Studies on the Biosynthesis of Microsomal Membrane Proteins

Abstract

The site of synthesis and mechanism of insertion into membranes of several microsomal polypeptides was studied using translation system programmed in vitro with polysomes or with mRNA extracted from free and membrane-bound rat liver polysomes. Primary translation products of cytochrome bs, NADH : cytochrome b5 oxidoreductase, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase were isolated by specific immunoprecipitation and compared with the mature proteins. The following observations were made :1. While cytochrome b5 and NADH : cytochrome bs oxidoreductase are synthesized in free polysomes, NADPH : cytochrome P-450 oxidoreductase and epoxide hydrolase are made in membrane-bound polysomes.2. In all cases the radioactively labelled products synthesized in vitro comigrated with the purified native polypeptides. Since these proteins are not glycoproteins, it can be inferred that in vivo they do not undergo proteolytic cleavage during or after translation.3. Levels of translatable mRNA coding for NADPH : cytochrome P-450 oxidoreductase or epoxide hydrolase were increased by phenobarbital or by various carcinogens respectively. 4. A comparison of amino-terminal segments of the native epoxide hydrolase and that synthesized in vitro showed that this protein retains an amino-terminal polypeptide sequence which resembles the transient insertion signal found in presecretory proteins.5. The amino-terminal region of NADPH : cytochrome P-450 oxidoreductase contains 7 leucine residues in its first 15 amino acids and can, therefore, be presumed to be the hydrophobic portion which anchors this polypeptide to the membrane.Mechanisms which may account for the spatial disposition of the membrane proteins and for their selective accumulation in endoplasmic reticulum membranes are discussed.There is now considerable interest in the mechanisms which serve to ensure the specific incorporation of newly synthesized polypeptides into the different organellar membranes which characterize the eukaryotic cell. The endoplasmic reticulum plays an important role in the biogenesis of membranes by providing sites for cotranslational insertion of a variety of polypeptides, which are synthesized by membrane-bound ribosomes and are subsequently transferred to other subcellar compartments (cf. [l]).In the hepatocyte and in many other cell types the endoplasmic reticulum constitutes the most extensive membrane system of the cell and contains membrane-associated enzymes which play an important role in metabolic and biosynthetic processes affecting a variety of substrates. The biosynthesis of integral membrane proteins which are characteristic of the endoplasmic reticulum raises interesting questions concerning the incorporation of the polypeptides into the membranes and the means which allow for their retention in the endoplasmic reticulum by distinguishing between them and those to be transferred to other membrane systems [2].To study these questions we have selected several proteins of the hepatocyte endoplasmic reticulum membranes which...

Protein Translocation into Peroxisomes

“…As stated above, PMP50 is localized to the ER and the peroxisomes and is synthesized on membrane-bound polysomes lending credence to the suggestion that cotranslational insertion of a protein into the ER might lead eventually to residence of the protein in the peroxisome (9). Alternatively, post-translational insertion of membrane proteins such as cytochrome b 5 (44,45), cytochrome b 5 reductase (46), and the SNARE protein, synaptobrevin (47), into the ER membrane suggests that the post-translational insertion of certain PMPs into the ER membrane is plausible. Recent results also support a role for vesicles and vesicular fusion events in peroxisome biogenesis.…”

Nucleotide sequence of the Escherichia coli motB gene and site-limited incorporation of its product into the cytoplasmic membrane

“…Since (i) there is no consensus signal sequence at the N terminus, (ii) 10 of 27 of the N-terminal amino acids are charged, and (iii) it is known from in vitro translation studies that the MotB protein is not cleaved posttranslationally (7), it seems unlikely that the N terminus of MotB is translocated to the outside of the membrane. It is possible, however, that the highly hydrophobic region A is an "insertion signal" similar to the C-terminal end of cytochrome b (5,34) and would form the first of two segments spanning the membrane. A nearby region (indicated as B in Fig.…”

Multiple Forms of Plant Cytochromes P-450

“…The orientations of the anchor sequences are related to the mechanisms of insertion of the proteins in the membrane. Cyt b5 and its reductase are translated on cytoplasmic ribosomes and inserted after synthesis is complete, whereas Cyt P-450 and NADPH:Cyt P-450 reductase are cotranslationally inserted on membrane bound ribosomes (23).…”