Mutational analysis of yeast vacuolar H sup + -ATPase

Journal Article · Fri Mar 01 04:00:00 EST 1991 · Proceedings of the National Academy of Sciences of the United States of America; (United States)

DOI:https://doi.org/10.1073/pnas.88.5.1938· OSTI ID:6397627

Noumi, Takato; Beltran, C; Nelson, H; Nelson, N ^[1]

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1. Roche Research Center, Nutley, NJ (United States)

Yeast mutants in which genes encoding subunits of the vacuolar H{sup +} -ATPase were interrupted were assayed for their vacuolar ATPase and proton-uptake activities. The vacuoles from the mutants lacking subunits A (72 kDa), B (57 kDa), or c (proteolipid, 16 kDa) were completely inactive in these reactions. Immunological studies revealed that in the absence of each one of those subunits the catalytic sector was not assembled. Labeling with N,N{prime} -({sup 14}C)dicyclohexylcarbodiimide showed the presence of the proteolipid in vacuoles of mutants in which genes encoding subunits of the catalytic sectors were interrupted. No labeling was detected in the mutant in which the gene encoding the proteolipid was interrupted. The authors conclude that of all the ATPase subunits only the proteolipid is assembled independently and it serves as a template for the assembly of the other subunits. Site-specific mutations were generated in the gene encoding the proteolipid. All of the drastic changes and replacements gave inactive proteins. About half of the single amino acid replacements gave active proteins. Replacing glutamic acid-137 by any of several amino acids, except for aspartic acid, abolished the activity of the enzyme. Other amino acids that may function in proton conductance were changed. It was found that glycine residues may replace amino acids with exchangeable protons.

• OSTI ID: 6397627
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (United States), Journal Name: Proceedings of the National Academy of Sciences of the United States of America; (United States) Vol. 88:5; ISSN 0027-8424; ISSN PNASA
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AMINO ACIDS
ASPARTIC ACID
ATP-ASE
BACTERIA
CARBOXYLIC ACIDS
CELL CONSTITUENTS
CELL MEMBRANES
ELECTROPHORESIS
ENZYMES
ESCHERICHIA COLI
EUMYCOTA
FUNGI
GENE REGULATION
GLUTAMIC ACID
HYDROLASES
IMMUNOLOGY
LIPIDS
LIPOPROTEINS
MEMBRANES
MICROORGANISMS
MOLECULAR STRUCTURE
MUTAGENESIS
ORGANIC ACIDS
ORGANIC COMPOUNDS
PHOSPHOHYDROLASES
PLANTS
PLASMIDS
PROTEINS
YEASTS