity (Fig. 2, b and d ) . However, when the pre2-1 mutation is combined with the p e l -I mutation, the resulting double mutants show phenotypes that are by far more dramatic than those observed for the p e l -1 single mutant (Fig. 2, a-d). In contrast to pre2-1 single mutants, pre2-2 mutants exhihit some canavanine sensitivity (Fig. 2d), but as pre2-I mutants, they are not heat-sensitive (Fig. 2 h ) . Combination of pre2-2 with prel-1 yields double mutants, which also show strengthening of the prel-]-induced phenotypes (Fig. 2, a-d). Remarkahly, pre2-l, which causes a less pronounced phenotype compared with pre2-2, induces a somewhat tighter phenotype when combined with prel-I as does pre2-2.
Analysis of the accumulation of uhiquitinated proteins in the mutant strains parallels their stress-dependent growth behavior. While no accumulat,ion is seen in pre2-I mutant cells upon stress application (not shown), pre2-2, p e l -I , a n d prel-l/pre2-2 mutants exhihit a 3-, 9-, and 18-fold increase, respectively, in the amount of high molecular weight uhiquitin-protein conjugates compared with wild type (Fig. 3). We consider this accumulation of uhiquitinated proteins in the pre mutant cells to result from reduced degradation. This view is supported by the finding that hydrolysis of short-lived Nend rule substrates, which are known to be destined for degradat.ion by attachment of uhiquitin chains, is blocked in these mutants (18).
An additional phenotype of t h e pre mutants rests in their capacity to hetker survive a heat shock of 52 "C when preincubated at 37 "C (not shown).
The Pre2 protein is shown to he an integral component of the yeast proteasome by two lines of evidence. 1) The reduction of chymotrypsin-like activity is a feature inherent to purified pre2 mutant proteasomes (17).
2) The Pre2 protein shows structural similarity to proteins from yeast (including Prel, see Fig. 4) and other organisms, which have been unambiguously identified as components of the proteasome particle. All ident,ified proteasomal genes encode suhunits with characteristic similarities and can he classified as a proteasomal gene family of ancient origin ( 5 , 31). P R E 2 clearly represents a member of this family.
An extraordinarily high degree of homology is found he- Proteins in cell extracts of heat-treated wild-type ( W T ) andprr mutant strains (homozygous diploids with the relevant genotypes as indicated) were separated hy SI)S-polyacrylamide gel electrophoresis on 10"; gels, hlotted onto nitrocellulose filters, and reacted with antibody recognizing uhiquitin-protein conjugates. Origin ( O R I ) of the gel and molecular weights of marker proteins are indicated. Quantification of the amount of immunoreactive ubiquitin-protein conjugates was done within the regions containing proteins with molecular masses higher than 110 kna. tween the Pre2 protein and the recently analyzed R I N G 1 0 gene product, which is encoded in the human MHC class I1 region. Not considering the poorlv conserved amino termini, a striking 60% identity and 80% similarity can he established in the entire remaining three-quarters of the I'RE2-and RINGIO-encoded proteins (Fig. 4). Because identity between proteasomal proteins of a given species generally does not exceed values of 40%, stronger homologies hetween two proteasome suhunits of different origin indicate corresponding funct,ions of the respective subunits within the heterologous protease complexes. Thus, the highlv conserved structures of the Pre2 and Ring10 proteins from evolutionarily distant organisms, i .~. yeast and man, predict some functional equivalence of these two suhunits.
The alignment of the Pre2 and Ring10 protein sequences in Fig. 4 includes other yeast (Prel and Pup1 (17, 2 8 ) ) and human (Ring12 and 6 (15, 32)) proteasome suhunits as well as t.he P-suhunit of the proteasome from the archaehacterium Thermoplasma acidophilum (31). These seven proteins can be grouped into the &subfamily of proteasome components, of which the archaehacterial P-suhunit is regarded as the ancestor (31). The only two t-ypes of suhunits, (rand 13, constituting the Thermoplasma proteasome, solely confer chvmotrypsinlike activity to the complex. This activity is thought to reside i n t h e [ h u h u n i t (31). Rernarkahly, the amino acid exchanges in thepre2-I (Gly-259 to Ser-259) and thepw2-2 (Ala-124 to Val-124) gene products both reside at positions highly conserved among the archaehacterial and eukaryotic proteasomal prot,eins compared in Fig. 4.
None of the yeast proteasomal suhunits involved in chymotr-vpsin-like activity, Prel and Pre2, exhihit any sequence similarity to known proteinases. By assuming some functional homology of Pre2 and Ring10, speculations about a new type of serine proteinase represented hv Ring10 ( 1 4 ) cannot he supported hy the Pre2 sequence data. No histidine exists in the Pre2 region corresponding to the Ring10 sequence stretch that was found to contain a histidine residue in a similar environment as the one present in the catalytic triad of suhtilisin-t-ype proteinases. Two recently cloned genes from the rat and the mouse (83, 34), which encode proteasomal suhunit,s (RC1 and MC13, respectively) highlv homologous to Ringlo, also lack a histidine at this posit ion. The chymotrypsin-like activity of the proteasome could he brought about hy a completely new proteinase t-ype, which might even he formed by interaction of two or more suhunits. The fact that mutations in two different suhunits of the yeast proteasome result in defective chymotrypsin-like activitv mav point t o a cooperation of distinct proteasomal proteins in activc-site formation.
Mutations in yeast proteasomal subunits have uncovered proteasome functions in a varietv of cellular events such as growth, differentiation, and stress response (this paper (17)). One molecular basis of action seems to reside in the capacity to degrade ubiquitinated proteins (this paper (17,18)). which are derived from different degradation pathways (18). In mammalian cells, the proteasome is also expected to exert a variety of different functions, a specialization of which is the h.ypot,hetical participation in antigen processing. The striking homology between the human MHC-encoded Ring10 protein and the proteasomal Pre2 suhunit, necessary for the chymotrypsin-like activity of the yeast protease complex, supports t,he involvement of human proteasomes in this latter process. Whether uhiquitin conjugation to proteins is a prerequisite for cleavage and suhsequent presentation of peptides as antigens has to he investigated.
In contrast to Pre2, the Ring10 suhunit does not seem t o nnqtL~TmTtVgIt1kdaVI"ErRViwnFIMhKng

HSE (NT)
x x x L A f K F r x Q U I V~l s Q g V f Rn6 (NT) mLAfKFQ.QYILILwD. HsRinglZ    HsRingZ2, and Hs6 (14, 16, 32)). Note that the Hs6 sequence is assembled with residues encoded by a cDNA lacking the 5'-end of the coding region and an overlapping amino-terminal sequence derived from amino acid sequencing. Some of the amino-terminal peptides ( N T ) obtained from amino acid sequencing of proteasomal subunits from man (Hsc (35)) and rat (Rn5, Rn6, and Rn7 (36)) are included ( x , unidentified residues). Sequences are shown in single-letter code; gaps (-) are inserted for optimal alignment. Amino acids are depicted in upper case letters if more than half of the residues in a row are conserved (gaps and n are regarded as mismatches) and are additionally depicted in boldface type if more than half of the residues are identical. Residues identical or conserved at corresponding positions in ScPreQ and HsRinglO are marked with double or sing& points, respectively. Similarity groupings are (IVLMAC) (EDQN) (HYWF) (ST) (RK) (PG).

: m l r a g a p t g D l p r a g o V n T~M A V o F D p g~8~~8 V V n R v f
Underlined regions in HsRinglO represent motifs including the proposed (14) active-site serine, histidine, and asparate (each doubly underlined) of a serine protease-type catalytic triad. Amino acid exchanges in the mutant proteins encoded by pre2-1, pre2-2, and p e l -1 are indicated. The prel-1 allele was cloned analogously to the pre2 alleles by gap repair. It shows a C to T mutation at nucleotide position 425 of the coding region. (Note that the ScPrel sequence differs at amino acid positions 183 and 188 from the published data (17), which turned out to be incorrect. The corrected amino acid and nucleotide sequences are available under accession number X56812 in the EMBL, GenBank, and DDBJ data bases.) be a constitutive proteasomal protein, because the complete absence of this MHC-encoded subunit from human cells does not impair their viability (11). Therefore, it may be assumed that MHC-encoded proteasome subunits substitute for constitutive components of similar structure. These constitutive components may provide the proteasome with similar catalytic specificity but they may change the intracellular biological function of the particle. Incorporation of RinglO could generate a proteasome that serves the specialized task of antigen processing, whereas an equivalent constitutive component may be contained in proteasomes involved in housekeeping functions, as is the enzyme complex of the unicellular yeast. The strong homology between the human Ring12 and the human 6-component (55% identity, see Fig. 4) favors this model. Subunit 6 could represent the constitutive subunit, which is replaced in antigen processing proteasomes by the Ring12 protein. Although until now no constitutive human proteasomal protein with outstanding homology to RinglO has been identified, the strong similarity of the amino-terminal peptide derived from purified human proteasomal subunit 6 (35) with part of the RinglO sequence (Fig. 4) suggests that other RinglO-related subunits are encoded in the human genome.