Arg-735 of the 100-kDa subunit a of the yeast V-ATPase is essential for proton translocation.
The vacuolar (H(+))-ATPases (V-ATPases) are ATP-dependent proton pumps that acidify intracellular compartments and pump protons across specialized plasma membranes. Proton translocation occurs through the integral V(0) domain, which contains five different subunits (a, d, c, c', and c"). Proton transport is critically dependent on buried acidic residues present in three different proteolipid subunits (c, c', and c"). Mutations in the 100-kDa subunit a have also influenced activity, but none of these residues has proven to be required absolutely for proton transport. On the basis of previous observations on the F-ATPases, we have investigated the role of two highly conserved arginine residues present in the last two putative transmembrane segments of the yeast V-ATPase a subunit (Vph1p). Substitution of Asn, Glu, or Gln for Arg-735 in TM8 gives a V-ATPase that is fully assembled but is totally devoid of proton transport and ATPase activity. Replacement of Arg-735 by Lys gives a V-ATPase that, although completely inactive for proton transport, retains 24% of wild-type ATPase activity, suggesting a partial uncoupling of proton transport and ATP hydrolysis in this mutant. By contrast, nonconservative mutations of Arg-799 in TM9 lead to both defective assembly of the V-ATPase complex and decreases in activity of the assembled V-ATPase. These results suggest that Arg-735 is absolutely required for proton transport by the V-ATPases and is discussed in the context of a revised model of the topology of the 100-kDa subunit a.
Kawasaki-Nishi
S
S
Department of Physiology, Tufts University School of Medicine, Boston, MA 02111, USA.
Nishi
T
T
Forgac
M
M
D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D001692:Biological Transport; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011522:Protons; D013329:Structure-Activity Relationship; D025262:Vacuolar Proton-Translocating ATPases / Q000737:chemistry* / Q000502:physiology; D015003:Yeasts / Q000201:enzymology*
D021122:Protein Subunits; D011522:Protons; D000255:Adenosine Triphosphate; D025262:Vacuolar Proton-Translocating ATPases