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Carboxy Terminally Truncated Forms of Ribophorin I Are Degraded in Pre-Golgi Compartments by a Calcium-Dependent Process Yung Shyeng Tsao, N

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Carboxy Terminally Truncated Forms of Ribophorin I Are Degraded in Pre-Golgi Compartments by a Calcium-Dependent Process Yung Shyeng Tsao, N Carboxy Terminally Truncated Forms of Ribophorin I Are Degraded in Pre-Golgi Compartments by a Calcium-dependent Process Yung Shyeng Tsao, N. Erwin Ivessa, Milton Adesnik, David D. Sabatini, and Gert Kreibich Department of Cell Biology, New York University School ofMedicine, New York 10016 Abstract. Two COOH terminally truncated variants of when added immediately after labeling, ionophores ribophorin I (RI), a type I transmembrane glycoprotein that inhibit vesicular flow out of the ER, such as car- of 583 amino acids that is segregated to the rough bonyl cyanide m-chlorophenylhydrazone (CCCP) and portions of the ER and is associated with the protein- monensin, suppressed the second phase of degradation translocating apparatus of this organelle, were expressed of 81332 . On the other hand, when CCCP was added in permanent HeLa cell transformants . Both variants, after the second phase of degradation of 81332 was ini- one membrane anchored but lacking part of the cyto- tiated, the degradation was unaffected. Moreover, in plasmic domain (RI,67) and the other consisting of the cells treated with brefeldin A the degradation of 81332 luminal 332 N112-terminal amino acids (RI332), were became monophasic, and took place with a half-life retained intracellularly but, in contrast to the endoge- intermediate between those of the two normal phases. nous long lived, full length ribophorin I (tv2 = 25 h), These results point to the existence of two subcellular were rapidly degraded (ti/2 < 50 min) by a nonlyso- compartments where abnormal ER proteins can be de- somal mechanism. The absence of a measurable lag graded. One is the ER itself and the second is a non- phase in the degradation of both truncated ribophorins lysosomal pre-Golgi compartment to which ER pro- indicates that their turnover begins in the ER itself. teins are transported by vesicular flow. A survey of the The degradation of R1467 was monophasic (t2 = 50 effects of a variety of other ionophores and protease min) but the rate of degradation of R1332 molecules in- inhibitors on the turnover of 81332 revealed that metallo- creased about threefold -50 min after their synthesis. proteases are involved in both phases of the turnover Seveal pieces of evidence suggest that the increase in and that the maintenance of a high Ca2+ concentration degradative rate is the consequence of the transport of is necessary for the degradation of the luminally trun- 81332 molecules that are not degraded during the first cated ribophorin. phase to a second degradative compartment . Thus, IKE any subcellular organelle, the ER contains proteins In recent years it has become apparent that many proteins that permanently reside within it and function in its that are normally transported from the ER to the Golgi ap- various biosynthetic and metabolic activities. How paratus, when improperly folded or incompletely assem- ever, it also contains proteins with other subcellular destina- bled, undergo degradation within the ER by a proteolytic tions that transit through the ER after they are inserted into system that has not yet been fully characterized (for review its membrane or translocated into its lumen during the course see Klausner and Sitia, 1990) . Many abnormal or unassem- oftheir synthesis on membrane-bound ribosomes (for review bled polypeptide subunits are degraded very rapidly, with see Sabatini and Àdesnik, 1989). Polypeptides synthesized half-lives of 4-60 min (Lippincott-Schwartz et al ., 1988; in the ER not only undergo various co- and posttranslational Stoller and Shields, 1989), whereas others, although appar- modifications within the organelle, but are also folded and ently not leaving the ER, are much more stable (Bonifacino in some cases oligomerized to form multisubunit proteins et al., 1989; Wileman et al., 1990a). For example, the a, ß within the ER itself (for review see Rose and Doms, 1988; and 6-chains of the T cell receptor (TCR),' type I trans- Hurtley and Helenius, 1989), apparently assisted by resident membrane polypeptides that remain in the ER when ex- which prevent the egress ofincompletely assembled proteins 1. Abbreviations used in this paper: BFA, brefeldin A; BiP, heavy chain products (Bole et al., 1986; deSilva et al., 1990; Siegel and binding protein; CCCP, carbonyl cyanide m-chlorophenylhydrazone; endo Walter, 1989). H, endoglycosidase H; RI332 and R1467, truncated forms of ribophorin I containing its 332 and 467 NH2-terminal amino acids, respectively ; TCR, Yung Shyeng Tsadspresentaddress is Schering-Plough Research, Union, NJ. T cell receptor. ® The Rockefeller University Press, 0021-9525/92/01/57/11 $2 .00 TheJournal of Cell Biology, Volume 116, Number 1, January 1992 57-67 57 pressed in transfected cells in the absence of other subunits Materials and Methods necessary for multimerization, undergo a rapid degradation, whereas the y and e subunits are stable, although they too Reagents are apparently retained in the ER (Lippincott-Schwartz et The pSP65 plasmid vector waspurchased fromPromega Biotech (Madison, al., 1988; Chen et al., 1988; Bonifacino et al., 1989; Wile- WI). Polymerases, restriction endonucleases, and mungbean nuclease were man et al., 1990x). In addition, a truncated variant of the a from New England Biolabs (Beverly, MA) and Bethesda Research Labora- chain of the TCR that contains only the luminal domain, tories (Gaithersberg, MD), nucleotides and endoglycosidase H (endo H) which is also retained in the ER, is much more stable than from Boehringer-Mannheim Biochemicals (Indianapolis, IN), and [35S]- methionine (1,100 Ci/mmol) from new England Nuclear (Boston, MA). the intact polypeptide. In fact, it has been shown that in the HBSS and MEM were from Whittaker Bioproducts Inc. (Walkersville, case of the TCR a chain the rapid degradation of the native MD), methionine-free RPMI 1,640 from Mediatech (Washington, DC), polypeptide depends on the presence of its specific trans- and FBS from Hazleton Biologics, Inc. (Lenexa, KS). Cell culture-tested membrane domain (Bonifacino et al., 1990) . Transmem- Geneticin (G 418), sodium butyrate, protein A-Sepharose CL-4B beads, brane portions of 3-hydroxy-3-methylglutaryl coenzyme A protease inhibitors, and ionophores were obtained from Sigma Chemical Co. (St. Louis, MO), Trasylol (Aprotinin) from Mobery Chemical Corp. reductase, a permanent resident protein of the ER that (New York), and En3Hance from New England Nuclear Research Products traverses the membrane several times, have also been shown (Boston, MA). When not specified, reagents were purchased from Fisher to play a critical role in determining the enhanced degrada- Scientific Co. (Pittsburgh, PA). Brefeldin A (BFA) was obtained as a gift tion rate of that protein under certain metabolic conditions from Dr. Richard Klausner (National Institute of Child Health and Human Development, Bethesda, MD). The anti-Bip antibody was kindly provided (Jingami et al ., 1987) . by Dr. D. Bole (HowardHughes Medical Institute, University of Michigan, It is not clear to what extent the natural turnover of normal Ann Arbor, MI). The rabbit polyclonal antibodies against rat liver ribopho- resident proteins of the ER involves the same degradative rin I have been previously described (Marcantonio et al ., 1984 ; Yu et system that destroys defective and incompletely assembled al ., 1990). polypeptide subunits of proteins that are normally exported from the ER. The turnover of some ER proteins, such as Plasmid Construction and Transfection ofHeLa Cells cytochrome P450 and its reductase, which accumulate at with Tlruncated Rat Ribophorin I Genes high levels after administration of some drugs and are The rat ribophorin IcDNA (Hamik-Ort et al ., 1987) was removed fromthe degraded after removal ofthe inducer, has been shown to in- plasmid vector pGEM-1 by digestion with EcoRl and HindIII, enzymes volve autophagy (Masaki et al., 1987), a process which re- which cleave at the 5' end of the cDNA and within the 3' untranslated region (residue 1964), respectively (Fig. 1 B), and subcloned into the plasmid vec- quires lysosomal functions and in hepatocytes appears to be tor pSP65. To produce the Ytruncations in the cDNA, a chemically synthe- the major mechanism for overall cellular protein degradation sized double-stranded oligonucleotide (Fig. 1 A) containing three termina- (Gordon and Seglen, 1982) . The ER degradation of abnor- tion codons and four restriction enzyme cleavage sites (NcoI at the Send, mal and incompletely assembled polypeptides, however, oc- HindIII at the 3' end, and Mlul and C1al in between) was cloned between curs by a different pathway, since it is not affected by drugs unique NcoI and HindIII sites within the 3'-noncoding region of the ribophorin I cDNA (Fig. 1 B) in the plasmid pSP65. When the ribophorin that block lysosomal proteolysis (Lippincott-Schwartz et al., I cDNA was cut in its coding region with one of the restriction enzymes 1988; Stoller and Shields, 1989). indicated on the map, it was possible to choose a cleavage site within the In this paper we studied the fate of two abnormal, COOH oligonucleotide so that cleavage at both sites, followed by mung terminally truncated variants of ribophorin I (RI), a resident bean-nuclease treatment and circularization, placed a terminationcodon in frame immediately after the ligation site. membrane glycoprotein of the ER that is segregated to the By using this strategy it is possible to generate cDNAs coding for car- rough portions of this organelle (Kreibich et al., 1978a,b). boxy terminally truncated forms of ribophorin I that contain, in addition This type I transmembrane protein is highly stable (t2 = to the signal peptide (residues -1 to -22), various N112-terminal portions of 25 h) (Rosenfeld et al ., 1984) and is part ofa supramolecular the mature ribophorin I sequence . These constructs were cloned into the complex that includes other integral ER membrane proteins expressionvector pSV2 tk neo, and permanent transformants ofHeLa cells were obtained after selection for growth in the presence of 0.4% geneticin that are components of the polypeptide translocation appara- (Compton et al ., 1989).
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