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

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)     

Up-regulation of ribophorin I after yellow head virus (YHV) challenge in black tiger shrimp Penaeus monodon

This work constitutes the second report from a continuing investigation of shrimp genes that may be involved in apoptosis associated death resulting from yellow head virus (YHV) infection. Here, we describe from the black tiger shrimp Penaeus monodon, a ribophorin I-like gene that is probably a subunit of the oligosaccharyltransferase complex (OST), a key enzyme in N-linked glycosylation that occurs in the endoplasmic reticulum. The OST complex also contains DAD1 (defender against apoptotic death 1) that has been reported to control apoptosis and that we have previously reported from P. monodon. The full length ribophorin I of P. monodon comprised 2157 bp with the ORF of 1806 bp corresponding to 601 deduced amino acids and three putative N-linked glycosylation sites. Analysis revealed hydrophobic properties implying that it could be a membrane protein. Tissue distribution analysis using real-time RT-PCR with SYBR Green revealed that ribophorin I was endogenously expressed in all examined tissues of normal shrimp. However, unlike DAD1 that was down-regulated after YHV challenge, ribophorin I expression was up-regulated and remained high until the moribund stage.     

Molecular cloning and functional expression of the gene for a cotton Delta-12 fatty acid desaturase (FAD2).

Two overlapping genomic clones spanning 16.5 kb of cotton DNA were found to encompass a Delta-12 fatty acid desaturase (FAD2-3) gene. A partial FAD2-3 cDNA clone was also analyzed. The FAD2-3 gene has one large intron of 2967 bp entirely within its 5'-untranslated region, only 12 bp upstream from the ATG initiation codon. Several potential promoter elements, including several light-responsive motifs, occur in the 5'-flanking region. The continuous FAD2-3 coding region is 1155 bp and would encode a protein of 384 amino acids. The polypeptide has four putative membrane-spanning helices, indicative of an integral membrane protein, and is most likely localized in the endoplasmic reticulum. Yeast cells transformed with a plasmid construct containing the cotton FAD2-3 coding region accumulate an appreciable amount of linoleic acid (18:2), not normally present in wild-type yeast cells, indicating that the gene encodes a functional FAD2 enzyme.     

Segregation of the polypeptide translocation apparatus to regions of the endoplasmic reticulum containing ribophorins and ribosomes. II. Rat liver microsomal subfractions contain equimolar amounts of ribophorins and ribosomes

Ribophorins I and II, two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum (ER) are thought to be part of the translocation apparatus for proteins made on membrane bound polysomes. To study the stoichiometry between ribophorins and membrane-bound ribosomes we have determined the RNA and ribophorin content in rat liver microsomes or in microsomal subfractions of different density (i.e., ribosome content). The specificity of antibodies against the ribophorins was demonstrated by Western blot analysis of rat liver rough microsomes separated by 2-dimensional gel electrophoresis. The ribophorin content of microsomal subfractions was determined by indirect immunoprecipitation and for ribophorin I by a radioimmune assay. In the latter assay a molar ratio of ribophorin I/ribosomes approaching one was calculated for total microsomes as well as in the gradient subfractions. We therefore suggest that ribophorins mediate the binding of ribosomes to endoplasmic reticulum membranes or play a role in co-translational process which depend on this binding, such as the insertion of nascent polypeptides into the membrane or their transfer into the cisternal lumen.     

Purification and functional characterization of membranes derived from the rough endoplasmic reticulum of Saccharomyces cerevisiae

Isolation and biochemical analysis of the components involved in protein translocation into the rough endoplasmic reticulum (ER) requires starting material highly enriched in membranes derived from this organelle. We have chosen to study the yeast Saccharomyces cerevisiae in order to profit from the ease of genetic manipulation. To date, however, no efficient scheme has been devised that allows the purification of functional rough ER-derived membranes from yeast, largely because proteins have yet to be identified that are rough ER-specific. In the experiments described here, we expressed the human rough ER marker ribophorin I to facilitate the analysis of subcellular fractionation. We found that the endoplasmic reticulum of yeast could be separated into two distinct domains by fractionation on continuous sucrose gradients. This procedure revealed a bimodal distribution of ER markers. The yeast homologue of the heavy chain-binding protein, BiP (encoded by the KAR2 gene), and the product of the SEC62 gene were present in two fractions having equilibrium densities of 1.146 and 1.192 g/ml, respectively. In contrast, our analysis showed that preprotein translocation activity and retention of the rough ER-specific protein ribophorin I were specific only to the membrane fraction with an equilibrium density of 1.192 g/ml. To prepare fractions highly enriched in translocation competent rough ER-derived membranes for analysis, we developed a density shift fractionation scheme that optimizes the purity of membranes containing human ribophorin I. Membranes obtained by this method were found to possess the majority of the appropriate functional markers, including ATP-independent preprotein binding, ribosome binding, and post-translational translocation. Mitochondria, the major contaminant of the 1.192 g/ml fraction, were significantly depleted in density-shifted membrane populations.     

Cloning and characterization of cDNA clones encoding membrane-bound and potentially secreted major histocompatibility class I receptors from walleye (Stizostedion vitreum)

Major histocompatibility (MH) gene polymorphism has been used to type populations of humans, mice, and fish. Walleye ( Stizostedion vitreum) comprise an economically important fishery in Lake Erie, but whether those in the western basin form a single population or separate shoal- and river-breeding populations is not known. To develop MH gene markers for use in defining their population structure, we constructed a head kidney cDNA library from which five full-length class I heavy-chain clones were isolated and sequenced. Although they came in roughly three sizes, 1300, 1400, and over 2000 bp, the clones all exhibited a high degree of sequence similarity to each other and to known teleost MH class I cDNAs in the area encoding the extracellular domains, but showed dramatic differences in their transmembrane and cytoplasmic domains. One clone had an AG repeat that eliminated the hydrophobicity of the transmembrane domain, indicating that it may encode a secreted class I receptor. The other four clones encode three distinctly different cytoplasmic domains. The two clones that encode the same cytoplasmic domain resemble those of the known teleost MH class I sequences the most. Southern blotting indicated that there were four copies of the gene present in the walleye genome. Northern blotting showed that class I MH genes are expressed in most tissues and mRNAs of all three size classes can be detected. A preliminary survey of the polymorphism of these genes indicates that they will provide useful markers for differentiating fish stocks.     

Organization of the human protein S genes

Human genomic clones that span the entire protein S expressed gene (PS alpha) and the 3' two-thirds of the protein S pseudogene (PS beta) have been isolated and characterized. The PS alpha gene is greater than 80 kilobases in length and contains 14 introns and 15 exons, as well as 6 repetitive "Alu" sequences. Exons I and XV contain 112 and 1139 bp 5' and 3' noncoding segments in addition to the amino and carboxyl termini, respectively. Exons I-VIII encode protein segments that are homologous to the vitamin K dependent clotting proteins and are bounded by introns whose position and type are identical with other members of this protein family. Exons IX-XV encode protein segments homologous to sex hormone binding globulin (SHBG) and are bounded by introns of identical type and position as in the SHBG gene. Genomic clones for the PS beta gene cover a distance of greater than 55 kilobases and contain segments corresponding to amino acids 46-635 of the mature protein and the 1.1-kb 3' noncoding region of the cDNA. The presence of multiple base changes in the coding portions of this gene, resulting in termination codons and frame shifts, suggests that it is a pseudogene. Comparison of DNA sequences for the two genes reveals 97% identity for coding and 3' noncoding, and 95.4% for intronic regions, suggesting divergence of the two genes is a relatively recent event.     

The 48-kDa subunit of the mammalian oligosaccharyltransferase complex is homologous to the essential yeast protein WBP1.

Oligosaccharyltransferase has been purified from canine microsomal membranes as a protein complex with three nonidentical subunits of 66, 63/64, and 48 kDa. The 66- and 63/64-kDa subunits were found to be identical to ribophorins I and II, respectively. The ribophorins are integral membrane glycoproteins that were previously shown to be localized exclusively to the rough endoplasmic reticulum. The 48-kDa subunit (OST48) of the oligosaccharyltransferase complex is not a glycoprotein and is not recognized by antibodies to either ribophorin. Here, we describe the characterization of a cDNA clone that encodes OST48. Like ribophorins I and II, OST48 was found to be an integral membrane protein, with the majority of the polypeptide located within the lumen of the endoplasmic reticulum. OST48 does not show significant amino acid sequence homology to either ribophorin I or II. A 45-kDa integral membrane protein, designated WBP1, from the yeast Saccharomyces cerevisiae was found to be 25% identical in sequence to OST48. Recently, WBP1 was shown to be essential for in vivo and in vitro expression of oligosaccharyltransferase activity in yeast. We conclude that OST48 and WBP1 are homologous gene products.     

C-terminal sequences can inhibit the insertion of membrane proteins into the endoplasmic reticulum of Saccharomyces cerevisiae.

We have constructed three gene fusions that encode portions of a membrane protein, arginine permease, fused to a reporter domain, the cytoplasmic enzyme histidinol dehydrogenase (HD), located at the C-terminal end. These fusion proteins contain at least one of the internal signal sequences of arginine permease. When the fusion proteins were expressed in Saccharomyces cerevisiae and inserted into the endoplasmic reticulum (ER), two of the fusion proteins placed HD on the luminal side of the ER membrane, but only when a piece of DNA encoding a spacer protein segment was inserted into the fusion joint. The third fusion protein, with or without the spacer included, placed HD on the cytoplasmic side of the membrane. These results suggest that (i) sequences C-terminal to the internal signal sequence can inhibit membrane insertion and (ii) HD requires a preceding spacer segment to be translocated across the ER membrane.     

Retention of subunits of the oligosaccharyltransferase complex in the endoplasmic reticulum

Membrane proteins of the endoplasmic reticulum (ER) may be localized to this organelle by mechanisms that involve retention, retrieval, or a combination of both. For luminal ER proteins, which contain a KDEL domain, and for type I transmembrane proteins carrying a dilysine motif, specific retrieval mechanisms have been identified. However, most ER membrane proteins do not contain easily identifiable retrieval motifs. ER localization information has been found in cytoplasmic, transmembrane, or luminal domains. In this study, we have identified ER localization domains within the three type I transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Together with DAD1, these membrane proteins form an oligomeric complex that has oligosaccharyltransferase (OST) activity. We have previously shown that ER retention information is independently contained within the transmembrane and the cytoplasmic domain of RII, and in the case of RI, a truncated form consisting of the luminal domain was retained in the ER. To determine whether other domains of RI carry additional retention information, we have generated chimeras by exchanging individual domains of the Tac antigen with the corresponding ones of RI. We demonstrate here that only the luminal domain of RI contains ER retention information. We also show that the dilysine motif in OST48 functions as an ER localization motif because OST48 in which the two lysine residues are replaced by serine (OST48ss) is no longer retained in the ER and is found instead also at the plasma membrane. OST48ss is, however, retained in the ER when coexpressed with RI, RII, or chimeras, which by themselves do not exit from the ER, indicating that they may form partial oligomeric complexes by interacting with the luminal domain of OST48. In the case of the Tac chimera containing only the luminal domain of RII, which by itself exits from the ER and is rapidly degraded, it is retained in the ER and becomes stabilized when coexpressed with OST48.     

Analysis of structural signals conferring localisation of pig OST48 to the endoplasmic reticulum.

Pig liver oligosaccharyltransferase (OST) is a heterooligomeric protein complex responsible for the co-translational transfer of GlcNAc2-Man9-Glc3 from Dol-PP onto specific asparagine residues in the nascent polypeptide. OST48, one of the catalytic subunits in this complex, exerts a typical type I membrane topology, containing a large luminal domain, a hydrophobic transmembrane domain and a short cytosolic peptide tail. Because OST48 is found within the endoplasmic reticulum (ER) when overexpressed in COS-1 cells, we carried out experiments to identify structural signals potentially capable of directing ER-targeting, using OST48 mutants and hybrid proteins consisting of individual OST48 domains and Man9-mannosidase. Immunofluorescence microscopy showed that OST48 mutants in which the C-terminal lysine-3 or lysine-5, but not lysine-7, had been replaced by leucine (OST48AK) could be detected on the cell surface. This indicates that these two lysine residues are sufficient for conferring ER-residency on OST48. The double-lysine motif operates only when exposed cytosolically, where it acts as a relocation signal rather than causing retention. OST48AK-3, when co-expressed in COS-1 cells together with myc-tagged ribophorin 1, was quantitatively retained in the ER. By contrast, co-expression in the presence of ribophorin I resulted in no reduction of cell surface fluorescence for the OMOdeltaK-5 chimera containing the cytosolic and transmembrane domain of OST48 attached to the C-terminus of the Man9-mannosidase luminal domain. Thus ER-localisation of OST48 is probably brought about by complex formation with ribophorin I and this most likely involves the luminal domains of both proteins. Consequently, the double-lysine motif in the cytosolic domain of OST48 is unlikely to have a primary function except being involved in re-capture of molecules which have escaped from the ER.     

Antiribophorin antibodies inhibit the targeting to the ER membrane of ribosomes containing nascent secretory polypeptides

Polyclonal antibodies directed against ribophorins I and II, two membrane glycoproteins characteristic of the rough endoplasmic reticulum, inhibit the cotranslational translocation of a secretory protein growth hormone into the lumen of dog pancreas or rat liver microsomes. As expected, site-specific antibodies to epitopes located within the cytoplasmic domain of ribophorin I, but not antibodies to epitopes in the luminal domain of this protein, were effective in inhibiting translocation. Since monovalent Fab fragments were as inhibitory as intact IgG molecules, ribophorins must be closely associated with the translocation site and, therefore, are likely to function at some stage in the translocation process. In all cases, the antibodies that inhibited translocation also caused a significant reduction in total protein synthesis and treatments that neutralized their capacity to inhibit translocation also prevented their inhibitory effect on protein synthesis. This would be expected if the antibodies blocked the membrane-mediated relief of the SRP-induced arrest of polypeptide elongation. The antibodies were effective only when added before translocation was allowed to begin. In this case, they prevented the targeting of active ribosomes containing mRNA and nascent chains to the ER membrane. Thus, ribophorins must either directly participate in targeting or be so close to the targeting site that the antibodies sterically blocked this early phase of the translocation process.