A Simple and Specific Procedure to Permeabilize the Plasma Membrane ofSchizosaccharomyces pombe

Abstract

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.

Functional Expression and Characterization of Schizosaccharomyces pombe Avt3p as a Vacuolar Amino Acid Exporter in Saccharomyces cerevisiae

“…The S . pombe wild-type, avt3 Δ, and avt3 Δ/pTN54- avt3 + cells were cultured in MM medium without thiamine, and vacuolar amino acid pools were prepared using the cupric ion method [33]. As shown in Table 1, the vacuolar levels of threonine, serine, asparagine, glutamine, glycine, alanine, and proline, as well as basic amino acids (histidine, arginine, and lysine), were obviously increased in the avt3 Δ cells compared with the wild-type cells.…”

“…pombe and S . cerevisiae [33,34]. The amino acid contents in the fractions were analyzed with an amino acid analyzer (Hitachi L-8800).…”

Vacuolar transporter Avt4 is involved in excretion of basic amino acids from the vacuoles of Saccharomyces cerevisiae

“…1,2) Vacuolar accumulation of basic amino acids has been reported in several fungal species, [17][18][19] implicating that it is a common feature among fungi. Under nitrogen starvation, basic amino acids (Table 2) as well as other amino acids (data not shown) rapidly disappeared from vacuoles, suggesting the existence of export system(s).…”

Vba2p, A Vacuolar Membrane Protein Involved in Basic Amino Acid Transport in Schizosaccharomyces pombe

“…5) In the fission yeast Schizosaccharomyces pombe, there are also large amounts of basic amino acids compartmentalized in the vacuoles, and small but significant amounts of acidic amino acids are retained in the organelles. 6) Our reverse genetics study suggested that Fnx1p and Fnx2p of S. pombe, two Vba2p homologs, are involved in the vacuolar uptake of lysine and isoleucine. 7) Based on phylogenetic analysis of the S. cerevisiae and S. pombe genome databases, we found homologs of S. cerevisiae AVT members, Avt3p and Avt5p, in S. pombe.…”