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Characterization of Schizosaccharomyces pombe mutants defective in vacuolar acidification and protein sorting

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Abstract

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.

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Acknowledgements

We are grateful to Dr. Yuko Giga-Hama for providing the S. pombe strain and to Dr. Taro Nakamura for providing the plasmids. This work was partly supported by the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R&D of New Industrial Science and Technology Frontiers by the Ministry of Economics, Trade and Industry (METI) and by a fellowship from the New Energy and Industrial Technology Development Organization (NEDO)

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  1. Department of Life Sciences, Faculty of Agriculture, Kagawa University, 761-0795, Miki-cho, Kagawa, Japan

    T. Iwaki, T. Goa, N. Tanaka & K. Takegawa

  2. Research Center, Asahi Glass Co. Ltd., 221-8755, Yokohama, Kanagawa, Japan

    T. Iwaki

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  1. T. Iwaki
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Iwaki, T., Goa, T., Tanaka, N. et al. Characterization of Schizosaccharomyces pombe mutants defective in vacuolar acidification and protein sorting. Mol Genet Genomics 271, 197–207 (2004). https://doi.org/10.1007/s00438-003-0971-7

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