We have previously shown that mutations in buried charged residues in the last two transmembrane helices of Vph1p (the 100-kDa subunit of the yeast V-ATPase) inhibit proton transport and ATPase activity (Leng, X. H., Manolson, M., Liu, Q., and Forgac, M. (1996) J. Biol. Chem. 271, 22487-22493). In this report we have further explored the function of this region of Vph1p (residues 721-840) using a combination of site-directed and random mutagenesis. Effects of mutations on stability of Vph1p, assembly of the V-ATPase complex, 9-amino-6-chloro-2-methoxyacridine quenching (as a measure of proton transport), and ATPase activity were assessed. Additional mutations were analyzed to test the importance of Glu-789 in TM7 and His-743 in TM6. Although substitution of Asp for Glu at position 789 led to a 50% decrease in 9-amino-6-chloro-2-methoxyacridine quenching, substitution of Ala at this position gave a mutant with 40% quenching relative to wild type, suggesting that a negative charge at this position is not absolutely essential for proton transport. Similarly, a positive charge is not essential at position His-743, since the H743Y and H743A mutants retain 20 and 60% of wild-type quenching, respectively. Interestingly, H743A approaches wild-type ATPase activity at elevated pH while the E789D mutant shows a slightly lower pH optimum than wild type, suggesting that these residues are in a location to influence V-ATPase activity. The low pumping activity of the double mutant (E789H/H743E) suggests that these residues do not form a simple ion pair. Random mutagenesis identified a number of additional mutations both inside the membrane (L739S and L746S) as well as external to the membrane (H729R and V803D) which also significantly inhibited proton pumping and ATPase activity. By contrast, a cluster of five mutations were identified between residues 800 and 814 in the soluble segment just COOH-terminal to TM7 which affected either assembly or stability of the V-ATPase complex. Two mutations (F809L and G814D) may also affect targeting of the 100-kDa subunit. These results suggest that this segment of Vph1p plays a crucial role in organization of the V-ATPase complex.
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Evidence ID | Analyze ID | Gene/Complex | Systematic Name/Complex Accession | Qualifier | Gene Ontology Term ID | Gene Ontology Term | Aspect | Annotation Extension | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Phenotype | Experiment Type | Experiment Type Category | Mutant Information | Strain Background | Chemical | Details | Reference |
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Evidence ID | Analyze ID | Gene | Gene Systematic Name | Disease Ontology Term | Disease Ontology Term ID | Qualifier | Evidence | Method | Source | Assigned On | Reference |
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Evidence ID | Analyze ID | Regulator | Regulator Systematic Name | Target | Target Systematic Name | Direction | Regulation of | Happens During | Regulator Type | Direction | Regulation Of | Happens During | Method | Evidence | Strain Background | Reference |
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Site | Modification | Modifier | Source | Reference |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Allele | Assay | Annotation | Action | Phenotype | SGA score | P-value | Source | Reference | Note |
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Evidence ID | Analyze ID | Interactor | Interactor Systematic Name | Interactor | Interactor Systematic Name | Assay | Annotation | Action | Modification | Source | Reference | Note |
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Complement ID | Locus ID | Gene | Species | Gene ID | Strain background | Direction | Details | Source | Reference |
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Evidence ID | Analyze ID | Dataset | Description | Keywords | Number of Conditions | Reference |
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