Roles Of V-ATPase Subunits In Autophagy In Saccharomyces Cerevisiae

Autophagy is a conserved intracellular degradation pathway,which promotes cell survival against stress by degrading autophagosome which contain proteins,damaged organelles and recycling nutrients.In higher eukaryotes,autophagy is involved in multiple physiological and pathological processes,such as development,immunity,programmed cell death,cancer and neurodegeneration and so on.V-ATPase is widespread on the eukaryotic cell membrane and organelle membrane from yeast to humans.It transports H+ into intracellular organelles or pumps out of the cell,thereby maintaining the pH of the cytoplasm and the acidity of some organelles.The functional V-ATPase relies on the hydrolysis of ATP to produce energy as the driving force against the concentration gradient.Stable intracellular environment is the necessity for the physiological activities of cells.V-ATPases are composed of 14 different subunits,just subunit "a" exists as two isoforms(Vphlp and Stvlp),Vphlp targets the pump to the vacuole while Stvlp targets it to the endosomal/Golgi subunit.In yeast,autophagosomes are degraded by the vacuolar hydrolase after they fuse with the vacuole.However,the hydrolase activity is dependent on the acidic environment maintained by the V-ATPase.Recently there have been reported that autophagy is related to V-ATPase complex or the expression levels of its subunits,but it is still unclear how the V-ATPase complex affects autophagy.In this study,we investigate the roles of V-ATPase subunits in autophagy.Two main results are showed below:1.The absence of subunits of V-ATPase affects autophagyThe localization and degradation of GFP-Atg8 and transport and maturation of prApel can be used to reflect the situation of autophagy in yeast.In the mutant with the absence of V-ATPase single subunit,some GFP-Atg8 cannot be transported into the vacuole and accumulated at the PAS,some other Atg8 transported into the vacuole cannot be degraded completely,especially in vph2??vma6? mutants.The maturation of Ape1 is also suppressed in the mutants with the absence of V-ATPase single subunit.Complemented with their wild type genes,autophagy defects in mutant cells were restored to be as in wild type.When two subunits of V-ATPase were absent at the same time,autophagy defects become severer than in the absence of V-ATPase single subunit,as shown by the degradation of GFP-Atg8 and the maturation of Ape1.In some mutations with the absence of V-ATPase single subunit,the transportation and localization of autophagy protein Atg9 was changed.Compared to the organell membrane localization of Atg9-3GFP peripheral to the vocuole in wild type under starvation,Atg9-GFP appears around the vacuolar membrane in vma6??vma3??vph2?and vmalOA mutants,which indicate that the absence of V-ATPase subunit affects the retrograde transportation of Atg9.When V-ATPase subunits were deleted from the ATG1 deletion mutant,Atg9-GFP appears as multiple dots,different from the sole dot at the PAS in ATG1 deletion cells.Except vph1? mutant cells,some Atg9-GFP dots cannot locate at the PAS in most V-ATPase mutant cells,especially in vma6A and vph2A mutant cells.V-ATPase mutants are sensitive to alkaline environment,but the sensitivity of the different mutants is not identical.We found that V-ATPase mutants' sensitivity to alkaline is proportional to the degradation of GFP-Atg8,so we speculate that the absence of the V-ATPase subunits affects autophagy by regulating the acidity of the vacuole.2.The absence of the "a" subunit of vacuole membrane and Golgi/endosomal membrane simultaneously affects autophagyVPH1 is the gene coding the "a" subunit of the vacuolar membrane,STV1 is the gene encoding the "a" subunit on the Golgi/endosomal membrane.The absence of either one will not make any effect to autophagy.When VPH1 and STV1 were deleted simultaneously,the degradation of GFP-Atg8 was blocked in the vph1?stv1? double mutant and autophagic bodies inside the vacuole cannot be degraded.In addition,Apel cannot be mature.When VPH1 and/or STV1 were complemented into the vph1?stvl? double mutants,the degradation of GFP-Atg8 and maturation of Ape1 were restored to the normal levels.In the vph1?stv1? double mutant,Atg9-3GFP forms dots close to the vacuolar,indicating the retrograde transportation of Atg9 may be affected.In the background of ATG1 deletion,the localization of Atg9-3GFP in vph1?stv1? mutant cells was seriously affected.Atg9 appears as multiple dots,and could not locate at the PAS properly.The vph1?stv1? double mutant was sensitive to alkaline.Single deletion of STV1 or VPH1 did not affect autophagy.But the doubel deletion of STV1 and VPH1 had a great influence on autophagy.We propose that the absence of the "a" subunit of V-ATPase made the functional V-ATPase decreased greatly in the vph1?stv1? double mutant so that the acidity of the vacuoles could not be maintained.Under this situation,autophagosomes could not be degraded by the vacuolar hydrolases to present the autophagy defect..Taken together,we showed that the absence of most V-ATPase subunits affects the normal autophagy in Saccharomyces cerevisiae.Although the single absence of Vphl and Stvl did not cause obvious autophagy,the simutaneous absence led to complete block of autophagy with accumulated autophagical bodies in the vacuole.This study provides a evidence for the roles of V-ATPase in autophagy.