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J Biol Chem, Vol. 274, Issue 49, 34903-34910, December 3, 1999

Patch Clamp Studies on V-type ATPase of Vacuolar Membrane of Haploid Saccharomyces cerevisiae
PREPARATION AND UTILIZATION OF A GIANT CELL CONTAINING A GIANT VACUOLE

Isamu Yabe, Ken-ichi Horiuchi^¶, Katsumi Nakahara^¶, Tetsuo Hiyama^¶, Tadae Yamanaka, Pi-Chao Wang, Kiyoshi Toda, Aiko Hirata, Yoshinori Ohsumi^**, Ryogo Hirata, Yasuhiro Anraku, and Iwao Kusaka

From the  Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, the ^¶ Department of Biochemistry and Molecular Biology, Saitama University, Urawa 338-8570, Japan, the  Department of Biology, Faculty of Science, The University of Tokyo, Tokyo 113-0033, Japan, the  Department of Polymer Engineering, National Institute of Materials and Chemical Research, 1-1 Higashi, Tsukuba, Ibaraki 305, Japan, and the ^** National Institute of Basic Biology, Okazaki 444-8585, Japan

A method for obtaining giant protoplasts of Escherichia coli (the spheroplast incubation (SI) method: Kuroda et al. (Kuroda, T., Okuda, N., Saitoh, N., Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mogi, T., Kusaka, I., Tsuchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapted to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to become as large as 20 µm in diameter and to contain an oversized vacuole inside. A patch clamp technique in the whole cell/vacuole recording mode was applied for the vacuole isolated by osmotic shock. At zero membrane potential, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/µm²) toward the inside of the vacuole. Bafilomycin A_1, a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K_i = 10 nM). Complete inhibition at higher concentrations indicated that any other ATP-driven transport systems were not expressed under the present incubation conditions. This current was not observed in the vacuoles prepared from a mutant that disrupted a catalytic subunit of the V-type ATPase (RH105(vma1::TRP)). The K_m value for the ATP dose response of the current was 159 µM and the H⁺/ATP ratio estimated from the reversible potential of the V-I curve was 3.5 ± 0.3. These values agreed well with those previously estimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion transporter in S. cerevisiae, and can also be used to investigate gene functions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.

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