Michael Hortsch

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)

Transfer of secretory proteins through the membrane of the endoplasmic reticulum.

Publisher Summary Secretory proteins are synthesized on ribosomes located in the cytoplasm and are released from the cell as a fairly homogeneous, highly concentrated population of molecules. Secretory proteins can be localized in the lumen of the endoplasmic reticulum (ER) very early in their existence. Translocation of secretory proteins from the cytoplasmic to the luminal side of the rough ER membrane is an essential feature of the transport of these molecules. The information for the association of the mRNAs encoding secretory proteins to the membrane is contained within secretory protein mRNAs itself. mRNA encoding a secretory protein contains the signal for its ultimate association with the membrane. All secretory protein mRNAs encode a transient N-terminal peptide. This short amino acid sequence provides a signal, which results in the formation of membrane-bound polysomes and, ultimately, the translocation across the membrane. This chapter presents the results of studies, which deal with various individual aspects of protein translocation. It also describes signal sequences, signal recognition particle, docking protein, ribosome binding, signal peptidase, and the cotranslational covalent modifications, which are carried out in the rough ER.

The human docking protein does not associate with the membrane of the rough endoplasmic reticulum via a signal or insertion sequence-mediated mechanism.

Docking protein (DP, or SRP receptor) is an essential component of the cellular machinery that mediates the targeting of nascent secretory and membrane proteins to the rough endoplasmic reticulum (ER). In this study we have investigated the nature of its own targeting to its site of function, the rough ER. Using an in vitro transcription-translation system we demonstrate that DP is not inserted into the membrane via a classical SRP/DP-mediated process (in contrast to human ribophorins), nor via hydrophobic insertion sequences (in contrast to cytochrome b5). Instead, we suggest that membrane assembly of DP is receptor-mediated; requiring the presence in the membrane of other proteins that mediate its targeting and insertion.