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J. Biol. Chem., Vol. 278, Issue 15, 12985-12991, April 11, 2003
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From the Department of Physiology, Tufts University School of
Medicine, Boston Massachusetts 02111
Subunit A is the catalytic nucleotide binding
subunit of the vacuolar proton-translocating ATPase (or
V-ATPase) and is homologous to subunit
Mutational Analysis of the Non-homologous Region of
Subunit A of the Yeast V-ATPase*
,
of the
F1F0 ATP synthase (or F-ATPase). Amino
acid sequence alignment of these subunits reveals a 90-amino acid
insert in subunit A (termed the non-homologous region) that is absent from subunit . To investigate the functional role of this region, site-directed mutagenesis has been performed on the VMA1
gene that encodes subunit A in yeast. Substitutions were performed on
13 amino acid residues within this region that are conserved in all
available A subunit sequences. Most of the 18 mutations introduced
showed normal assembly of the V-ATPase. Of these, one (R219K) greatly
reduced both proton transport and ATPase activity. By contrast, the
P217V mutant showed significantly reduced ATPase activity but higher
than normal levels of proton transport, suggesting an increase in
coupling efficiency. Two other mutations in the same region (P223V and
P233V) showed decreased coupling efficiency, suggesting that changes in
the non-homologous region can alter coupling of proton transport and
ATP hydrolysis. It was previously shown that the V-ATPase must possess
at least 5-10% activity relative to wild type to undergo in
vivo dissociation in response to glucose withdrawal. However,
four of the mutations studied (G150A, D157E, P177V, and P223V) were
partially or completely blocked in dissociation despite having greater
than 30% of wild type levels of activity. These results suggest that
changes in the non-homologous region can also alter in vivo
dissociation of the V-ATPase independent of effects on activity.
*
This work was support in part by National Institutes of
Health Grant GM 34478 (to M. F.). E. coli strains were
provided through National Institutes of Health Grant DK34928.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Present address: Division of Biological Sciences, Institute of
Scientific and Industrial Research, Osaka University, Osaka 567-0047, Japan.
§
To whom correspondence should be addressed. Dept. of Physiology,
Tufts University School of Medicine, 136 Harrison Ave., Boston MA
02111. Tel.: 617-636-6939; Fax: 617-636-0445; E-mail:
michael.forgac@tufts.edu.
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