Abstract
We have isolated the proteolipid subunit from the vacuolar ATPase of Neurospora crassa, using ion-exchange chromatography. We have also isolated several cDNA clones and the corresponding genomic DNA that encodes this subunit. The derived protein sequence indicates that the polypeptide is composed of 161 amino acid residues with an Mr of 16 328 kDa. The gene encoding the proteolipid, named vma-3, is unusual in several respects. It contains four introns and, unlike other fungal genes, has non-coding regions that are as large as the coding regions. The 3′ untranslated regions of the cDNAs were quite heterogeneous, with polyadenylation sites more than 300 by apart. Analysis of the mRNA indicates that two size classes of transcripts are produced, differing in the length of the 3′ untranslated region. Mapping of the vma-3 gene showed that it is closely linked, but not adjacent to, vma-1, the gene encoding the 67 kDa subunit of the vacuolar ATPase. This raises the possibility that in N. crassa some of the vacuolar ATPase genes may be clustered.
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Bowman BJ, Allen R, Wechser MA, Bowman EJ (1988) Isolation of genes encoding the Neurospora vacuolar ATPase. J Biol Chem 263:14002–14007
Bowman BJ, Dschida WJ, Harris T, Bowman EJ (1989) The vacuolar ATPase of Neurospora crassa contains an F1-like structure. J Biol Chem 264:15606–15612
Bowman BJ, Dschida WJ, Bowman EJ (1992a) Vacuolar ATPase of Neurospora crassa: electron microscopy, gene characterization, and gene inactivation/mutation. J Exp Biol 172:57–66
Bowman BJ, Vázquez-Laslop N, Bowman EJ (1992b) The vacuolar ATPase of Neurospora crassa. J Bioenerg Biomembr 24:361–370
Bowman EJ (1983) Comparison of the vacuolar ATPase of Neurospora crassa with the mitochondrial and plasma membrane ATPaes. J Biol Chem 258:15238–15244
Bowman EJ, Bowman BJ (1988) Purification of vacuolar membranes, mitochondria, and plasma membranes from Neurospora crassa and modes of discriminating among the different H+-ATPases. Methods Enzymol 157:562–573
Bowman EJ, Knock TE (1992) Structures of the genes encoding the α and β subunits of the Neurospora crassa mitochondrial ATP synthase. Gene 114:157–163
Bowman EJ, Tenney K, Bowman BJ (1988) Isolation of genes encoding the Neurospora crassa vacuolar ATPase. J Biol Chem 263:13994–14001
Callis J, Fromm M, Walbot V (1987) Introns increase gene expression in cultured maize cells. Genes Dev 1:1183–1200
Dow JAT, Goodwin SF, Kaiser K (1992) Analysis of the gene encoding a 16-kDa proteolipid subunit of the vacuolar H+-ATPase from Manduca sexta midgut and tubules. Gene 122:355–360
Dschida WJ, Bowman BJ (1992) Structure of the vacuolar ATPase from Neurospora crassa as determined by electron microscopy. J Biol Chem 267:18783–18789
Fillingame RH (1990) Molecular mechanics of ATP synthesis by F1Fo-type H+-transporting ATP synthase. In: Krulwich TA (ed) The Bacteria, vol XII, Bacterial Energetics. Academic Press, New York, pp 345–391
Forgac M (1989) Structure and function of vacuolar class of ATP-driven proton pumps. Physiol Rev 69:765–795
Gilbert W (1985) Genes-in-pieces revisited. Science 228:823–824
Gurr SJ, Unkles SE, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (ed) Gene Structure in Eukaryotic Microbes. IRL Press, Oxford, pp 93–139
Hasebe M, Hanada H, Moriyama Y, Maeda M, Futai M (1992) Vacuolar type H+-ATPase genes: presence of four genes including pseudogenes for the 16-kDa proteolipid subunit in the human genome. Biochem Biophys Res Commun 183:856–863
Kyte J, Doolittle RF (1982) A simple method for displaying the hydropathic character of a protein. J Mol Biol 157:105–132
Lai S, Watson JC, Hansen JN, Sze H (1991) Molecular cloning and sequencing of cDNAs encoding the proteolipid subunit of the vacuolar H+-ATPase from a higher plant. J Biol Chem 266:16078–16084
Mandel M, Moriyama Y, Hulmes JD, Pan Y-CE, Nelson H, Nelson N (1988) cDNA sequence encoding the 16-kDa proteolipid of chromaffin granules implies gene duplication in the evolution of H+-ATPases. Proc Natl Acad Sci USA 85:5521–5524
Matsudaira PT (1989) A Practical Guide to Protein and Peptide Purification for Microsequencing. Academic Press, New York
Metzenberg RL, Grotelueschen J (1992) Restriction polymorphism maps of Neurospora crassa: update Fungal Genet Newsletter 39:50–58
Nelson H, Nelson N (1989) The progenitor of ATP synthases was closely related to the current vacuolar H+-ATPase. FEBS Lett 247:147–153
Noumi T, Beltrán C, Nelson H, Nelson N (1991) Mutational analysis of yeast vacuolar H+-ATPase. Proc Natl Acad Sci USA 88:1938–1942
Oakley BR, Rinehart JE, Mitchell BL, Oakley CE, Carmona C, Gray GS, May GS (1987) Cloning of the riboB locus of Aspergillus nidulans. Gene 1:385–400
Perkins DD, Radford A, Newmeyer D, Björkman M (1982) Chromosomal loci of Neurospora crassa. Microbiol Rev 46:426–570
Pitkin J, Davis RH (1990) The genetics of polyamine synthesis in Neurospora crassa. Arch Biochem Biophys 278:386–391
Sanger F, Nicklen S, Oulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74:5463–5467
Schneider E, Altendorf K (1987) Bacterial adenosine 5′-triphosphate synthase (F1Fo): purification and reconstitution of Fo complexes and biochemical functional characterization of their subunits. Microbiol Rev 51:477–497
Sebald W, Graf T, Lukins HB (1979) The dicyclohexylcarbodiimide-binding protein for the mitochrondrial ATPase complex from Neurospora crassa and Saccharomyces cerevisiae. Eur J Biochem 93:587–599
Sista H, Bowman BJ (1992) Characterization of the ilv-2 gene from Neurospora crassa encoding alpha-keto-hydroxyacyl reductoisomerase. Gene 120:115–118
Sun S-Z, Xie X-S, Stone DK (1987) Isolation and reconstitution of the dicyclohexylcarbodiimide-sensitive protein pore of the clathrin-coated vesicle proton translocating complex. J Biol Chem 262:14790–14794
Sutton R, Apps DK (1981) Isolation of a DCCD-binding protein from bovine chromaffin-granule membranes. FEBS Lett 130:103–106
Umemoto N, Yoshihisa T, Hirata R, Anraku Y (1990) Roles of the VM A3 gene product, subunit c of the vacuolar membrane H+-ATPase on vacuolar acidification and protein transport. J Biol Chem 265:18447–18453
Viebrock A, Perz A, Sebald W (1982) The imported preprotein of the proteolipid subunit of the mitochondrial ATP synthase from Neurospora crassa. Molecular cloning and sequencing of the mRNA. EMBO J 1:565–571
Vollmer SJ, Yanofsky C (1986) Efficient cloning of genes in Neurospora crassa. Proc Natl Acad Sci USA 83:1–5
Zhang J, Myers M, Forgac M (1992) Characterization of the Vo domain of the coated vesicle (H+)-ATPase. J Biol Chem 267:9773–9778
Zimniak L, Dittrich P, Gogarten JP, Kibak H, Taiz L (1988) The cDNA sequence of the 69-kDa subunit of the carrot vacuolar H+-ATPase. J Biol Chem 263:9102–9112
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Communicated by C.A.M.J.J. van den Hondel
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Sista, H., Wechser, M.A. & Bowman, B.J. The proteolipid subunit of the Neurospora crassa vacuolar ATPase: isolation of the protein and the vma-3 gene. Molec. Gen. Genet. 243, 82–90 (1994). https://doi.org/10.1007/BF00283879
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DOI: https://doi.org/10.1007/BF00283879