Cargando…
The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate
Yeast vacuoles are acidified by the v-type H(+)-ATPase (V-ATPase) that is comprised of the membrane embedded V(O) complex and the soluble cytoplasmic V(1) complex. The assembly of the V(1)-V(O) holoenzyme on the vacuole is stabilized in part through interactions between the V(O) a-subunit ortholog V...
Autores principales: | , , , , , , , , , |
---|---|
Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
American Society for Biochemistry and Molecular Biology
2022
|
Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9706634/ https://www.ncbi.nlm.nih.gov/pubmed/36334632 http://dx.doi.org/10.1016/j.jbc.2022.102672 |
_version_ | 1784840548004134912 |
---|---|
author | Zhang, Chi Feng, Yilin Balutowski, Adam Miner, Gregory E. Rivera-Kohr, David A. Hrabak, Michael R. Sullivan, Katherine D. Guo, Annie Calderin, Jorge D. Fratti, Rutilio A. |
author_facet | Zhang, Chi Feng, Yilin Balutowski, Adam Miner, Gregory E. Rivera-Kohr, David A. Hrabak, Michael R. Sullivan, Katherine D. Guo, Annie Calderin, Jorge D. Fratti, Rutilio A. |
author_sort | Zhang, Chi |
collection | PubMed |
description | Yeast vacuoles are acidified by the v-type H(+)-ATPase (V-ATPase) that is comprised of the membrane embedded V(O) complex and the soluble cytoplasmic V(1) complex. The assembly of the V(1)-V(O) holoenzyme on the vacuole is stabilized in part through interactions between the V(O) a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)). PI(3,5)P(2) also affects vacuolar Ca(2+) release through the channel Yvc1 and uptake through the Ca(2+) pump Pmc1. Here, we asked if H(+) and Ca(2+) transport activities were connected through PI(3,5)P(2). We found that overproduction of PI(3,5)P(2) by the hyperactive fab1(T2250A) mutant augmented vacuole acidification, whereas the kinase-inactive fab1(EEE) mutant attenuated the formation of a H(+) gradient. Separately, we tested the effects of excess Ca(2+) on vacuole acidification. Adding micromolar Ca(2+) blocked vacuole acidification, whereas chelating Ca(2+) accelerated acidification. The effect of adding Ca(2+) on acidification was eliminated when the Ca(2+)/H(+) antiporter Vcx1 was absent, indicating that the vacuolar H(+) gradient can collapse during Ca(2+) stress through Vcx1 activity. This, however, was independent of PI(3,5)P(2), suggesting that PI(3,5)P(2) plays a role in submicromolar Ca(2+) flux but not under Ca(2+) shock. To see if the link between Ca(2+) and H(+) transport was bidirectional, we examined Ca(2+) transport when vacuole acidification was inhibited. We found that Ca(2+) transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca(2+) transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P(2). |
format | Online Article Text |
id | pubmed-9706634 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2022 |
publisher | American Society for Biochemistry and Molecular Biology |
record_format | MEDLINE/PubMed |
spelling | pubmed-97066342022-11-30 The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate Zhang, Chi Feng, Yilin Balutowski, Adam Miner, Gregory E. Rivera-Kohr, David A. Hrabak, Michael R. Sullivan, Katherine D. Guo, Annie Calderin, Jorge D. Fratti, Rutilio A. J Biol Chem Research Article Yeast vacuoles are acidified by the v-type H(+)-ATPase (V-ATPase) that is comprised of the membrane embedded V(O) complex and the soluble cytoplasmic V(1) complex. The assembly of the V(1)-V(O) holoenzyme on the vacuole is stabilized in part through interactions between the V(O) a-subunit ortholog Vph1 and the lipid phosphatidylinositol 3,5-bisphosphate (PI(3,5)P(2)). PI(3,5)P(2) also affects vacuolar Ca(2+) release through the channel Yvc1 and uptake through the Ca(2+) pump Pmc1. Here, we asked if H(+) and Ca(2+) transport activities were connected through PI(3,5)P(2). We found that overproduction of PI(3,5)P(2) by the hyperactive fab1(T2250A) mutant augmented vacuole acidification, whereas the kinase-inactive fab1(EEE) mutant attenuated the formation of a H(+) gradient. Separately, we tested the effects of excess Ca(2+) on vacuole acidification. Adding micromolar Ca(2+) blocked vacuole acidification, whereas chelating Ca(2+) accelerated acidification. The effect of adding Ca(2+) on acidification was eliminated when the Ca(2+)/H(+) antiporter Vcx1 was absent, indicating that the vacuolar H(+) gradient can collapse during Ca(2+) stress through Vcx1 activity. This, however, was independent of PI(3,5)P(2), suggesting that PI(3,5)P(2) plays a role in submicromolar Ca(2+) flux but not under Ca(2+) shock. To see if the link between Ca(2+) and H(+) transport was bidirectional, we examined Ca(2+) transport when vacuole acidification was inhibited. We found that Ca(2+) transport was inhibited by halting V-ATPase activity with Bafilomycin or neutralizing vacuolar pH with chloroquine. Together, these data show that Ca(2+) transport and V-ATPase efficacy are connected but not necessarily through PI(3,5)P(2). American Society for Biochemistry and Molecular Biology 2022-11-02 /pmc/articles/PMC9706634/ /pubmed/36334632 http://dx.doi.org/10.1016/j.jbc.2022.102672 Text en © 2022 The Authors https://creativecommons.org/licenses/by/4.0/This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Research Article Zhang, Chi Feng, Yilin Balutowski, Adam Miner, Gregory E. Rivera-Kohr, David A. Hrabak, Michael R. Sullivan, Katherine D. Guo, Annie Calderin, Jorge D. Fratti, Rutilio A. The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title | The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title_full | The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title_fullStr | The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title_full_unstemmed | The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title_short | The interdependent transport of yeast vacuole Ca(2+) and H(+) and the role of phosphatidylinositol 3,5-bisphosphate |
title_sort | interdependent transport of yeast vacuole ca(2+) and h(+) and the role of phosphatidylinositol 3,5-bisphosphate |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9706634/ https://www.ncbi.nlm.nih.gov/pubmed/36334632 http://dx.doi.org/10.1016/j.jbc.2022.102672 |
work_keys_str_mv | AT zhangchi theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT fengyilin theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT balutowskiadam theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT minergregorye theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT riverakohrdavida theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT hrabakmichaelr theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT sullivankatherined theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT guoannie theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT calderinjorged theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT frattirutilioa theinterdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT zhangchi interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT fengyilin interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT balutowskiadam interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT minergregorye interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT riverakohrdavida interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT hrabakmichaelr interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT sullivankatherined interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT guoannie interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT calderinjorged interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate AT frattirutilioa interdependenttransportofyeastvacuoleca2andhandtheroleofphosphatidylinositol35bisphosphate |