Background: Nhx1/Vps44 is proposed to be a Class E gene involved in formation of the multivesicul... more Background: Nhx1/Vps44 is proposed to be a Class E gene involved in formation of the multivesicular body (MVB). However, this hypothesis has not been tested. Results: Nhx1 is not required for cargo sorting or MVB formation and shows synthetic phenotypes with select ESCRT mutants. Conclusion: Nhx1 functions independently of the ESCRT pathway. Significance: Nhx1 may have a post-ESCRT role in endosome membrane fusion.
Here we are employing a new approach called the String Method with swarmsof-trajectories to study... more Here we are employing a new approach called the String Method with swarmsof-trajectories to study transition pathways for various zwitterionic and anionic molecules across both WT and mutant OmpF porins. Results of this work will therefore assist in the design of new antibiotics that are more effective in the treatment of bacterial infections. [Supported by NIH grant GM062342].
The complexity of intracellular compartments in eukaryotic cells probably evolved to provide dist... more The complexity of intracellular compartments in eukaryotic cells probably evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers, represented by 28 CHX genes in ...
Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transm... more Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v)) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v) (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pH(v) under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v) dysregulation in a neo1(ts) mutant restored viability whereas cholesterol accumulation in human NPC1(-/-) fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.
The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct envi... more The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in Arabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHX15-CHX20, in yeast and bacterial cells engineered to lack multiple cation-handling mechanisms. Of these, CHX16-CHX20 were implicated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17-CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K(+)- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX20 elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K(+) uptake systems, we provide evidence for K(+) ((86)Rb) transport mediated by CHX17 and CHX20. Finally, we show that CHX17 and CHX20 affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX20-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17-CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K(+) and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival.
PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to... more PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, GR (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca ...
PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to... more PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, GR (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca ...
Background: Nhx1/Vps44 is proposed to be a Class E gene involved in formation of the multivesicul... more Background: Nhx1/Vps44 is proposed to be a Class E gene involved in formation of the multivesicular body (MVB). However, this hypothesis has not been tested. Results: Nhx1 is not required for cargo sorting or MVB formation and shows synthetic phenotypes with select ESCRT mutants. Conclusion: Nhx1 functions independently of the ESCRT pathway. Significance: Nhx1 may have a post-ESCRT role in endosome membrane fusion.
Here we are employing a new approach called the String Method with swarmsof-trajectories to study... more Here we are employing a new approach called the String Method with swarmsof-trajectories to study transition pathways for various zwitterionic and anionic molecules across both WT and mutant OmpF porins. Results of this work will therefore assist in the design of new antibiotics that are more effective in the treatment of bacterial infections. [Supported by NIH grant GM062342].
The complexity of intracellular compartments in eukaryotic cells probably evolved to provide dist... more The complexity of intracellular compartments in eukaryotic cells probably evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers, represented by 28 CHX genes in ...
Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transm... more Protons, the smallest and most ubiquitous of ions, are central to physiological processes. Transmembrane proton gradients drive ATP synthesis, metabolite transport, receptor recycling and vesicle trafficking, while compartmental pH controls enzyme function. Despite this fundamental importance, the mechanisms underlying pH homeostasis are not entirely accounted for in any organelle or organism. We undertook a genome-wide survey of vacuole pH (pH(v)) in 4,606 single-gene deletion mutants of Saccharomyces cerevisiae under control, acid and alkali stress conditions to reveal the vacuolar pH-stat. Median pH(v) (5.27±0.13) was resistant to acid stress (5.28±0.14) but shifted significantly in response to alkali stress (5.83±0.13). Of 107 mutants that displayed aberrant pH(v) under more than one external pH condition, functional categories of transporters, membrane biogenesis and trafficking machinery were significantly enriched. Phospholipid flippases, encoded by the family of P4-type ATPases, emerged as pH regulators, as did the yeast ortholog of Niemann Pick Type C protein, implicated in sterol trafficking. An independent genetic screen revealed that correction of pH(v) dysregulation in a neo1(ts) mutant restored viability whereas cholesterol accumulation in human NPC1(-/-) fibroblasts diminished upon treatment with a proton ionophore. Furthermore, while it is established that lumenal pH affects trafficking, this study revealed a reciprocal link with many mutants defective in anterograde pathways being hyperacidic and retrograde pathway mutants with alkaline vacuoles. In these and other examples, pH perturbations emerge as a hitherto unrecognized phenotype that may contribute to the cellular basis of disease and offer potential therapeutic intervention through pH modulation.
The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct envi... more The complexity of intracellular compartments in eukaryotic cells evolved to provide distinct environments to regulate processes necessary for cell proliferation and survival. A large family of predicted cation/proton exchangers (CHX), represented by 28 genes in Arabidopsis thaliana, are associated with diverse endomembrane compartments and tissues in plants, although their roles are poorly understood. We expressed a phylogenetically related cluster of CHX genes, encoded by CHX15-CHX20, in yeast and bacterial cells engineered to lack multiple cation-handling mechanisms. Of these, CHX16-CHX20 were implicated in pH homeostasis because their expression rescued the alkaline pH-sensitive growth phenotype of the host yeast strain. A smaller subset, CHX17-CHX19, also conferred tolerance to hygromycin B. Further differences were observed in K(+)- and low pH-dependent growth phenotypes. Although CHX17 did not alter cytoplasmic or vacuolar pH in yeast, CHX20 elicited acidification and alkalization of the cytosol and vacuole, respectively. Using heterologous expression in Escherichia coli strains lacking K(+) uptake systems, we provide evidence for K(+) ((86)Rb) transport mediated by CHX17 and CHX20. Finally, we show that CHX17 and CHX20 affected protein sorting as measured by carboxypeptidase Y secretion in yeast mutants grown at alkaline pH. In plant cells, CHX20-RFP co-localized with an endoplasmic reticulum marker, whereas RFP-tagged CHX17-CHX19 co-localized with prevacuolar compartment and endosome markers. Together, these results suggest that in response to environmental cues, multiple CHX transporters differentially modulate K(+) and pH homeostasis of distinct intracellular compartments, which alter membrane trafficking events likely to be critical for adaptation and survival.
PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to... more PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, GR (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca ...
PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to... more PMR1, a P-type ATPase cloned from the yeast Saccharomyces cerevisiae, was previously localized to the Golgi, and shown to be required for normal secretory processes (Antebi, A., and Fink, GR (1992) Mol. Biol. Cell 3, 633-654). We provide biochemical evidence that PMR1 is a Ca ...
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Papers by Rajini Rao