The Na+/H+ exchanger Nhx1p regulates the initiation of Saccharomyces cerevisiae vacuole fusion.
Nhx1p is a Na(+)(K(+))/H(+) antiporter localized at the vacuolar membrane of the yeast Saccharomyces cerevisiae. Nhx1p regulates the acidification of cytosol and vacuole lumen, and is involved in membrane traffic from late endosomes to the vacuole. Deletion of the gene leads to aberrant vacuolar morphology and defective vacuolar protein sorting. These phenotypes are hallmarks of malfunctioning vacuole homeostasis and indicate that membrane fusion is probably altered. Here, we investigated the role of Nhx1p in the regulation of homotypic vacuole fusion. Vacuoles isolated from nhx1Δ yeast showed attenuated fusion. Assays configured to differentiate between the first round of fusion and ongoing rounds showed that nhx1Δ vacuoles were only defective in the first round of fusion, suggesting that Nhx1p regulates an early step in the pathway. Although fusion was impaired on nhx1Δ vacuoles, SNARE complex formation was indistinguishable from wild-type vacuoles. Fusion could be rescued by adding the soluble SNARE Vam7p. However, Vam7p only activated the first round of nhx1Δ vacuole fusion. Once fusion was initiated, nhx1Δ vacuoles appeared behave in a wild-type manner. Complementation studies showed that ion transport function was required for Nhx1p-mediated support of fusion. In addition, the weak base chloroquine restored nhx1Δ fusion to wild-type levels. Together, these data indicate that Nhx1p regulates the initiation of fusion by controlling vacuole lumen pH.
Qiu
Quan-Sheng
QS
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
Fratti
Rutilio A
RA
D002738:Chloroquine / Q000494:pharmacology; D005816:Genetic Complementation Test; D006863:Hydrogen-Ion Concentration; D017136:Ion Transport / Q000187:drug effects / Q000235:genetics; D008561:Membrane Fusion / Q000187:drug effects / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000502:physiology*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D017384:Sequence Deletion / Q000235:genetics; D017923:Sodium-Hydrogen Exchangers / Q000235:genetics / Q000378:metabolism*; D050825:Synaptosomal-Associated Protein 25 / Q000494:pharmacology; D014617:Vacuoles / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*
C492183:NHX1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D017923:Sodium-Hydrogen Exchangers; D050825:Synaptosomal-Associated Protein 25; C494205:VAM7 protein, S cerevisiae; D002738:Chloroquine