The VPH1 gene encodes a 95-kDa integral membrane polypeptide required for in vivo assembly and activity of the yeast vacuolar H(+)-ATPase.

Yeast vacuolar acidification-defective (vph) mutants were identified using the pH-sensitive fluorescence of 6-carboxyfluorescein diacetate (Preston, R. A., Murphy, R. F., and Jones, E. W. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 7027-7031). Vacuoles purified from yeast bearing the vph1-1 mutation had no detectable bafilomycin-sensitive ATPase activity or ATP-dependent proton pumping. The peripherally bound nucleotide-binding subunits of the vacuolar H(+)-ATPase (60 and 69 kDa) were no longer associated with vacuolar membranes yet were present in wild type levels in yeast whole cell extracts. The VPH1 gene was cloned by complementation of the vph1-1 mutation and independently cloned by screening a lambda gt11 expression library with antibodies directed against a 95-kDa vacuolar integral membrane protein. Deletion disruption of the VPH1 gene revealed that the VPH1 gene is not essential for viability but is required for vacuolar H(+)-ATPase assembly and vacuolar acidification. VPH1 encodes a predicted polypeptide of 840 amino acid residues (molecular mass 95.6 kDa) and contains six putative membrane-spanning regions. Cell fractionation and immunodetection demonstrate that Vph1p is a vacuolar integral membrane protein that co-purifies with vacuolar H(+)-ATPase activity. Multiple sequence alignments show extensive homology over the entire lengths of the following four polypeptides: Vph1p, the 116-kDa polypeptide of the rat clathrin-coated vesicles/synaptic vesicle proton pump, the predicted polypeptide encoded by the yeast gene STV1 (Similar To VPH1, identified as an open reading frame next to the BUB2 gene), and the TJ6 mouse immune suppressor factor.     

Molecular structure of a gene, VMA1, encoding the catalytic subunit of H(+)-translocating adenosine triphosphatase from vacuolar membranes of Saccharomyces cerevisiae

Subunit a of the vacuolar membrane H(+)-translocating adenosine triphosphatase of the yeast Saccharomyces cerevisiae contains a catalytic site for ATP hydrolysis. N-terminal sequences of six tryptic peptides of the subunit were determined. Based on the peptide sequence information, a 39-base oligonucleotide probe was synthesized, and the gene encoding the subunit (VMA1) was isolated from a genomic DNA library by hybridization. The nucleotide sequence of the gene predicts a polypeptide of 1,071 amino acids with a calculated molecular mass of 118,635 daltons, which is much larger than the value 67 kDa estimated on sodium dodecyl sulfate-polyacrylamide gels. N- and C-terminal regions of the deduced sequence (residues 1-284 and 739-1,071) are very similar to those of the catalytic subunits of carrot (69 kDa) and Neurospora crassa (67 kDa) vacuolar membrane H(+)-ATPases (62 and 73% identity over 600 residues, respectively). The homologous regions also show about 25% sequence identity over 400 residues with beta-subunits of F0F1-ATPases. In contrast, the internal region containing 454 amino acid residues (residues 285-738) shows no detectable sequence similarities to any known ATPase subunits and instead is similar to a yeast endonuclease encoded by the HO gene. None of the six tryptic peptides is located in this internal region. Northern blotting analysis detected a single mRNA of 3.5 kilobases, indicating that the gene has no introns. Although the reason for the discrepancy in molecular mass is unclear at present, these results suggest that a novel processing mechanism, which might involve a post-translational excision of the internal region followed by peptide ligation, operates on the yeast VMA1 product. The VMA1 gene has proven to be the same gene as the TFP1 gene (Shih, C.-K., Wagner, R., Feinstein, S., Kanik-Ennulat, C., and Neff, N. (1988) Mol. Cell. Biol. 8, 3094-3103) whose dominant mutant allele (TFP1-408) confers a dominant trifluoperazine resistance and Ca2(+)-sensitive growth. This and our findings suggest that the vacuolar membrane H(+)-ATPase participates in maintenance of cytoplasmic Ca2+ homeostasis.     

Identification of a new gene in an operon for cellulose biosynthesis in Acetobacter xylinum

DNA sequencing of the region downstream of the cellulose synthase catalytic subunit gene of Acetobacter xylinum led to the identification of an open reading frame coding for a polypeptide of 86 kDa. The deduced amino acid sequence of this polypeptide matches from position 27 to 40 with the N-terminal amino acid sequence determined for a 93 kDa polypeptide that copurifies with the cellulose synthase catalytic subunit during purification of cellulose synthase. The cellulose synthase catalytic subunit gene and the gene encoding the 93 kDa polypeptide, along with other genes probably, are organized as an operon for cellulose biosynthesis in which the first gene is the catalytic subunit gene and the second gene codes for the 93 kDa polypeptide. The function of the 93 kDa polypeptide is not clear at present, however it appears to be tightly associated with the cellulose synthase catalytic subunit. Sequence analysis of the polypeptide shows that it is a membrane protein with a signal sequence at the N-terminal end and a transmembrane helix in the C-terminal region for anchoring it into the membrane.     

Physiological consequence of disruption of the VMA1 gene in the riboflavin overproducer Ashbya gossypii

The vacuolar ATPase subunit A structural gene VMA1 of the biotechnologically important riboflavin overproducer Ashbya gossypii was cloned and disrupted to prevent riboflavin retention in the vacuolar compartment and to redirect the riboflavin flux into the medium. Cloning was achieved by polymerase chain reaction using oligonucleotide primers derived form conserved sequences of the Vma1 proteins from yeast and filamentous fungi. The deduced polypeptide comprises 617 amino acids with a calculated molecular mass of 67.8 kDa. The deduced amino acid sequence is highly similar to that of the catalytic subunits of Saccharomyces cerevisiae (67 kDa), Candida tropicalis (67 kDa), and Neurospora crassa (67 kDa) with 89, 87, and 60% identity, respectively, and shows about 25% identity to the beta-subunit of the FoF1-ATPase of S. cerevisiae and Schizosaccharomyces pombe. In contrast to S. cerevisiae, however, where disruption of the VMA1 gene was conditionally lethal, and to N. crassa, where viable disruptants could not be isolated, disruption of the VMA1 gene in A. gossypii did not cause a lethal phenotype. Disruption of the AgVMA1 gene led to complete excretion of riboflavin into the medium instead of retention in the vacuolar compartment, as observed in the wild type.     

Isolation of genes encoding the Neurospora vacuolar ATPase. Analysis of vma-1 encoding the 67-kDa subunit reveals homology to other ATPases

The vacuolar membrane of Neurospora crassa contains a H+-translocating ATPase composed of at least three subunits with approximate molecular weights of 70,000, 60,000, and 15,000. Both genomic and cDNA clones encoding the largest subunit, which appears to contain the active site of the enzyme, have been isolated and sequenced. The gene for this subunit, designated vma-1, contains six small introns (60-131 base pairs) and encodes a hydrophilic protein of 607 amino acids, Mr 67,121. Within the sequence is a putative nucleotide-binding region, consistent with the proposal that this subunit contains the site of ATP hydrolysis. This 67-kDa polypeptide shows high homology (62% identical residues overall and 84% in the middle of the protein) to the analogous polypeptide of a higher plant vacuolar ATPase. The hypothesis that the vacuolar ATPase is related to F0F1 ATPases is strongly supported by the finding of considerable homology between the 67-kDa subunit of the Neurospora vacuolar ATPase and both the alpha and beta subunits of F0F1 ATPases.     

Molecular cloning of the gene encoding an outer-membrane-associated beta-N-acetylglucosaminidase involved in chitin degradation system of Alteromonas sp. strain O-7

The gene encoding beta-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial beta-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

Cloning and sequencing of the cellulose synthase catalytic subunit gene of Acetobacter xylinum

The gene for the catalytic subunit of cellulose synthase from Acetobacter xylinum has been cloned by using an oligonucleotide probe designed from the N-terminal amino acid sequence of the catalytic subunit (an 83 kDa polypeptide) of the cellulose synthase purified from trypsin-treated membranes of A. xylinum. The gene was located on a 9.5 kb HindIII fragment of A. xylinum DNA that was cloned in the plasmid pUC18. DNA sequencing of approximately 3 kb of the HindIII fragment led to the identification of an open reading frame of 2169 base pairs coding for a polypeptide of 80 kDa. Fifteen amino acids in the N-terminal region (positions 6 to 20) of the amino acid sequence, deduced from the DNA sequence, match with the N-terminal amino acid sequence obtained for the 83 kDa polypeptide, confirming that the DNA sequence cloned codes for the catalytic subunit of cellulose synthase which transfers glucose from UDP-glucose to the growing glucan chain. Trypsin treatment of membranes during purification of the 83 kDa polypeptide cleaved the first 5 amino acids at the N-terminal end of this polypeptide as observed from the deduced amino acid sequence, and also from sequencing of the 83 kDa polypeptide purified from membranes that were not treated with trypsin. Sequence analysis suggests that the cellulose synthase catalytic subunit is an integral membrane protein with 6 transmembrane segments. There is no signal sequence and it is postulated that the protein is anchored in the membrane at the N-terminal end by a single hydrophobic helix. Two potential N-glycosylation sites are predicted from the sequence analysis, and this is in agreement with the earlier observations that the 83 kDa polypeptide is a glycoprotein [13]. The cloned gene is conserved among a number of A. xylinum strains, as determined by Southern hybridization.     

Polysaccharide lyase: molecular cloning, sequencing, and overexpression of the xanthan lyase gene of Bacillus sp. strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520-2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4 degrees C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

A conserved gene encoding the 57-kDa subunit of the yeast vacuolar H+-ATPase

The peripheral (catalytic) sector of vacuolar H+-ATPases contains five different polypeptides denoted as subunits A-E in order of decreasing molecular masses from 72 to 33 kDa. The gene encoding subunit B (57 kDa) of yeast vacuolar H+-ATPase was cloned on a 5-kilobase pair genomic DNA fragment and sequenced. Four open reading frames were identified in the sequenced DNA. One of them encodes a protein of 504 amino acids with a calculated Mr of 56,557. Hydropathy plot revealed no apparent transmembrane segments. Southern analysis demonstrated that a single gene encodes this polypeptide in the yeast genome. The amino acid sequence exhibits extensive identity with the homologous protein from the plant Arabidopsis (77%). This polypeptide also contains regions of homology with the alpha subunits of H+-ATPases from mitochondria, chloroplasts, and bacteria. However, less similarity was detected when it was compared with the beta subunits of those enzymes. The implication of these phenomena on the evolution of proton pumps is discussed.     

Subunit composition of vacuolar membrane H(+)-ATPase from mung bean

The vacuolar H(+)-ATPase from mung bean hypocotyls was solubilized from the membrane with lysophosphatidycholine and purified by QAE-Toyopearl column chromatography. The purified ATPase was active only in the presence of exogenous phospholipid and was inhibited by nitrate, dicyclohexyl carbodiimide and Triton X-100, but not by vanadate or azide. Dodecyl sulfate/polyacrylamide gel electrophoresis of the purified ATPase yielded ten polypeptides of molecular masses of 68 kDa, 57 kDa, 44 kDa, 43 kDa, 38 kDa, 37 kDa 32 kDa, 16 kDa, 13 kDa and 12 kDa. All polypeptides remained in the peak activity fraction after glycerol density gradient centrifugation. Nine of them, excluding the 43-kDa polypeptide, comigrated in a polyacrylamide gradient gel in the presence of 0.1% Triton X-100. The 16-kDa polypeptide could be labeled with [14C]dicyclohexylcarbodiimide. The amino-terminal amino acid sequence of the isolated 68-kDa polypeptide generally agreed with that deduced from the cDNA for the carrot 69-kDa subunit [Zimniak, L., Dittrich, P., Gogarten, J. P., Kibak, H. & Taiz, L. (1988) J. Biol. Chem. 263, 9102-9112]. Thus, mung bean vacuolar H(+)-ATPase seems to consist of nine distinct subunits.     

Molecular Cloning of the Gene Encoding an Outer-Membrane-Associated β-N-Acetylglucosaminidase Involved in Chitin Degradation System of Alteromonas sp. Strain O-7

The gene encoding β-N-acetylglucosaminidase (GlcNAcaseA) was cloned using PCR with degenerate oligonucleotide primers from the partial amino acid sequence of the enzyme. The gene encoded a polypeptide of 863 amino acids with a predicted molecular mass of 97 kDa. A characteristic signal peptide, which was present at the amino-terminus of the precursor protein, contained four amino acids (Ala-Gly-Cys-Ser) identical in sequence and location to the processing and modification sites of the outer membrane lipoprotein of Escherichia coli, indicating that the mature GlcNAcaseA is a lipoprotein the N-terminal cysteine residue of which would be modified by the fatty acid that anchors the protein in the membrane. The predicted amino acid sequence of GlcNAcaseA showed similarity to bacterial β-N-acetylglucosaminidases belonging to the family 20 glycosyl hydrolases.     

The amino acid sequence of the 9 kDa polypeptide and partial amino acid sequence of the 20 kDa polypeptide of mitochondrial NADH:ubiquinone oxidoreductase.

Mitochondrial NADH:ubiquinone oxidoreductase (complex I) is the most complicated enzyme in the respiratory chain and is composed of at least 26 distinct polypeptides. Two hydrophilic subfractions of bovine heart complex I were systematically resolved into individual polypeptides by chromatography. Three polypeptides (51, 24, and 9 kDa) were isolated from the flavoprotein fraction (FP) of complex I, and the complete amino acid sequence of the 9 kDa polypeptide was determined. The 9 kDa polypeptide is composed of 75 amino acids with a molecular weight of 8,437. This protein exhibits no obvious sequence similarity to other proteins. The iron-sulfur protein fraction (IP) of complex I was separated into eight polypeptides, 75, 49, 30, 20, 18, 15, 13 kDa-A, and 13 kDa-B. The 20 kDa polypeptide was recognized as a novel component of IP for the first time. The N-terminal and several peptide sequences of the 20 kDa polypeptide were determined. Comparison of the sequences revealed significant sequence similarities of the 20 kDa polypeptide to the psbG gene products encoded in the chloroplast genome. The conserved sequence in these proteins was also found in the small subunit of the nickel-containing hydrogenases. These results suggest that complex I is related to other redox enzyme complexes.     

Roles of the VMA3 gene product, subunit c of the vacuolar membrane H(+)-ATPase on vacuolar acidification and protein transport. A study with VMA3-disrupted mutants of Saccharomyces cerevisiae

VMA3, a structure gene of the vacuolar membrane H(+)-ATPase subunit c of Saccharomyces cerevisiae, has been cloned and characterized. The VMA3 gene encodes a hydrophobic polypeptide with 160 amino acids as reported previously by Nelson and Nelson (Nelson, H., and Nelson, N. (1989) FEBS Lett. 247, 147-153). Peptide sequence analysis indicated that the VMA3 gene product lacks N-terminal methionine and does not have a cleavable signal sequence. To investigate functional and structural roles of the subunit c for vacuolar acidification and protein transport to the vacuole, haploid mutants with the disrupted VMA3 gene were constructed. The vma3 mutants can grow in nutrient-enriched medium, but they have completely lost the vacuolar membrane H(+)-ATPase activity and the ability of vacuolar acidification in vivo. The subunit c was found to be indispensable for the assembly of subunits a and b of the H(+)-ATPase complex. The disruption of the VMA3 gene causes yeast cells with considerable lesions in vacuolar biogenesis and protein transport to the vacuole and inhibits endocytosis of lucifer yellow CH completely.     

Primary structure and processing of the Candida tsukubaensis alpha-glucosidase. Homology with the rabbit intestinal sucrase-isomaltase complex and human lysosomal alpha-glucosidase.

The nucleotide sequence of a 4.39-kb DNA fragment encoding the alpha-glucosidase gene of Candida tsukubaensis is reported. The cloned gene contains a major open reading frame (ORF 1) which encodes the alpha-glucosidase as a single precursor polypeptide of 1070 amino acids with a predicted molecular mass of 119 kDa. N-terminal amino acid sequence analysis of the individual subunits of the purified enzyme, expressed in the recombinant host Saccharomyces cerevisiae, confirmed that the alpha-glucosidase precursor is proteolytically processed by removal of an N-terminal signal peptide to yield the two peptide subunits 1 and 2, of molecular masses 63-65 kDa and 50-52 kDa, respectively. Both subunits are secreted by the heterologous host S. cerevisiae in a glycosylated form. Coincident with its efficient expression in the heterologous host, the C. tsukubaensis alpha-glucosidase gene contains many of the canonical features of highly expressed S. cerevisiae genes. There is considerable sequence similarity between C. tsukubaensis alpha-glucosidase, the rabbit sucrase-isomaltase complex (proSI) and human lysosomal acid alpha-glucosidase. The cloned DNA fragment from C. tsukubaensis contains a second open reading frame (ORF 2) which has the capacity to encode a polypeptide of 170 amino acids. The function and identity of the polypeptide encoded by ORF 2 is not known.     

Polysaccharide Lyase: Molecular Cloning, Sequencing, and Overexpression of the Xanthan Lyase Gene of Bacillus sp. Strain GL1

When grown on xanthan as a carbon source, the bacterium Bacillus sp. strain GL1 produces extracellular xanthan lyase (75 kDa), catalyzing the first step of xanthan depolymerization (H. Nankai, W. Hashimoto, H. Miki, S. Kawai, and K. Murata, Appl. Environ. Microbiol. 65:2520–2526, 1999). A gene for the lyase was cloned, and its nucleotide sequence was determined. The gene contained an open reading frame consisting of 2,793 bp coding for a polypeptide with a molecular weight of 99,308. The polypeptide had a signal peptide (2 kDa) consisting of 25 amino acid residues preceding the N-terminal amino acid sequence of the enzyme and exhibited significant homology with hyaluronidase of Streptomyces griseus (identity score, 37.7%). Escherichia coli transformed with the gene without the signal peptide sequence showed a xanthan lyase activity and produced intracellularly a large amount of the enzyme (400 mg/liter of culture) with a molecular mass of 97 kDa. During storage at 4°C, the purified enzyme (97 kDa) from E. coli was converted to a low-molecular-mass (75-kDa) enzyme with properties closely similar to those of the enzyme (75 kDa) from Bacillus sp. strain GL1, specifically in optimum pH and temperature for activity, substrate specificity, and mode of action. Logarithmically growing cells of Bacillus sp. strain GL1 on the medium with xanthan were also found to secrete not only xanthan lyase (75 kDa) but also a 97-kDa protein with the same N-terminal amino acid sequence as that of xanthan lyase (75 kDa). These results suggest that, in Bacillus sp. strain GL1, xanthan lyase is first synthesized as a preproform (99 kDa), secreted as a precursor (97 kDa) by a signal peptide-dependent mechanism, and then processed into a mature form (75 kDa) through excision of a C-terminal protein fragment with a molecular mass of 22 kDa.     

A novel pathway of import of alpha-mannosidase, a marker enzyme of vacuolar membrane, in Saccharomyces cerevisiae

We have investigated the vacuolar delivery of alpha-mannosidase, a marker enzyme of the vacuolar membrane in the yeast Saccharomyces cerevisiae, and found that the enzyme has several unique characteristics in its biosynthesis and vacuolar delivery. alpha-Mannosidase has no typical signal sequence (Yoshihisa, T., and Anraku, Y. (1989) Biochem. Biophys. Res. Commun. 163, 908-915) but is located on the inner surface of the vacuolar membrane. The enzyme is synthesized as a 107-kDa polypeptide and converted to a 73-kDa polypeptide. Although the conversion depends on a vacuolar processing protease, proteinase A, it is much slower (t1/2 = 10 h) than the proteinase A-dependent processing of other vacuolar proteins. None of Asn-X-Thr/Ser sites on the 107-kDa alpha-mannosidase or on two alpha-mannosidase-invertase fusion proteins that are localized inside the vacuole receives N-linked oligosaccharide, whereas those sites on a carboxypeptidase Y-alpha-mannosidase fusion protein are N-glycosylated. The newly synthesized alpha-mannosidase is normally delivered to the vacuole and converted to the 73-kDa polypeptide even when the secretory pathway is blocked by a subset of sec mutations. These characteristics are different from those of other vacuolar proteins targeted to the vacuole via the secretory pathway. We conclude that alpha-mannosidase is delivered to the vacuole in a novel pathway separate from the secretory pathway.     

Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803

Summary Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803. Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic crossreactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved. Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein. The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen. Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed. These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.     

Analysis of a cDNA encoding arginine decarboxylase from oat reveals similarity to the Escherichia coli arginine decarboxylase and evidence of protein processing

Summary Arginine decarboxylase is the first enzyme in one of the two pathways of putrescine synthesis in plants. We purified arginine decarboxylase from oat leaves, obtained N-terminal amino acid sequence, and then used this information to isolate a cDNA encoding oat arginine decarboxylase. Comparison of the derived amino acid sequence with that of the arginine decarboxylase gene from Escherichia coli reveals several regions of sequence similarity which may play a role in enzyme function. The open reading frame (ORF) in the oat cDNA encodes a 66 kDa protein, but the arginine decarboxylase polypeptide that we purified has an apparent molecular weight of 24 kDa and is encoded in the carboxyl-terminal region of the ORF. A portion of the cDNA encoding this region was expressed in E. coli, and a polyclonal antibody was developed against the expressed polypeptide. The antibody detects 34 kDa and 24 kDa polypeptides on Western blots of oat leaf samples. Maturation of arginine decarboxylase in oats appears to include processing of a precursor protein.     

A vacuolar ATPase and pyrophosphatase in Acetabularia acetabulum.

Vacuole-rich fractions were isolated from Acetabularia acetabulum by Ficoll step gradient centrifugation. The tonoplast-rich vesicles showed ATP-dependent and pyrophosphate-dependent H(+)-transport activities. ATP-dependent H(+)-transport and ATPase activity were both inhibited by the addition of a specific inhibitor of vacuolar ATPase, bafilomycin B1. A 66 kDa polypeptide present in the preparation cross-reacted with the anti-IgG fractions against the alpha and beta subunits of Halobacterium halobium ATPase and with the antibody against the A subunit (68 kDa subunit) of mung bean vacuolar ATPase. A 56 kDa polypeptide present in the vacuole preparation showed cross-reactivity with the antibody against the B subunit (57 kDa) of mung bean vacuolar ATPase but not with the anti-beta subunit of H. halobium ATPase. A 73 kDa polypeptide cross-reacted with the antibody against inorganic pyrophosphatase of mung bean vacuoles. These results suggest that vacuolar membrane of A. acetabulum equipped energy transducing systems similar to those found in other plant vacuoles.     

Purification and complete sequence of a small proteolipid associated with the plasma membrane H(+)-ATPase of Saccharomyces cerevisiae.

The purified plasma membrane H(+)-ATPase of Schizosaccharomyces pombe and Saccharomyces cerevisiae display, in addition to the catalytic subunit of 100 kDa, a highly mobile component, soluble in chloroform/methanol. Chloroform/methanol extraction of S. cerevisiae plasma membranes led to isolation of a low molecular weight proteolipid identical to that present in purified H(+)-ATPase. NH2-terminal amino acid sequencing revealed a 38-residue polypeptide with a calculated molecular mass of 4250 Da. The polypeptide lacks the first two NH2-terminal amino acids as compared with the deduced sequence of the PMP1 gene (for plasma membrane proteolipid) isolated by hybridization with an oligonucleotide probe corresponding to an internal amino acid sequence of the proteolipid. The polypeptide is predicted to contain an NH2-terminal transmembrane segment followed by a very basic hydrophilic domain.