In vitro biosynthesis and membrane insertion of gamma-glutamyl transpeptidase.
gamma-Glutamyl transpeptidase consists of two polypeptide chains anchored to the kidney brush-border membrane only through a short hydrophobic domain near the NH2-terminal end of the heavy subunit. The two subunits were reported to derive from a single polypeptide precursor by tissue labeling experiments. We have investigated the first steps of GGT biosynthesis and processing in a cell-free system. mRNA was prepared from kidney and enriched in specific sequences by a preparative gel electrophoresis. In vitro translation resulted in the synthesis of a single polypeptide (Mr = 63,000) specifically immunoprecipitated by antibodies raised against the mature dimeric enzyme. Incubation with microsomal membranes resulted in the appearance of a glycosylated form of the propeptide (Mr = 78,000). This latter form was cotranslationally segregated into microsomes and was sensitive to endoglycosidase H. Purified Escherichia coli leader peptidase did not process the primary gamma-glutamyl transpeptidase chain. This ectoprotein therefore appears to be inserted in the phospholipid bilayer without cleavage of a signal peptide, similar to most integral membrane proteins so far studied.
D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel; D007668:Kidney / Q000201:enzymology*; D046911:Macromolecular Substances; D008861:Microsomes / Q000201:enzymology; D008871:Microvilli / Q000201:enzymology*; D008970:Molecular Weight; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D012333:RNA, Messenger / Q000235:genetics / Q000302:isolation & purification; D011817:Rabbits; D051381:Rats; D012156:Reticulocytes / Q000378:metabolism; D005723:gamma-Glutamyltransferase / Q000235:genetics* / Q000302:isolation & purification
D046911:Macromolecular Substances; D012333:RNA, Messenger; D005723:gamma-Glutamyltransferase