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

A method for obtaining giant protoplasts of Escherichia coli (the spheropla st incubation (SI) method: Kuroda ct al. (Kuroda, T., Okuda, N., Saitoh, N. , Hiyama, T., Terasaki, Y., Anazawa, H., Hirata, A., Mos, T., Kusaka, I., T suchiya, T., and Yabe, I. (1998) J. Biol. Chem. 273, 16897-16904) was adapt ed to haploid cells of Saccharomyces cerevisiae. The yeast cell grew to bec ome as large as 20 mu m in diameter and to contain an oversized vacuole ins ide. A patch clamp technique in the whole cell/vacuole recording mode was a pplied for the vacuole isolated by osmotic shock. At zero membrane potentia l, ATP induced a strong current (as high as 100 pA; specific activity, 0.1 pA/mu m(2)) toward the inside of the vacuole. Bafilomycin A(1), a specific inhibitor of the V-type ATPase, strongly inhibited the activity (K-i = 10 n M). Complete inhibition at higher concentrations indicated that any other A TP-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(Delta vma1::TRP)). The K-m value for the ATP dose response of the current was 15 9 mu M 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 e stimated by measuring the V-type ATPase activity biochemically. This method can potentially be applied to any type of ion channel, ion pump, and ion t ransporter in S. cerevisiae, and can also be used to investigate gene funct ions in various organisms by using yeast cells as hosts for homologous and heterogeneous expression systems.