Cysteine proteinase inhibitors cause the accumulation of ubiquitin ...

170 of myosin light chain phosphorylation (e.g. England, 1983). In contrast, we have observed an increase in the phosphorylation of myosin light chain in response to both SK&F 94 120 and isoprenaline. Interestingly, it appeared that SK&F 94 120 caused a relatively greater phosphorylation of myosin light chain. These data are consistent with SK&F 94120 exerting its inotropic effects in guinea-pig hcart by inhibition of phosphodiesterase, and the subsequent increase in the levels of cyclic AMP-dependent protein phosphorylation. Although qualitatively the phosphorylation patterns were similar for both SK&F 94 120 and isoprenaline. quantitative differences were observed, particularly in the phosphorylation of myosin light chain.

BIOCHEMICAL SOCIETY TRANSACTIONS England, P. J. (1976) Hiochrm. J . 1 6 0 . 2 9 5 3 0 4 England, P. J. ( 198.7)in Cordicrc Metdwlisrn (Drake-Holland, A . J. & Noble, M. I. M., eds.), pp. 365-389, Wiley, Chichester Gilboe, D. P.. Larson, K. L. & Nuttall, F. Q. ( 1 972) Atia/. Hioclirm. 47.20-27 Murray. K. J., England. P. J. & Reeves. M. L. ( I 987) Br. J . I%armrrcol. 92, 75 5 P Murray, K. J., England, P. I., Lynham, J. A,, Mills, D. & Reeves. M. L. ( 1 988) Bioclrem. Soc. Trans. 16. 355 Reeves, M. L., Leigh, B. K. & England. P. J. ( 1987) Nioclrem. J. 241. 535-54 I

lieceived I 7 June I988

Cysteine proteinase inhibitors cause the accumulation of ubiquitin-polypeptide conjugates in lysosomes of 3T3-Ll fibroblasts FERGUS J. DOHERTY, NATASHA U. OSBORN, JULIE A. WASSELL and R. JOHN MAYER Department of Biochemistry, Uiiiversir)iof Nottirighnm Medical School, Queen S Medicul Centre, Notringham NG7 2UH, U.K. Proteolytic inhibitors may aid the identification of protein degradation intermediates by permitting their accumulation within the cell. We have shown that the cysteine and serine proteinase inhibitor leupeptin results in the accumulation of insoluble aggregates of long-lived polypeptides in 3T3-Ll fibroblasts [ 11. Insolubility was judged by failure to extract following sequential addition of the detergent Triton X-100 (I"/o, w/v, in 0.1 M-Hepes, pH 6.9, 1.5 mM-MgC12)and salt (0.3 M-KCI). Here we report the effects of E-64, a more specific inhibitor of cysteine proteinases, and its ability to result in polypeptide aggregation. We have analysed the eomposition of inhibitor-induced aggregates. Cells were pulsechased to follow the degradation of long-lived polypeptides as described previously [l]. Under these conditions it was found that the effect of E-64 was largely non-additive to that of ammonium chloride suggesting that it acts on lysosomal proteolysis of long-lived polypeptides (results not shown). E-64 resulted in an increase in radiolabelled insoluble polypeptides which was concomitant with the decrease in polypeptides degraded, suggesting that some of the polypeptides that failed to be degraded due to the inhibition of proteolysis formed aggregates, or were aggregated as a prelude to lysosomal proteolysis. Pepstatin, an inhibitor of aspartate proteinases did not result in insoluble polypeptide accumulation and ammonium chloride was less effective than the specific cysteine proteinase inhibitors (results not shown). Analysis of the detergent- and salt-insoluble fraction from control and E-64-treated cells by SDS/polyacrylamide-gel electrophoresis demonstrated that numerous polypeptide species appeared in this fraction in the presence of E-64 and that many of these were disulphide cross-linked (results not shown). Western blotting analysis with a rabbit antibody raised to synthetic ubiquitin-globulin conjugates [2] demonstrated an increase (75% by densitometry) of high molecular mass ( > 30 kDa) ubiquitin conjugates in E-64-treated wholecell extracts compared to control cells. A much smaller increase (20%) was seen with ammonium-chloride-treated cells. There was also a marked (2-fold) increase in ubiquitin monomer in both E-64- and ammonium-chloride-inhibited cells. Only in cells treated with E-64 was there an increase in high molecular mass ubiquitin conjugates in the detergent-

and salt-insoluble fraction. The migration of these conjugates was unaffected by lack of prior reduction with mercaptoethanol. Homogenization and fractionation on Nycodenz density gradients [ 11 showed that insoluble radiolabelled polypeptides which accumulated in the presence of E-64 were co-localized with the denser ( 1.16- I .18 g/ml) peak of acid phosphatase enzyme activity, a marker for lysosomes. Dotimmunoblotting revealed that ubiquitin conjugates could be dctected in E-64-treated cells as two peaks, in fractions from the top of Nycodenz density gradients containing the cytosolic marker enzyme lactate dehydrogenase and in fractions containing the lysosomal marker enzyme acid phosphatase. In control cells, anti-ubiquitin conjugate immunopositive material was found in much smaller amounts and restricted to cytosolic fractions (Fig. 1).The greater sensitivity of this antibody to ubiquitin conjugates compared with ubiquitin monomer [2, 31 and the results described above suggest that a large part of the increase in immunopositive material seen

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Fig. I. Dot-immunoblot detection of ubiquiliri conjugates in Nycodenz density gradients fractions of 3713-LI cells Cells were cultured for 48 h in thc absence ( A) and presence ( A ) of E-64 (0.5 mM) and after homogenization and fractionation on Nycodenz gradients [ I ] , aliquots were heated in SDS (1%, W / V ) and mercaptoethanol (5%, v/v), dot-blotted on to nitrocellulose and probed with rabbit anti-ubiquitin conjugate antibody. Bound antibody was detected with the avidin-biotin-alkaline phosphatase system (Vector Laboratories, Peterborough, U.K.) and quantified by reflectance densitometry. Acid phosphatase (0). 1989

627th MEETING, NOTTINGHAM in the denser lysosome fractions of cells treated with E-64 is due to an increase in ubiquitin conjugates in these fractions. Ubiquitin has been suggested to be involved in the marking of proteins for non-lysosomal degradation [4]. Our results suggest that ubiquitination and/or aggregation may: ( a ) serve to ‘isolate’ polypeptides whose degradation by the lysosomal system has been prevented; ( 6 ) ‘mark’ polypeptides for lysosomal degradation; ( c )be a feature of some normally ubiquitinated proteins, e.g. cell surface proteins [ 5 , 6, 71 which are degraded lysosomally and accumulate in the lysosome when lysosomal proteolysis is blocked. I . Doherty, F. J., Wassell, J. A. & Mayer, R. J. (1987) Biochem. J. 241,793-800 2. Haas, A. L. & Bright, P. M. (1985) J. Biol. Chem. 260, 12464-12473

171 3. Hershko, A,, Eytan, E. & Ciechanover, A. & Haas, A. L. (1982) J. Biol. Chem. 251,13964-13970 4. Ciechanover, A,, Finley, D. & Varshavsky, A. ( 1 984) J. Cell. Biochem. 24,27-58 5. Leung, D. W., Spencer, S. A,, Cachianes, G., Hammonds, R. G., Collins, C., Henzel, W. J., Barnard, R., Waters, M. J. & Wood, W. I. (1987) Nulure( London) 330,537-543 6. Yarden, Y., Escobedo, J. A., Kuang, W.-J., Yang-Feng, T. L., Tremble, D. P. M., Chen, E. Y., Ando, M. E., Harkins, R. N., Francke, U., Fried, V. A,, Ullrich, A. & Williams, L. T. (1986) Nature(London)323,226-232 7. Meyer, E. M., West, C. M. & Chau, V. (1986) J. Biol. Chem. 261, 14365-14368

Received 17 June 1988

Immunochemical demonstration of ubiquitin conjugates in fibroblasts treated with cysteine proteinase inhibitors FERGUS J. DOHERTY,* NATASHA U. OSBORN,* JULIE A. WASSELL? LAJOS LASZLO? and R. JOHN MAY ER* *Department of Biochemistry, University of Nottingham Medical School, Queen’s Medical Centre, Nottingham NG7 2UH, U.K. and ?Department of General Zoology, Elte University, Puskin U.3, H-1088, Budapest, Hungary An increase in ubiquitin conjugates has been detected by Western and dot-immunoblotting in 3T3-Ll fibroblasts treated with the specific cysteine proteinase inhibitor E-64, which inhibits lysosomal proteolysis [l].This finding was surprising as ubiquitination is thought to signal nonlysosomal proteolysis of substrate proteins [2]. Density gradient fractionation of E-64-treated cells suggested that ubiquitin conjugates accumulate in a lysosomal fraction in inhibitor-treated cells. To further characterize the cytomorphological site of ubiquitin conjugate accumulation we probed cells immunohistochemically with an antibody raised against synthetic, denatured ubiquitin conjugates [3,4] using a biotinylated anti-rabbit second antibody and avidinalkaline phosphatase complex detection system (Vector Laboratories, Peterborough, U.K.). Control cells exhibited ubiquitin-positive staining of the nucleus, consistent with the presence of ubiquitinated histones, but poor staining of the cytoplasm (Fig. l a ) . The poor cytoplasmic staining confirms the relative specificity of this antibody for conjugated as opposed to free ubiquitin, which is present to a high level in the cytoplasm of many cell types [5].In contrast, cells treated for 48 h with E-64 contained large amounts of punctate immunoreactive material in the cytoplasm, consistent with the accumulation of conjugates in some form of vesicle (Fig. 1b).Similar distributions of ubiquitin conjugates were seen in cells treated for 48 h with the inhibitor of cathepsins B and L, Z-Phe-Ala-CHN,. Chloroquine, a cathepsin B inhibitor and a lysosomotropic agent, was an effective promoter of ubiquitin conjugate accumulation, while ammonium chloride was much less effective (not shown). The very low level of immunohistochemically detectable ubiquitin conjugates in control cells (Fig. l a ) is surprising as they were readily detectable in control cells following electrophoresis and Western blotting (not shown) and may be due to the antibody being specific for highly denatured ubiquitin conjugates, i.e. after heating in SDS, or present intracellularily in a denatured, aggregated form in inhibitortreated cells [ 11.

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Fig. 1. Irnrnunohistochemical demonstration of ubiquitin conjugates in 3T3-LI cells Cells were cultured for 48 h in the absence ( a )or presence ( b ) of E-64 (0.5 mM), fixed with formaldehyde and after blocking with non-immune goat serum probed with rabbit anti-ubiquitin conjugate antibody. Antibody was detected with the avidin-biotin-alkaline phosphatase detection system (Vector Laboratories, Peterborough, U.K.).