2 PATHWAYS FOR THE DEGRADATION OF THE H2 SUBUNIT OF THE ASIALOGLYCOPROTEIN RECEPTOR IN THE ENDOPLASMIC-RETICULUM

An intermediate of 35 kD accumulates transiently during ER degradation of the H2 subunit of the asialoglycoprotein receptor; it is derived b y an endoproteolytic cleavage in the exoplasmic domain near the transm embrane region. In the presence of cycloheximide all of the precursor H2 is converted to this intermediate, which is degraded only after cyc loheximide is removed (Wikstrom, L., and H. F. Lodish. 1991. J. Cell B iol. 113:997-1007). Here we have generated mutants of H2 that do not f orm the 35-kD fragment, either in transfected cells or during in vitro translation reactions in the presence of pancreatic microsomes. In tr ansfected cells the kinetics of ER degradation of these mutant protein s are indistinguishable from that of wild-type H2, indicating the exis tence of a second pathway of ER degradation which does not involve for mation of the 35-kD fragment. Degradation of H2 in the ER by this alte rative pathway is inhibited by TLCK or TPCK, but neither formation nor degradation of the 35-kD fragment is blocked by these reagents. As de termined by NH2-terminal sequencing of the 35-kD fragment, formed eith er in transfected cells or during in vitro translation reactions in th e presence of pancreatic microsomes, the putative cleavage sites are b etween small polar, uncharged amino acid residues. Substitution of the residues NH2- or COOH-terminal to the cleavage site by large hydropho bic or charged ones decreased the amount of 35-kD fragment formed and in some cases changed the putative cleavage site. Cleavage can also be affected by amino acid substitutions (e.g., to proline or glycine) wh ich change protein conformation. Therefore, the endoprotease that gene rates the 35-kD fragment has specificity similar to that of signal pep tidase. H2a and H2b are isoforms that differ only by a pentapeptide in sertion in the exoplasmic juxtamembrane region of H2a. 100% of H2a is degraded in the ER, but up to 30% of H2b folds properly and matures to the cell surface. The sites of cleavage to form the 35-kD fragment ar e slightly different in H2a and H2b. Two mutant H2b proteins, with eit her a glycine or proline substitution at the position of insertion of the pentapeptide in H2a, have metabolic fates similar to that of H2a. These mutations are likely to change the protein conformation in this region. Thus the conformation of the juxtamembrane domain of the H2 pr otein is important in determining its metabolic fate within the ER.