Michelle E. Maxson, Y. M. Abbas, J. Wu, S. Grinstein, J. Rubinstein
{"title":"Detection and quantification of the vacuolar H+ATPase using the Legionella effector protein SidK","authors":"Michelle E. Maxson, Y. M. Abbas, J. Wu, S. Grinstein, J. Rubinstein","doi":"10.1101/2021.07.29.454369","DOIUrl":null,"url":null,"abstract":"Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with AlexaFluor-568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.","PeriodicalId":343306,"journal":{"name":"The Journal of Cell Biology","volume":"44 1","pages":"0"},"PeriodicalIF":0.0000,"publicationDate":"2021-07-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"10","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"The Journal of Cell Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/2021.07.29.454369","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 10
Abstract
Acidification of secretory and endocytic organelles is required for proper receptor recycling, membrane traffic, protein degradation, and solute transport. Proton-pumping vacuolar ATPases (V-ATPases) are responsible for this luminal acidification, which increases progressively as secretory and endocytic vesicles mature. An increasing density of V-ATPase complexes is thought to account for the gradual decrease in pH, but available reagents have not been sufficiently sensitive nor specific to test this hypothesis. We introduce a new probe to localize and quantify V-ATPases in eukaryotic cells. The probe is derived from SidK, a Legionella pneumophila effector protein that binds to the V-ATPase A subunit. We generated plasmids encoding fluorescent chimeras of SidK1-278, and labeled recombinant SidK1-278 with AlexaFluor-568 to visualize and quantify V-ATPases with high specificity in live and fixed cells, respectively. We show that V-ATPases are acquired progressively during phagosome maturation, that they distribute in discrete membrane subdomains, and that their density in lysosomes depends on the subcellular localization of the lysosome.