Abstract
Pmc1p, the Ca(2+)-ATPase of budding yeast related to plasma membrane Ca(2+)-ATPases of animals, is transcriptionally up-regulated in response to signaling by the calmodulin-calcineurin-Tcn1p/Crz1p signaling pathway. Little is known about post-translational regulation of Pmc1p. In a genetic screen for potential negative regulators of Pmc1p, a vacuolar v-SNARE protein, Nyv1p, was recovered. Cells overproducing Nyv1p show decreased Ca(2+) tolerance and decreased accumulation of Ca(2+) in the vacuole, similar to pmc1 null mutants. Overexpression of Nyv1p had no such effects on pmc1 mutants, suggesting that Nyv1p may inhibit Pmc1p function. Overexpression of Nyv1p did not decrease Pmc1p levels but decreased the specific ATP-dependent Ca(2+) transport activity of Pmc1p in purified vacuoles by at least 2-fold. The effect of Nyv1p on Pmc1p function is likely to be direct because native immunoprecipitation experiments showed that Pmc1p coprecipitated with Nyv1p. Complexes between Nyv1p and its t-SNARE partner Vam3p were also isolated, but these complexes lacked Pmc1p. We conclude that Nyv1p can interact physically with Pmc1p and inhibit its Ca(2+) transport activity in the vacuole membrane. This is the first example of a Ca(2+)-ATPase regulation by a v-SNARE protein involved in membrane fusion reactions.
Highlights
Ca2ϩ plays an important role as a signaling molecule for cell growth
Extensive studies have revealed that all three Ca2ϩ transporters in yeast are regulated by a signaling network involving calcineurin, a protein phosphatase that becomes activated by binding Ca2ϩ and calmodulin upon elevation of [Ca2ϩ]c
Vacuoles isolated from the same strain but overexpressing NYV1 strain exhibited a Pmc1p- and ATP-dependent Ca2ϩ transport activity with apparent Km similar to the control; the maximal transport activity was diminished to ϳ43%
Summary
PMCA Naϩ/Ca2ϩ exchanger SPCA Synaptobrevin/VAMP Syntaxin SNAP25 Synaptobrevins Synaptobrevins VGCC, ␣1 subunit None detected portant under these and other conditions. No negative regulators of Pmc1p have been identified. We screened for negative regulators of Pmc1p in vivo and identified Nyv1p, a transmembrane v-SNARE protein in the vacuole membrane, as a likely candidate. We found no obvious role of Pmc1p or Nyv1p in vacuole morphology or fusion in vivo. Nyv1p bound to Pmc1p and inhibited its Ca2ϩ transport activity in vivo and in vitro. A new role of Nyv1p in yeast may be the regulation of Pmc1p activity and Ca2ϩ homeostasis
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