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In situ study of K + transport into the vacuole of Saccharomyces cerevisiae
Author(s) -
MartínezMuñoz Gloria A.,
Peña Antonio
Publication year - 2005
Publication title -
yeast
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.923
H-Index - 102
eISSN - 1097-0061
pISSN - 0749-503X
DOI - 10.1002/yea.1238
Subject(s) - saccharomyces cerevisiae , biology , vacuole , in situ , saccharomyces , yeast , biophysics , microbiology and biotechnology , genetics , physics , cytoplasm , meteorology
Permeable spheroplasts were prepared from two strains of Saccharomyces cerevisiae by incubating with zymolyase without a permeabilizing agent. The loss of the plasma membrane barrier was confirmed by the nucleotide release, the activity of glucose 6‐phosphate dehydrogenase with external substrates and by the effects on respiration of mitochondrial substrates and ADP. Mitochondrial integrity was maintained, as shown by respiration with lactate, pyruvate, glucose and ethanol, and its acceleration by ADP showed a coupled respiration. Potassium uptake into the vacuole was measured with a selective electrode and found to be taken up effectively by spheroplasts only in the presence of Mg‐ATP; it was reverted by CCCP and PCP and inhibited by bafilomycin A 1 , but not by sodium vanadate or sodium azide. Potassium ions did not alter ΔΨ of the vacuole, followed with oxonol V, but caused vacuolar alkalinization, as followed with pyranine. The increase of vacuolar pH was non‐selective and observed at 50–200 m M of several monovalent cations. Isolated vacuoles with pyranine inside showed similar changes of the internal pH in the presence of KCl. Results indicate that some strains do not require a permeabilizing agent to directly access the vacuole in spheroplasts prepared with zymolyase. The hypothesis about the existence of a K + /H + antiporter in the vacuolar membrane of S. cerevisiae is discussed. Copyright © 2005 John Wiley & Sons, Ltd.