Proc. Nati. Acad. Sci. USA Vol. 89, pp. 4928-4932, June 1992 Immunology Proteasomes are regulated by interferon y: Implications for antigen processing (major hbitocompatlbility complex/class I moecules/lymphokines) YOUNG YANG, JAMES B. WATERS, KLAUS FROH, AND PER A. PETERSON Department of Immunology, The Scripps Research Institute, La Jolla, CA 92037 Communicated by Frank J. Dixon, February 27, 1992

ABSTRACT Class I major histocompatibility complex strengthened by the findings, presented in this communica- (MHC) molecules present antigenic peptides of cytoplasmic tion, that several proteasomal subunits, including MHC- origin to T cells. As the lengths ofthese peptides seem stried encoded subunits, are regulated by interferon y (IFN--y) and to eight or nine amino acids, an unusual proteolytic system that the incorporation of several more subunits into protea- must play a role in antigen processing. Proteasomes, a major somes appears to depend on the expression of the MHC- extralysosomal proteolytic system, are responsible for the encoded proteasomal subunits. Moreover, the pattern of degradation of cytoplasmic proteins. We demonstrate that expression of IFN-y-regulated subunits suggests complexi- several proteasomal subunits, including MHC-encoded sub- ties in the regulation of proteasomes with respect to its units, are regulated by interferon y. These data and the finding subunit composition, subcellular localization, and its incor- that MHC-encoded and other interferon -regulated protea- poration into larger ubiquitin-related proteolytic complexes. somal subunits are uniquely associated with proteasomes Possible functions for the MHC-encoded and IFN-y- strongly suggest that the immune system has recruited protea- regulated proteasomal subunits in antigen processing are somes for antigen processing. discussed. Major histocompatibility complex (MHC) class I molecules MATERIALS AND METHODS are believed to primarily obtain antigenic peptides from Cell Cultures. HeLa cells (ATCC CCL185) were grown in proteins synthesized within the cell (1). The recent observa- Dulbecco's modified Eagle's medium (DMEM; GIBCO) sup- tion that a putative transmembrane peptide transporter (2-6) plemented with 8% fetal calf serum, 2 mM glutamine, peni- is essential for peptide loading onto class I molecules sug- cillin (100 ,ug/ml), and streptomycin (100 pug/ml). Spleno- gests that antigenic peptides, which are acquired by class I cytes offour different H-2 haplotypes (H-2k, H-2s, H-2d, and molecules in a pre-Golgi compartment (7-9), may arise in the H-2q), T1 and T2 cells (24), and murine astrocytes were cytoplasm. However, the proteolytic system responsible for grown in RPMI 1640 medium. generating class I MHC binding peptides has remained enig- Metabolic Radiag and Immunoprecipitaton. Meta- matic. bolic labeling of cells was carried out as described (25) with The majority ofproteolytic activity in the cytoplasm can be the following modifications. Unless otherwise stated, cells attributed to the activity of proteasomes (10, 11). This major were treated with IFN-y (2500 units/ml) for 96 hr prior to the extralysosomal proteolytic system (10) relies on a complex metabolic labeling. Pulse media contained 0.15 mCi of series of enzymatic events where proteins become targeted L-[35S]methionine per ml and 0.15 mCi of L-[35S]cystine per for degradation by covalent conjugation to the polypeptide ml (Amersham; 1 Ci = 37 GBq) in methionine- and cystine- ubiquitin (12, 13). The ubiquitinated protein is subsequently deficient DMEM. Cells were routinely labeled for 4 hr degraded into small peptides and free amino acids in an followed by chase periods of different lengths of time (up to ATP-dependent process by 26S ubiquitin-dedicated protea- 36 hr) in the presence of normal culture medium. Immuno- some complexes. This 26S proteasome complex contains the precipitations (25), SDS/PAGE (26), and fluorography (27) 19S form of the proteasome as its major proteolytic compo- were carried out as described (25). First-dimension nonequi- nent (14). The 19S proteasome also exists in free form in the librium pH gradient gel electrophoresis (using Ampholines cell and may represent an independent and separately regu- pH 3.5-10) was done as described (28). lated proteolytic system (14, 15). The proteasome displays Antisera and Materials. Rabbit anti-human and anti-rat several distinct peptidase activities, being able to cleave on proteasome sera were kindly provided by A. Ichihara (29). the carboxyl side of basic, acidic, and neutral amino acids, IFN-y from human T lymphocytes was obtained from Boeh- which suggests it can generate a wide variety ofpeptides from ringer Mannheim. Mouse IFN-y was obtained from Genen- diverse protein substrates (10, 11). In addition, the enzymatic tech and Amgen Biologicals. activity of the proteasome is regulated by a number of Homogenization of HeLa Cells. Two milliliters of isotonic different compounds, including nucleotides and polycations sucrose solution was added to HeLa cell monolayer. Cells (16, 17). These observations make the proteasome an attrac- were scraped from dishes and homogenized directly by 45 tive candidate as a proteolytic generator ofantigenic peptides passages through a precision clearance between the wall of a of cytoplasmic origin for class I MHC molecules (18). This metal chamber and a metal ball bearing of a Dounce homog- suggestion is further supported by the recent findings that enization vessel (30). During homogenization, the homoge- two proteasomal subunits are encoded in the MHC region nates were monitored using phase-contrast microscopy to (19-23). assess the number of broken cells, the number of intact and The view that the proteasome is a proteolytic generator of clean nuclei, and the absence of cytoplasmic clumps. antigenic peptides for class I MHC molecules is further Ammonium Sulfate Fractionation of HeLa Cell Homoge- nates. Homogenized HeLa cells, untreated and IFN-y- The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: MHC, major histocompatibility complex; IFN-y, in accordance with 18 U.S.C. §1734 solely to indicate this fact. interferon 'y; 2D, two-dimensional.

4928 Downloaded by guest on September 27, 2021 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4929 treated, were centrifuged to remove nuclei and cell debris, peared completely (spots 2, 10, 15, and 16) following IFN-y and the resultant supernatants were subjected to ammonium treatment. In addition, the intensity of spot 9 increased sulfate fractionation as described (31). 26S proteasomes considerably as a consequence of the IFN-y treatment. present in the supernatant were selectively precipitated with Experiments identical to those described above were also ammonium sulfate at 38% saturation; 19S proteasomes pre- carried out with a murine astrocyte cell line. The subunit sent in the supernatant after the 38% fractionation step were pattern of murine proteasomes was very similar to that precipitated at 60%o ammonium sulfate (32). observed for human proteasomes and the effect of IFN-y on Fractionation of Microsomes and Cytosol. Untreated and the astrocyte proteasomes was analogous to that ofthe HeLa IFN--treated HeLa cells were labeled for 4 hr, homoge- cell proteasomes (not shown). Thus, the subunit composition nized, and subjected to differential centrifugation. The ho- of proteasomes from two species is regulated by IFN-y. mogenates were first spun at 13,000 rpm for 30 min to remove If the IFN-y-regulated proteasomal subunits are involved nuclei and cell debris. The resulting supernatants were then in the generation ofantigenic peptides, one might assume that spun at 100,000 x g for 30 min to pellet microsomes. cells that express high levels of class I MHC molecules may Proteasomes in the supernatant fractions (cytosolic fraction) also constitutively express high levels ofthe IFN-y-regulated and in the pellets (crude microsomal fraction) were separately proteasomal subunits. For this reason we chose to examine immunoprecipitated and analyzed by two-dimensional (2D) proteasomes from murine splenocytes. In addition, we could gel electrophoresis. also determine if proteasomal subunits displayed structural polymorphisms indicative of many MHC-encoded proteins. Proteasomes from biosynthetically labeled splenocytes de- RESULTS rived from four mouse strains representing the H-2 haplo- Proteasomal Subunit Composition Is Regulated by IFN-y. types d, k, s, and q were immunoprecipitated with an The assembly of class I MHC molecules in the endoplasmic antiserum specific for rat proteasomes and subjected to 2D reticulum in transfected cells that overexpress class I heavy PAGE. The typical patterns of the proteasomal subunits are chains and f32-microglobulin is limited by the availability of depicted in Fig. 2. All but one ofthe proteasomal polypeptide peptides that can bind to class I MHC molecules. This spots occurred at corresponding positions for all four haplo- restriction can be overcome by treating the cells with IFN-y types. The exception was spot 9, which is likely to corre- (E. Song, Y.Y., M. R. Jackson, and P.A.P., unpublished spond to one of the MHC-encoded subunits (19-23) and work). IFN-y induces changes in the level of expression of a which obviously occurs in at least two allelic forms (21). The number ofgene products affecting various cellular responses splenocyte proteasomal subunit pattern was intermediate that appear to be aimed at defending the cell against viral between those of untreated and IFN-y-treated HeLa cells infections (33). We reasoned that it was likely that some ofthe inasmuch as spots 2, 10, 15, and 16 were weak or absent, components responsible for generating class I antigenic pep- whereas spots b, d, and 9 were prominent. Thus, we conclude tides might also be regulated by IFN-y. that only one proteasomal subunit, which is likely to be We focused our interest on the proteasome and examined encoded in the MHC, displayed structural polymorphism and whether its subcellular distribution and subunit composition that some IFN-y-regulated proteasomal polypeptides are were noticeably affected by IFN-,y treatment. Proteasomes constitutively expressed in splenocytes. The latter finding is from IFN-y-treated and untreated HeLa cells were immuno- consistent with splenocytes being excellent antigen- precipitated with an antiserum specific for human protea- presenting cells. somes and subjected to 2D PAGE. Typical autoradiograms Incorporation of MHC-Encoded and IFN-r-Regulated Sub- are depicted in Fig. 1. The proteasome consists of several units into Proteasomes. The data described above show that individual subunits whose apparent molecular masses range IFN-'y regulates more proteasomal subunits than the two that from =18,000 to -35,000 daltons (10, 11). However, the are known to be encoded by the MHC region (19-23). To number ofproteasomal subunits and their stoichiometry have examine if the MHC-encoded and IFN-y-regulated protea- not been unambiguously determined. The schematic diagram somal subunits control the recruitment of other polypeptides shown in Fig. 1C shows that by immunoprecipitation we into the proteasome, we examined the proteasomal subunit could isolate proteasomes, which resolved into a total of 25 compositions oftwo human lymphoblastoid cell lines, T1 and polypeptide spots by 2D gel electrophoresis. Spots 1-20 were T2, which differ with regard to their MHC regions (24) such reproducibly obtained with immunoprecipitated proteasomes that T2 cells lack the MHC-encoded proteasomal subunits. from untreated HeLa cells. The proteasomal subunit pattern Biosynthetically labeled proteasomes from untreated and obtained after IFN--y treatment of the HeLa cells was clearly IFN-y-treated T1 and T2 cells were immunoprecipitated and different from the pattern observed for proteasomes from analyzed by 2D PAGE. The typical proteasomal subunit untreated cells. Five novel spots were apparent, denoted a-e patterns are depicted in Fig. 3. Although the overall subunit in Fig. 1, and four spots were greatly diminished or disap- compositions were very similar to those observed for un- A B c .40 - - 0 - /16 .w do"w o0 13 1 6 4w~ v -CDfo 40 .111. -4%/0110 10 9 c b

E03

FIG. 1. Some subunits of human proteasomes are regulated by IFN-y. Proteasomes of HeLa cells, untreated (A) or IFN-y-treated (B) before labeling, were immunoprecipitated with antibodies against human proteasomes and subjected to 2D PAGE. The solid arrows denote proteasomal subunits whose intensities were increased to a minor (thin arrows) or major (bold arrows) extent after treatment with IFN-'y. Open arrows denote proteasomal subunits whose intensities were reduced after treatment with IFN-y. (C) The schematic diagram is a composite ofA and B. Filled spots indicate proteasomal subunits whose intensities increased; hatched spots indicate those whose intensities decreased following the IFN-y treatment. Downloaded by guest on September 27, 2021 4930 Immunology: Yang et aL Proc. Natl. Acad. Sci. USA 89 (1992)

H-2s H-2d therefore, conceivable that IFN-y controls several discrete steps in the proteasomal degradation of polypeptides (see below). -M lw 4w Protesomal Subunits Are Dis- 4w, IFN-r-Regulated Uniquely 40 4w The -W tributed Between the 19S and the 26S Proteasones. AM - 040.- 40 *.,&. 40 OR W proteasome exists in two molecular forms (the 19S protea- lc. 4k* IC), some and the 26S proteasome). As the 26S proteasome seems to degrade proteins extensively to yield very small peptides, which cannot bind to class I molecules, or free amino acids, H-2q H-2 k elimination of functional 26S proteasomes might make more peptides available for the class I molecules. If the MHC- encoded and IFN--regulated subunits facilitate the genera- Ab M. qw t -. I At tion of antigenic peptides, the IFN--regulated subunits 4w 1-0 might impede the formation of 26S proteasomes or may do 401i- 4w omi- 40 go died 41b inactivate their proteolytic activity. Therefore, to examine 1c), 1:2.- whether the IFN-y-regulated proteasomal subunits were dis- tributed differently between the 19S and 26S proteasomes, biosynthetically labeled proteasomes from untreated and IFN-y-treated HeLa cells were fractionated into those that FIG. 2. 2D gel electrophoresis of proteasomal subunits of differ- were precipitated at 38% ammonium sulfate saturation (26S ent H-2 haplotypes. Splenocytes of the indicated H-2 haplotypes were metabolically labeled for 4 hr. Proteasomes were immunopre- proteasomes) and those that were precipitated between 38% cipitated with antibodies against rat proteasomes and analyzed by 2D and 60% saturation (19S proteasomes). The ammonium sul- gel electrophoresis. The proteasomal subunit that displayed allelic fate-precipitated materials were dissolved, dialyzed, immu- variation is denoted by the arrows. noprecipitated with an antiserum specific for human protea- somes, and analyzed by 2D PAGE. Fig. 4 summarizes the treated and IFN-y-treated HeLa cells (see Fig. 1), several results. differences were also apparent. Several additional polypep- The subunit pattern of the 26S proteasome from untreated tides, denoted p, q, r, and s, were present in T1 cells. In HeLa cells lacked components 2, 9, 10, and 11, which were addition, the IFN--induced polypeptides a and e were present in the 19S proteasome. Differences between the constitutively expressed in untreated T1 cells. After IFN-y subunit patterns of the 19S and 26S proteasomes were more treatment, the T1 proteasomes also displayed differences pronounced after IFN-ytreatment. Fig. 4 C and D shows that from those of IFN-y-treated HeLa cells. The spots denoted IFN--induced subunits a and e were associated with the 26S 2, 10, 15, and 16 were more prominent in T1 than in HeLa proteasome, whereas those denoted b, c, and d were exclu- cells and polypeptide d was not induced in T1 cells. More- sively confined to the 19S proteasome. In addition, the 19S over, polypeptide c disappeared from the T1 cells but was proteasome isolated from IFN-y-treated HeLa cells lacked induced in the HeLa cells. subunits 2, 3, and 10. The 26S proteasome did not seem to The proteasomal subunit patterns of untreated and IFN- lack any additional subunits following IFN-y treatment. y-treated T1 cells displayed multiple differences. Polypep- Thus, the subunit compositions of the 19S and 26S protea- tides 14, c, p, r, and s disappeared, whereas increased somes differ from each other prior to and after IFN-y amounts of 9, 13, 15, 16, a, b, and e were observed. Some of treatment. Moreover, the presence of IFN--regulated pro- these polypeptides also differed between proteasomes of teasomal subunits seems mutually exclusive, as they are untreated T1 and T2 cells. Thus, polypeptides 9, 15, a, and c either incorporated into the 19S or the 26S proteasome. were absent from T2 cells. With IFN-y treatment T2 protea- Suelular Distribution of Proteasomial Subunits. Since somes displayed the expected differences with the exception peptides derived from cytoplasmic proteins must enter the that polypeptide b disappeared. Thus, it can be concluded endoplasmic reticulum to become accessible to class I mol- that in qualitative terms T1 and T2 proteasomes differed with ecules, we examined whether proteasomes present in a regard to polypeptides 9, 15, a, b, and c. These data dem- microsomal fraction had a different subunit pattern than onstrate that the incorporation of all but one of the IFN-- those existing in the cytoplasm, which would be consistent regulated subunits into proteasomes was dependent on one or with targeting of proteasomes to the MHC-encoded peptide more genes, which is (are) likely to be MHC-encoded pro- transporter. Biosynthetically labeled proteasomes from a teasomal subunits. Since polypeptide 9 is IFN-y-regulated, crude microsomal fraction and a cytosolic fraction of HeLa structurally polymorphic, and absent from untreated and cells were immunoprecipitated and subjected to 2D PAGE. IFN--treated T2 cells, we concluded that subunit 9 is Fig. 5, which displays typical autoradiograms, shows that the encoded within the MHC. This conclusion is supported by proteasomes present in the microsomal fraction were en- the finding that the polypeptide, detected from HeLa cells riched for subunits 5 and 14 and lacked subunits 6, 7, 13, and transfected with the MHC-encoded proteasomal subunit 20. Following IFN-y treatment, proteasomes present in the RING10 (23) cDNA, is identical to polypeptide 9 as revealed crude microsomal fraction contained more of the IFN-y- by 2D PAGE (unpublished data). Furthermore, the observa- regulated subunits b and c than did proteasomes present in tion that IFN-'y-regulated subunit b is absent from untreated the cytosolic fraction. In contrast, the IFN--regulated sub- and IFN-y-treated T2 cells in conjunction with the finding units a and e were predominantly confined to proteasomes that subunit b is identical to the polypeptide detected from present in the cytosolic fraction. The difference in the subunit HeLa cells transfected with the other MHC-encoded protea- compositions of the 19S and 26S proteasomes and the dif- somal subunit RING12 (22) cDNA (unpublished data) ference in the subunit compositions of cytoplasmic and strongly suggests that subunit b is encoded in the MHC. microsomal proteasomes reveal that multiple types of pro- If the MHC-encoded subunits facilitate the generation of teasomes must exist. antigenic peptides, then the other IFN-y-regulated polypep- tides might have contributing but independent effects. This suggestion is supported by the observation the IFN-y had DISCUSSION virtually identical effects on T1 and T2 proteasomes despite The fact that the proteasome is essential for the homeostasis the lack of the MHC-encoded subunits in T2 cells. It is, of cells in conjunction with its presence in lower eukaryotes Downloaded by guest on September 27, 2021 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 89 (1992) 4931

10 15 16C Q a4 1919 ib 2q 1442 01 _ 4sP 0 M o. 0 2l1 12 6 0 A o ~.9 c to p

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FIG. 3. 2D gel comparisons of proteasomal subunits from the cell lines T1 and T2. Proteasomes from untreated T1 (A) and T2 (B) cells as well as proteasomes from IFN-y-treated T1 (D) and T2 (E) cells were immunoprecipitated with antibodies against human proteasomes and analyzed by 2D gel electrophoresis. Positions for proteasomal subunits present in T1 cells but absent from T2 cells are circled. (C and F) The schematic diagrams are composites of A and B and of C and D, respectively. Filled spots indicate proteasomal subunits whose intensities increased; hatched spots indicate those whose intensities decreased in T2 compared to T1 cells. argue for the proteasome having evolutionarily predated the It has been shown that disruption of two proteasomal immune system. It is not surprising that the immune system subunit genes in Saccharomyces cerevisiae profoundly af- might have recruited the proteasome for the purpose of fected the growth characteristics of cells (34-36). Since the generating antigenic peptides that can be presented by class growth characteristics of the T1 and T2 cells were not I molecules. The findings that the proteasomal subunit pat- noticeably different, it can be assumed that the housekeeping tern was greatly affected by IFN-y treatment and that MHC- functions of proteasomes were not adversely affected by the lack of IFN-y-regulated and the MHC-encoded subunits. It encoded and other IFN-y-regulated proteasomal subunits are may, therefore, be assumed that the latter subunits alter the uniquely associated with proteasomes are, therefore, fully activity ofthe proteasomal system such that the generation of consistent with the proteasome being involved in antigen antigenic peptides becomes facilitated. The MHC-encoded presentation. The view that proteasomes play a role in subunit 9 was distributed among cytosolic and microsomal as antigen processing is further strengthened by our preliminary well as 19S and 26S proteasomes. However, the MHC- experiments showing that proteasome-treated ovalbumin sta- encoded subunit b was only present in 19S microsomal bilized empty MHC Kb molecules and that the major product proteasomes. This unique distribution of subunit b among of the proteasome-processed ovalbumin was an octapeptide proteasomes may imply that subunit b-containing protea- (unpublished data). somes are dedicated to the production of antigenic peptides.

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FIG. 4. Subunit compositions of26S and 19S proteasomes. Untreated (A and B) and IFN-y-treated (C and D) HeLa cells were homogenized and centrifuged at 13,000 rpm for 30 min to remove nuclei and cell debris. The resultant supernatants were subjected to ammonium sulfate fractionation as described (31). 26S proteasomes present in the supernatant were selectively precipitated with ammonium sulfate at 38% saturation, whereas 19S proteasomes present in the supernatant after the 38% fractionation step were precipitated at 60%o ammonium sulfate (32). The dialysates of HeLa cell 19S (A and C) and 26S (B and D) proteasomes were separately immunoprecipitated with antibodies against human proteasomes and analyzed by 2D PAGE. Proteasomal subunits differing between the 19S and the 26S proteasomes are circled. Downloaded by guest on September 27, 2021 177A032 Immunology: Yang et al. Proc. Natl. Acad. Sci. USA 89 (1992)

A B genes (Y.Y., unpublished work), which are closely linked to the class II loci. Thus, it is conceivable that MHC-encoded and IFN--regulated proteasomal subunits may affect gen- . II. eration of class I binding peptides such that self-proteins a become substrates or novel peptides are generated from self-proteins. Absence of tolerance to such peptides may contribute to the induction of autoimmune disease. A*e We thank E. Song and T. Spies for providing unpublished data and valuable suggestions, A. Ichihara for kindly providing antibodies, P. Cresswell for kindly providing T1 and T2 cells, and D. Uranowski for C1 D expert secretarial assistance. This work was supported by grants Ab from the National Institutes of Health and K.F. is the recipient of a r" fellowship from the Deutsche Forschungsgemeinschaft. Mb .

.M. 4b 0 - - m 410 W *e 1. Townsend, A., Gotch, F. M. & Davey, J. (1985) Cell 42, 457-467. 41* 2. Monaco, J. J., Cho, S. & Attaya, M. (1990) Science 250,1723-1726. 3. Deverson, E. V., Gow, I. R., Coadwell, W. J., Monaco, J. J., Butcher, G. W. & Howard, J. C. (1990) Nature (London) 348, 738-741. 4. Trowsdale, J., Hanson, I., Mockridge, I., Beck, S., Townsend, A. & Kelly, A. (1990) Nature (London) 348, 741-744. FIG. 5. Subunit compositions of microsomal and cytosolic pro- 5. Spies, T., Bresnahan, M., Bahram, S., Arnold, D., Blanck, G., Mellins, E., Pious, D. & DeMars, R. (1990) Nature (London) 348, teasomes from untreated and IFN--treated HeLa cells. Untreated 744-747. (A and B) and IFN--treated (C and D) HeLa cells were labeled for 6. Spies, T. & DeMars, R. (1991) Nature (London) 351, 323-324. 4 hr, homogenized, and subjected to differential centrifugation. The 7. Yewdell, J. W. & Bennink, J. R. (1989) Science 244, 1072-1075. homogenates were first spun at 13,000 rpm for 30 min to remove 8. Nuchtern, J. G., Bonifacino, J. S., Biddison, W. E. & Klausner, nuclei and cell debris. The resulting supernatants were then spun at R. D. (1989) Nature (London) 330, 223-226. 100,000 x g for 30 min to pellet microsomes. The proteasomes in the 9. Townsend, A., Ohlen, C., Bastin, 3., Ljunggren, H.-G., Foster, L. supernatant fractions (cytosolic fraction, A and D) and in the pellets & Karre, K. (1989) Nature (London) 340, 443 448. (crude microsomal fraction, B and D) were separately immunopre- 10. Orlowski, M. (1990) Biochemistry 29, 10289-10298. cipitated with antibodies against human proteasomes and analyzed 11. Rivett, J. A. (1989) Arch. Biochem. Biophys. 26, 1-8. by 2D gel electrophoresis. Filled arrows indicate subunits that 12. Hershko, A. & Ciechanover, A. (1982) Annu. Rev. Biochem. 51, 335-364. predominantly appeared in the cytosolic fractions; open arrows 13. Rechsteiner, M. (1991) Cell 66, 615-618. denote spots that predominantly occurred in the microsomal frac- 14. Driscoll, J. & Goldberg, A. L. (1990)J. Biol. Chem. 265, 4789-4792. tions. 15. Ganoth, D., Leshinsky, E., Eytan, E. & Hershko, A. (1988) J. Biol. Chem. 263, 12412-12419. Several complementary mechanisms might contribute to 16. Driscoll, J. & Goldberg, A. L. (1989) Proc. Natl. Acad. Sci. USA rendering the proteasome dedicated to generating peptides 86, 787-791. suitable for binding to class I molecules. (i) The IFN-- 17. Mellgren, R. L. (1990) Biochim. Biophys. Acta 1040, 28-34. 18. Parham, P. (1990) Nature (London) 348, 674-675. regulated subunits might inactivate or impede the formation 19. Martinez, C. K. & Monaco, J. J. (1991) Nature (London) 353, of 26S proteasomes such that more peptides of the correct 664-667. length are available for the class I molecules. Our observation 20. Brown, M. G., Driscoll, J. & Monaco, J. J. (1991) Nature (London) that some IFN-y-regulated subunits are exclusively associ- 353, 355-357. 21. Ortiz-Navarrete, V., Seelig, A., Gernold, M., Kloet- ated with either the 19S or the 26S particles would be Frentzel, S., zel, P. M. & Hammerling, G. J. (1991) Nature (London) 353, consistent with this view. (ii) One or more of the IFN-y- 662-664. regulated subunits may target proteasomes to a locality in the 22. Kelly, A., Powis, S. H., Glynne, R., Radley, E., Beck, S. & cell where antigenic peptides are preferably generated. Thus, Trowsdale, J. (1991) Nature (London) 353, 667-668. unless a specific transport system exists, which accumulates 23. Glynne, R., Powis, S. H., Beck, S., Kelly, A., Kerr, L.-A. & peptides and transfers them to the peptide transporters in Trowsdale, J. (1991) Nature (London) 353, 357-360. the 24. Salter, R. D., Howell, D. N. & Cresswell, P. (1985) Immunogenet- endoplasmic reticulum, proteasomes associated with the ics 21, 235-237. endoplasmic reticulum might mainly be responsible for gen- 25. Jackson, M. R., Nilsson, T. & Peterson, P. A. (1990) EMBO J. 9, erating antigenic peptides. The observation that proteasomes 3153-3162. present in a crude microsomal fraction differ in their subunit 26. Blobel, G. & Dobberstein, B. (1975) J. Cell Biol. 67, 835-851. composition from those present in the supernatant would 27. Bonner, W. & Laskey, R. A. (1974) Eur. J. Biochem. 46, 83-88. be 28. Jones, P. P. (1980) in Selected Methods in Cellular Immunology, consistent with this view. (iii) The MHC-encoded and IFN- eds. Mishell, B. P. & Shigii, S. P. (Freeman, San Francisco), pp. -regulated subunits might directly affect the catalytic prop- 398 440. erties of the proteasomal system. The proteasome is believed 29. Tanaka, K., Ichihara, A., Waxman, L. & Goldberg, A. L. (1986) J. to contain two independent, but simultaneously active, cat- Biol. Chem. 261, 15197-15203. 30. Howell, K. E., Devaney, E. & Gruenberg, J. (1989) Trends Bio- alytic centers (37). The distance between these centers may chem. Sci. 14, 44-47. be affected by the IFN-y-regulated subunits such that octa- 31. Rivett, J. A. (1985) J. Biol. Chem. 260, 12600-12612. or nonapeptide generation is favored. 32. Waxman, L., Fan, J. M. & Goldberg, A. L. (1987) J. Biol. Chem. The present observations raise a number of important 262, 2451-2457. questions about the system giving rise to antigenic peptides 33. Taniguchi, T. (1988) Annu. Rev. Immunol. 6, 439 464. for class I molecules. It is conceivable that 34. Emori, Y., Tsukahara, T., Kawasaki, H., Ishiura, S., Sugita, H. & regulatory mech- Suzuki, K. (1991) Mol. Cell. Biol. 11, 344-353. anisms exist that render antigenic peptides more likely to 35. Fujiwara, T., Tanaka, K., Orino, E., Yoshimura, T., Kimatori, A., become available to class I molecules than peptides derived Tamura, T., Chung, C.-H., Nakari, T., Yamaguchi, K., Shiu, S., from self-proteins, which, in turn, may have bearing on our Kakizuka, A., Nakanishi, S. & Ichihara, A. (1990)J. Biol. Chem. 27, understanding of autoimmunity. The well-established fact 16604-16613. that many autoimmune diseases are 36. Tanaka, K. & Ichihara, A. (1989) Biochem. Biophys. Res. Commun. genetically linked to 159, 1309-1315. alleles of class II genes may have to be revisited in light of the 37. Dick, L. R., Moomaw, C. R., DeMartino, G. N. & Slaughter, C. A. polymorphism demonstrated by transporter and proteasomal (1991) Biochemistry 30, 2725-2734. Downloaded by guest on September 27, 2021