Carboxy terminally truncated forms of ribophorin I are degraded in pre-Golgi compartments by a calcium-dependent process.

Abstract

Abstract. Two COOH terminally truncated variants of ribophorin I (RI), a type I transmembrane glycoprotein of 583 amino acids that is segregated to the rough portions of the ER and is associated with the proteintranslocating apparatus of this organelle, were expressed in permanent HeLa cell transformants . Both variants, one membrane anchored but lacking part of the cytoplasmic domain (RI,67) and the other consisting of the luminal 332 N112-terminal amino acids (RI332), were retained intracellularly but, in contrast to the endogenous long lived, full length ribophorin I (tv2 = 25 h), were rapidly degraded (ti/2 < 50 min) by a nonlysosomal mechanism . The absence of a measurable lag phase in the degradation of both truncated ribophorins indicates that their turnover begins in the ER itself. The degradation of R1467 was monophasic (t 2 = 50 min) but the rate of degradation of R1332 molecules increased about threefold -50 min after their synthesis. Seveal pieces of evidence suggest that the increase in degradative rate is the consequence of the transport of 81332 molecules that are not degraded during the first phase to a second degradative compartment . Thus, IKE any subcellular organelle, the ER contains proteins that permanently reside within it and function in its various biosynthetic and metabolic activities. How ever, it also contains proteins with other subcellular destinations that transit through the ER after they are inserted into its membrane or translocated into its lumen during the course oftheir synthesis on membrane-bound ribosomes (for review see Sabatini and Àdesnik, 1989). Polypeptides synthesized in the ER not only undergo various co-and posttranslational modifications within the organelle, but are also folded and in some cases oligomerized to form multisubunit proteins within the ER itself (for review see Rose and Doms, 1988;Hurtley and Helenius, 1989), apparently assisted by resident proteins which prevent the egress ofincompletely assembled products (Bole et al., 1986; deSilva et al., 1990;Siegel and Walter, 1989). when added immediately after labeling, ionophores that inhibit vesicular flow out of the ER, such as carbonyl cyanide m-chlorophenylhydrazone (CCCP) and monensin, suppressed the second phase of degradation of 81332 . On the other hand, when CCCP was added after the second phase of degradation of 81332 was initiated, the degradation was unaffected. Moreover, in cells treated with brefeldin A the degradation of 81332 became monophasic, and took place with a half-life intermediate between those of the two normal phases. These results point to the existence of two subcellular compartments where abnormal ER proteins can be degraded . One is the ER itself and the second is a nonlysosomal pre-Golgi compartment to which ER proteins are transported by vesicular flow. A survey of the effects of a variety of other ionophores and protease inhibitors on the turnover of 81332 revealed that metalloproteases are involved in both phases of the turnover and that the maintenance of a high Ca 2+ concen...

Misfolded major histocompatibility complex class I molecules accumulate in an expanded ER-Golgi intermediate compartment.

“…Several polypeptides including the asialoglycoprotein receptor, ribophorin, or T cell receptor subunits are known to be degraded by a nonlysosomal endoproteolytic cleavage that takes place in the ER itself or in ER-derived subcompartments (Amara et al, 1989;Wikstrom and Lodish, 1993;Tsao et al, 1992;Klausner and Sitia, 1990;Bonifacino and Lippincott-Schwartz, 1991). Our observations on thymic epithelial cells show that the ER-Golgi intermediate station is complex and can be induced to expand, in response to the quantity and quality of passenger proteins.…”

Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast

“…3). A lag phase and biphasic kinetics have also been observed for ER degradation of mammalian secretory proteins (Le et al, 1990;LippincottSchwartz et al, 1988;Tsao et al, 1992). …”

“…Recent studies on the T-cell receptor have identified the ER itself as the compartment where protein degradation occurs (Stafford and Bonifacino, 1991). A study on a carboxy-terminally truncated version of ribophorin I describes characteristics for the degradative event which indicate the existence of a second, a preGolgi compartment, where proteins in the secretory pathway can be degraded (Tsao et al, 1992). The so-called 'ER degradation' (Lippincott-Schwartz et al, 1988) is not only limited to membrane proteins.…”

Endoplasmic reticulum: a dynamic patchwork of specialized subregions.

“…Yet, degradation concerns specifically the proteins (both membrane-bound and luminal) that have failed to fold or assemble properly and spares nascent proteins. The lag observed between synthesis and degradation might reflect migration into a specialized subcompartment (Tsao et al, 1992). Whether the latter is physically separated from the ER is doubtful, however, as degradation occurs within permeabilized cells even in the absence of cytosol and ATP, which are essential for vesicular transport (Stafford and Bonifacino, 1991;Wilkstrom and Lodish, 1992).…”