Characterization of Schizosaccharomyces pombe mutants defective in vacuolar acidification and protein sorting.
The vacuolar H+-ATPases (V-ATPases) are ATP-dependent proton pumps responsible for acidification of intracellular compartments in eukaryotic cells. To investigate the functional roles of the V-ATPase in Schizosaccharomyces pombe, the gene vma1 encoding subunit A or vma3 encoding subunit c was disrupted. Both deletion mutants lost the capacity for vacuolar acidification in vivo, and showed sensitivity to neutral pH or high concentrations of divalent cations including Ca2+. The delivery of FM4-64 to the vacuolar membrane and accumulation of Lucifer Yellow CH were strongly inhibited in the vma1 and vma3 mutants. Moreover, deletion of the S. pombe vma1+ or vma3+ gene resulted in pleiotropic phenotypes consistent with lack of vacuolar acidification, including the missorting of vacuolar carboxypeptidase Y, abnormal vacuole morphology, and mating defects. These findings suggest that V-ATPase is essential for endocytosis, ion and pH homeostasis, and for intracellular targeting of vacuolar proteins and vacuolar biogenesis in S. pombe.
Iwaki
T
T
Department of Life Sciences, Faculty of Agriculture, Kagawa University, 761-0795 Miki-cho, Kagawa, Japan.
Goa
T
T
Tanaka
N
N
Takegawa
K
K
D043402:Cathepsin A / Q000378:metabolism; D017931:DNA Primers; D030541:Databases, Genetic; D004705:Endocytosis / Q000502:physiology; D005456:Fluorescent Dyes / Q000378:metabolism; D017353:Gene Deletion; D006863:Hydrogen-Ion Concentration; D015151:Immunoblotting; D007546:Isoquinolines / Q000378:metabolism; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics*; D010957:Plasmids / Q000235:genetics; D021381:Protein Transport; D011726:Pyridinium Compounds / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012568:Schizosaccharomyces / Q000201:enzymology / Q000235:genetics*; D017386:Sequence Homology, Amino Acid; D025262:Vacuolar Proton-Translocating ATPases / Q000235:genetics* / Q000502:physiology*; D014617:Vacuoles / Q000648:ultrastructure
D017931:DNA Primers; C092350:FM 4-64; D005456:Fluorescent Dyes; D007546:Isoquinolines; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; C017475:lucifer yellow; D043402:Cathepsin A; D025262:Vacuolar Proton-Translocating ATPases