Human ribophorins I and II: the primary structure and membrane topology of two highly conserved rough endoplasmic reticulum-specific glycoproteins.

Abstract

Ribophorins I and II represent proteins that are postulated to be involved in ribosome binding. They are abundant, highly‐conserved glycoproteins located exclusively in the membranes of the rough endoplasmic reticulum. As the first step in the further characterization of the structure and function of these proteins, we have isolated and sequenced full‐length human cDNA clones encoding ribophorins I and II using probes derived from a human liver expression library cloned into pEX1. The authenticity of the clones was verified by overlaps in the protein sequence of N‐terminal and several internal fragments of canine pancreatic ribophorins I and II. The cDNA clones hybridize to mRNA species of 2.5 kb in length, and encode polypeptides of 68.5 and 69.3 kd, respectively. Primary sequence analysis, coupled with biochemical studies on the topology, indicates that both ribophorins are largely luminally disposed, spanning the membrane once and having 150 and 70 amino acid long cytoplasmically disposed C termini, respectively. Both are synthesized as precursors having cleavable signal sequences of 23 (ribophorin I) and 22 (ribophorin II) amino acids. The topology suggested by the primary structure has been confirmed biochemically using proteolytic enzymes and anti‐ribophorin antibodies. Proteolysis of intact microsomes with a variety of enzymes resulted in a reduction in the apparent mol. wt of ribophorin I that would correspond to a loss of its 150‐amino acid cytoplasmic tail. In the case of ribophorin II, it is completely resistant to such proteolysis which is consistent with its luminal disposition and fairly hydrophobic C terminus.(ABSTRACT TRUNCATED AT 250 WORDS)

Interactions among Subunits of the Oligosaccharyltransferase Complex

“…Likewise, it appears that RI and RII, which are glycosylated in the native state, but not in the yeast two-hybrid system, do not need to be glycosylated to interact with OST48. Evidence that N-glycosylation of RII may not be required for its proper oligomerization and functioning is provided by the fact that, in vivo, only about half of the molecules are glycosylated (6).…”

“…It was found that the OST forms an oligomeric complex that sediments in a sucrose gradient as a 10 S particle. This complex was composed of three integral membrane proteins, of which ribophorin I (RI) (5,6) and OST48 (7) are type I transmembrane proteins with most of their polypeptide chains facing the lumen of the ER (Fig. 1).…”

“…1). The third member of the complex, ribophorin II (RII), also has a large luminal domain, but the disposition of the transmembrane and cytoplasmic domains is less clear: although this region bears three hydrophobic domains (6,8), only the one most proximal to the N terminus is of sufficient length and hydrophobicity to function as a typical transmembrane domain and RII may, therefore, also have a type I disposition, like RI and OST48 (Fig. 1).…”

Protein translocation into proteoliposomes reconstituted from purified components of the endoplasmic reticulum membrane

“…Antibodies were newly made against the following peptides: a peptide of the α subunit of the SRP receptor corresponding to positions 137-150 in the amino acid sequence [23] plus a C-terminal cysteine (KKFEDSEKAKKPVRC), a peptide corresponding to the N-terminal 9 amino acids of Sec61β plus a C-terminal cysteine (PGPTPSGTNC), a peptide corresponding to the N-terminal 10 amino acids of RAMP4 plus a C-terminal cysteine (VAKQRIRMANC), a peptide corresponding to the C-terminal 10 amino acids of ribophorin I [5] plus an N-terminal cysteine (CTKIDHILDAL), and a peptide corresponding to the N-terminal 10 amino acids of canine p34 (unpublished data) plus a C-terminal cysteine (TKAGSKGGNLC). These peptides were coupled to keyhole limpet hemocyanin that had been activated with sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate (sulfo-SMCC).…”

Structure of the mammalian oligosaccharyl-transferase complex in the native ER protein translocon

“…The catalytic OST subunit is present in two paralogous forms, STT3A and B, joined by at least six accessory subunits of poorly understood function: ribophorin I (RibI), ribophorin II (RibII), OST48, DAD1, N33 or IAP, and OST4. Sequence analysis and proteinase protection assays 4 suggest that all OST subunits are integral membrane proteins and many possess substantial lumenal domains, adding up to nearly 200 kDa 2 ( Supplementary Fig. 1).…”