The translocation, folding, assembly and redox-dependent degradation of secretory and membrane proteins in semi-permeabilized mammalian cells (original) (raw)
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The Journal of Cell Biology, 1989
Salt-extracted microsomal membranes (K-RM) contain an activity that is capable of releasing the signal recognition particle (SRP)-mediated elongation arrest of the synthesis of secretory polypeptides (Walter, P., and G. Blobel, 1981, J. Cell Biol., 91:557-561). This arrestreleasing activity was shown to be a function of an integral microsomal membrane protein, termed the SRP receptor (Gilmore, R., P. Walter, and G. Blobel, 1982, J. Cell BioL, 95:470-477). We attempted to solubilize the arrest-releasing activity of the SRP receptor by mild protease digestion of K-RM using either trypsin or elastase. We found, however, that neither a trypsin, nor an elastase "solubilized" supernatant fraction exhibited the arrest-releasing activity. Only when either the trypsin-or elastase-derived supernatant fraction was combined with the trypsinized membrane fraction, which by itself was also inactive, was the arrest-releasing activity restored. Release of the elongation arrest was followed by the translocation of the secretory protein across the microsomal membrane and the removal of the signal peptide. Thus, although we have been unable to proteolytically sever the arrest-releasing activity from K-RM and thereby to uncouple the release of the elongation arrest from the process of chain translocation, we have been able to proteolytically dissect and reconstitute the arrest-releasing activity. Furthermore, we found that the arrest-releasing activity of the SRP receptor can be inactivated by alkylation of K-RM with N-ethylmaleimide.
A membrane component of the endoplasmic reticulum that may be essential for protein translocation
The Embo Journal, 1989
We have purified a glycosylated, membrane-spanning protein of relative molecular mass -34 000 (Mr -34 K) from canine microsomes that appears to be essential for protein translocation across the endoplasmic reticulum (ER) as shown by the inhibitory action of antibodies directed against it and of monovalent Fab-fragments produced from them. The ER membrane contains at least as many molecules of the 34 K membrane protein as bound ribosomes. The protein can be detected immunologically in tissues of various organisms, indicating an universal function.
The Journal of cell biology, 1985
Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL RNA. It was previously shown to promote the co-translational translocation of secretory proteins across the endoplasmic reticulum by (a) arresting the elongation of the presecretory nascent chain at a specific point, and (b) interacting with the SRP receptor, an integral membrane protein of the endoplasmic reticulum which is active in releasing the elongation arrest. Recently a procedure was designed by which the particle could be disassembled into its protein and RNA components. We have further separated the SRP proteins into four homogeneous fractions. When recombined with each other and with 7SL RNA, they formed fully active SRP. Particles missing specific proteins were assembled in the hope that some of these would retain some functional activity. SRP(-9/14), the particle lacking the 9-kD and 14-kD polypeptides, was fully active in promoting ...
Biochemical Society Transactions, 1984
Vectorial discharge of proteins at the endoplasmic reticulum The ER of metazoa has mechanisms that achieve vectorial translocation of secretory proteins into the ER lumen from ribosomes attached to the ER membrane, in the course of elongation (Adelman et al., 1973). A clue to why secretory (and also plasma-membrane and lysosomal) proteins are specifically made on ribosomes bound to membranes was found when it was discovered that the mRNA species for many of these proteins, when translated in cell-free systems, yield proteins that are slightly larger than their mature counterparts. The additional parts are transient N-terminal 'signal' sequences of between 15 and 30 residues in length (Milstein et al., 1972). During protein synthesis in intact cells, or when translation is performed in the presence of microsomal membranes, signals are generally cleaved off, either during translation or shortly after its completion, by an endoprotease (Mollay et al., 1982), confined to the lumenal side of the ER (Jackson & Blobel, 1977). Although not strictly homologous, signal sequences share common structural features (Devillers-Thiery et al., 1975 ; Austen, 1979). They contain stretches of between nine and 24 consecutive. uncharged, hydrophobic amino acids. A preponderance contain basic residues within a few residues of the initiating methionine, and a mixture of acidic and basic residues at the C-terminal end (Austen & Ridd, 1981). The sequence where signal peptidase cleaves to release the N-terminus (Y) of the mature protein can be expressed as-AX -BY where re B, which provides the carboxy of the cleaved peptide bond, is a small uncharged residue, generally glycine, alanine, serine, cysteine or threonine, and A is generally uncharged. X and Y may be any residue. Empirical methods of secondary structure prediction indicate that the central hydrophobic cores of signals prefer to adopt a repeating structure, a-helix more often than extended or /3-structure, and that /3-turns may often occur close to the cleavage sites (Austen, 1979; Garnier et al., 1980). Are protein translocators required? Interest in the role of microsomal proteins in the translocation process was stimulated by the signal hypothesis (Blobel & Dobberstein, 19756), which proposed that
Characterization of molecules involved in protein translocation using a specific antibody
The Journal of Cell Biology, 1982
The vectorial translocation of nascent proteins through the membrane of the rough endoplasmic reticulum has been shown to require a specific membrane-bound protein whose cytoplasmic domain can be proteolytically cleaved and isolated as an active peptide of mol wt 60,000 (Meyer and Dobberstein, 1980, J. Cell Biol . 87:503-508) . Rabbit antibodies raised against this peptide were used to further characterize the membrane-bound molecule. Immunoprecipitation of solubilized, radiolabeled rough microsomal proteins yielded a single polypeptide of mol wt 72,000, representing the membrane-bound protein from which the 60,000-mol wt peptide was proteolytically derived . The antibody could also be used to remove exclusively the 60,000-mol wt peptide, and thus the translocation activity, from elastase digests tested in a reconstituted system . Moreover, immunoprecipitation of elastase extracts alkylated with [t4 C] N-ethylmaleimide selected a single species of mol wt 60,000 .