Abstract
Endosomal Na+/H+ exchangers are important for salt and osmotolerance, vacuolar pH regulation, and endosomal trafficking. We show that the C terminus of yeast Nhx1 interacts with Gyp6, a GTPase-activating protein for the Ypt/Rab family of GTPases, and that Gyp6 colocalizes with Nhx1 in the endosomal/prevacuolar compartment (PVC). The gyp6 null mutant exhibits novel phenotypes consistent with loss of negative regulation of Nhx1, including increased tolerance to hygromycin, increased vacuolar pH, and decreased plasma membrane potential. In contrast, overexpression of Gyp6 increases sensitivity to hygromycin, decreases vacuolar pH, and results in a slight missorting of vacuolar carboxypeptidase Y to the cell surface. We conclude that Gyp6 is a negative regulator of Nhx1-dependent trafficking out of the PVC. Taken together with its GTPase-activating protein-dependent role as a negative regulator of Ypt6-mediated retrograde traffic to the Golgi, we propose that Gyp6 coordinates upstream and downstream events in the PVC to Golgi pathway. Our findings provide a possible molecular link between intraendosomal pH and regulation of vesicular trafficking.
Publication types
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Base Sequence
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Biological Transport, Active
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Cation Transport Proteins / chemistry
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Cation Transport Proteins / genetics
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Cation Transport Proteins / metabolism*
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DNA, Fungal / genetics
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Endosomes / metabolism
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GTPase-Activating Proteins / genetics
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GTPase-Activating Proteins / metabolism*
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Genes, Fungal
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Hydrogen-Ion Concentration
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Hygromycin B / pharmacology
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Models, Biological
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Models, Molecular
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Mutation
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Protein Binding
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Recombinant Proteins / genetics
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Recombinant Proteins / metabolism
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Saccharomyces cerevisiae / drug effects
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Saccharomyces cerevisiae / genetics
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Saccharomyces cerevisiae / metabolism*
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Saccharomyces cerevisiae Proteins / chemistry
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Saccharomyces cerevisiae Proteins / genetics
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Saccharomyces cerevisiae Proteins / metabolism*
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Sequence Deletion
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Sodium-Hydrogen Exchangers / chemistry
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Sodium-Hydrogen Exchangers / genetics
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Sodium-Hydrogen Exchangers / metabolism*
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Two-Hybrid System Techniques
Substances
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Cation Transport Proteins
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DNA, Fungal
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GTPase-Activating Proteins
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GYP6 protein, S cerevisiae
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NHX1 protein, S cerevisiae
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Recombinant Proteins
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Saccharomyces cerevisiae Proteins
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Sodium-Hydrogen Exchangers
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Hygromycin B