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 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 micrometer 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/micrometer(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 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(Deltavma1::TRP)). The K(m) value for the ATP dose response of the current was 159 microM 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.
Yabe
I
I
Institute of Molecular and Cellular Biosciences, The University of Tokyo, Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan. yabe@bio.t.u-tokyo.ac.jp
Horiuchi
K
K
Nakahara
K
K
Hiyama
T
T
Yamanaka
T
T
Wang
P C
PC
Toda
K
K
Hirata
A
A
Ohsumi
Y
Y
Hirata
R
R
Anraku
Y
Y
Kusaka
I
I
D000900:Anti-Bacterial Agents / Q000494:pharmacology; D000963:Antimetabolites / Q000378:metabolism; D003847:Deoxyglucose / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015726:Giant Cells / Q000166:cytology / Q000201:enzymology* / Q000648:ultrastructure; D006238:Haploidy; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007425:Intracellular Membranes / Q000201:enzymology*; D007477:Ions; D007700:Kinetics; D018942:Macrolides; D008564:Membrane Potentials / Q000187:drug effects; D008854:Microscopy, Electron; D008856:Microscopy, Fluorescence; D018408:Patch-Clamp Techniques; D006180:Proton-Translocating ATPases / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism / Q000648:ultrastructure; D013997:Time Factors; D025262:Vacuolar Proton-Translocating ATPases; D014617:Vacuoles / Q000201:enzymology*
D000900:Anti-Bacterial Agents; D000963:Antimetabolites; D004791:Enzyme Inhibitors; D007477:Ions; D018942:Macrolides; C040929:bafilomycin A1; D003847:Deoxyglucose; D025262:Vacuolar Proton-Translocating ATPases; D006180:Proton-Translocating ATPases