A simple and specific procedure to permeabilize the plasma membrane of Schizosaccharomyces pombe.
Cu(2+)-treatment is a useful technique in selectively permeabilizing the fungal plasma membrane. We describe herein a practical application with Schizosaccharomyces pombe. Incubation of cells with 0.5 mM CuCl(2) at 30 degrees C for 20 min induced efficient leakage of cytosolic constituents. The kinetic characteristics of the calcium and amino acid flux from Cu(2+)-treated S. pombe cells suggested that the Cu(2+) treatment permeabilized the plasma membrane without loss of vacuolar function. As a further application of the method, the amino acid contents of Cu(2+)-treated and untreated cells were also determined. The amino acid pool of Cu(2+)-treated wild-type cells was enriched in basic amino acids but not in acidic amino acids, as is characteristic of the vacuolar amino acid pool of fungi, including Saccharomyces cerevisiae and Neurosporra crassa. The amino acid pool of the S. pombe V-ATPase mutant vma1Delta was also successfully determined. We conclude that the vacuolar amino acid pool of S. pombe can be measured using Cu(2+)-treated cells. The method is simple, inexpensive, and rapid relative to the isolation of vacuolar vesicles, making it useful in estimating vacuolar pools and transport across the vacuolar membrane.
Chardwiriyapreecha
Soracom
S
Laboratory of Molecular Physiology and Genetics, Faculty of Agriculture, Ehime University, 3-5-7 Tarumi, Matsuyama 790-8566, Japan.
Hondo
Kana
K
Inada
Hiroko
H
Chahomchuen
Thippayarat
T
Sekito
Takayuki
T
Iwaki
Tomoko
T
Kakinuma
Yoshimi
Y
D000596:Amino Acids / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002463:Cell Membrane Permeability / Q000187:drug effects*; D003300:Copper / Q000494:pharmacology*; D007700:Kinetics; D012568:Schizosaccharomyces / Q000166:cytology* / Q000201:enzymology / Q000378:metabolism; D025262:Vacuolar Proton-Translocating ATPases / Q000378:metabolism
D000596:Amino Acids; D003300:Copper; D025262:Vacuolar Proton-Translocating ATPases; C029892:cupric chloride; D002118:Calcium