Biosynthesis of the plasma membrane H+-ATPase of Neurospora crassa.
The plasma membrane H+-ATPase of Neurospora is a 100-kDa integral membrane protein which appears, on the basis of hydropathy analysis of its amino acid sequence, to span the lipid bilayer at least eight times. To investigate the assembly and processing of the ATPase, a full-length cDNA has been constructed for use in in vitro transcription and translation experiments. Comparison of three different forms of the ATPase (nascent protein, nascent protein cotranslationally inserted into membranes, and mature protein) revealed no difference in electrophoretic mobility. Furthermore, the nascent and mature forms gave identical peptide patterns after partial proteolysis with Staphylococcus aureus V8 protease, suggesting that the ATPase does not contain an NH2-terminal signal peptide which is cleaved upon membrane insertion. Consistent with this interpretation, the NH2-terminal peptide has been purified from a tryptic digest of the ATPase and found to lack only the initiator methionine residue; the penultimate alanine is acetylated based on analysis by fast atom bombardment mass spectroscopy. Although the ATPase contains one potential site of N-linked glycosylation, its electrophoretic mobility was unchanged following digestion with endoglycosidase H and it did not incorporate [3H]mannose or bind concanavalin A. Thus, the Neurospora plasma membrane-ATPase appears to undergo minimal post-translational processing, and its membrane insertion is probably mediated by internal sequences.
D000595:Amino Acid Sequence; D001483:Base Sequence; D002462:Cell Membrane / Q000201:enzymology; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004271:DNA, Fungal / Q000235:genetics; D005796:Genes; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D009710:Nucleotide Mapping; D006180:Proton-Translocating ATPases / Q000096:biosynthesis / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics
D004271:DNA, Fungal; D012333:RNA, Messenger; D004262:DNA Restriction Enzymes; D006180:Proton-Translocating ATPases