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Abstract
To investigate the function of subunit D in the vacuolar H(+)-ATPase (V-ATPase) complex, random and site-directed mutagenesis was performed on the VMA8 gene encoding subunit D in yeast. Mutants were selected for the inability to grow at pH 7.5 but the ability to grow at pH 5.5. Mutations leading to reduced levels of subunit D in whole cell lysates were excluded from the analysis. Seven mutants were isolated that resulted in pH-dependent growth but that contained nearly wild-type levels of subunit D and nearly normal assembly of the V-ATPase as assayed by subunit A levels associated with isolated vacuoles. Each of these mutants contained 2-3 amino acid substitutions and resulted in loss of 60-100% of proton transport and 58-93% of concanamycin-sensitive ATPase activity. To identify the mutations responsible for the observed effects on activity, 14 single amino acid substitutions and 3 double amino acid substitutions were constructed by site-directed mutagenesis and analyzed as described above. Six of the single mutations and all three of the double mutations led to significant (>30%) loss of activity, with the mutations having the greatest effects on activity clustering in the regions Val(71)-Gly(80) and Lys(209)-Met(221). In addition, both M221V and the double mutant V71D/E220V led to significant uncoupling of proton transport and ATPase activity, whereas the double mutant G80D/K209E actually showed increased coupling efficiency. Both a mutant showing reduced coupling and a mutant with only 6% of wild-type proton transport activity showed normal dissociation of the V-ATPase complex in vivo in response to glucose deprivation. These results suggest that subunit D plays an important role in coupling of proton transport and ATP hydrolysis and that only low rates of turnover of the enzyme are required to support in vivo dissociation.
Highlights
Processes as receptor-mediated endocytosis, intracellular targeting of lysosomal enzymes, protein processing and degradation, viral entry, and coupled transport of small molecules, such as neurotransmitters [1,2,3,4,5,6,7]
Both M221V and the double mutant V71D/E220V led to significant uncoupling of proton transport and ATPase activity, whereas the double mutant G80D/K209E showed increased coupling efficiency. Both a mutant showing reduced coupling and a mutant with only 6% of wild-type proton transport activity showed normal dissociation of the V-ATPase complex in vivo in response to glucose deprivation. These results suggest that subunit D plays an important role in coupling of proton transport and ATP hydrolysis and that only low rates of turnover of the enzyme are required to support in vivo dissociation
Mutations have been identified in the ␥ subunit that lead to uncoupling of ATP hydrolysis and proton transport by the F-ATPases [31, 32]
Summary
V-ATPase, vacuolar proton-translocating adenosine triphosphatase; F-ATPase, F1F0-ATP synthase; ACMA, 9-amino-6-chloro-2-methoxyacridine; DSP, disuccinimidyl propionate; PBS, phosphate-buffered saline; YEP, yeast extract peptone; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; C12E9, polyoxyethylene-9-lauryl ether. Sequencing and analysis of mutants isolated by this procedure together with analysis of subsequent mutations constructed by site-directed mutagenesis have led us to suggest that subunit D plays a role in coupling of proton transport and ATP hydrolysis by the V-. We have further analyzed the activity and coupling requirements for in vivo dissociation of the VATPase, a process believed to play an important role in regulation of V-ATPase activity in the cell [39, 40]
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