ATP-Dependent Export of Neutral Amino Acids by Vacuolar Membrane Vesicles ofSaccharomyces cerevisiae

Abstract

Amino acid analysis of Saccharomyces cerevisiae cells indicated that neutral amino acids such as glycine and alanine were probably excluded from the vacuoles, and that vacuolar H þ -ATPase (V-ATPase) was involved in the vacuolar compartmentalization of these amino acids. We found that vacuolar membrane vesicles export neutral amino acids in an ATP-dependent manner. This is important in identifying vacuolar transporters for neutral amino acids.Key words: Saccharomyces cerevisiae; vacuole; amino acid transportVacuoles are the largest organelles in the budding yeast Saccharomyces cerevisiae, occupying about 25% of the cell volume. They serve as storage compartments for a variety of amino acids.1,2) A majority of the cellular basic amino acids are localized in the vacuoles. In contrast, glutamate, the most abundant amino acid in the cytosol, is almost entirely excluded from the vacuoles.1,2) While other amino acids are also compartmentalized in the vacuoles, their amounts are much lower than those of the basic amino acids. These differences in the concentration of free amino acids between vacuolar and cytosolic pools suggest the existence of active amino acid transport systems on the vacuolar membrane. ATP-dependent amino acid uptake, driven by the proton electrochemical gradient generated by the vacuolar H þ -ATPase (V-ATPase), has been observed in purified vacuolar membrane vesicles.3,4) A kinetic analysis of the vesicles suggested that several systems independently operate in this activity. 4)However, amino acid export from the vacuoles is not well characterized, because observation of export activity by the vesicles was limited to acidic amino acids. 5)Here we found that neutral amino acids are exported by vacuolar membrane vesicles in an ATP-dependent manner.The S. cerevisiae strains used in this study were X2180-1B (MAT SUC2 mal mel gal2 CUP1), BY4741 (MATa his3Á1 leu2Á0 met15Á0 ura3Á0), and mutants derived from these strains. Cells were cultured in YPD medium. For the analysis of cellular amino acid compositions, 10 OD 660 units of cells were harvested at early logarithmic phase and washed twice with AA buffer 6) (2.5 mM potassium phosphate buffer pH 6.0, 0.6 M sorbitol, and 10 mM glucose). To prepare a wholecell fraction, the washed cells were boiled in distilled water for 15 min at 20 OD 660 units of cells/mL. After centrifugation the supernatants were collected. To prepare a vacuolar fraction, the cupric ion treatment method was used.7) Washed cells were resuspended in AA buffer containing 0.2 mM CuCl 2 at 3.3 OD 660 units of cells/mL and incubated for 10 min at 30 C. Cell suspensions were collected by centrifugation and washed once with 3 mL of AA buffer. The cell pellets were resuspended in distilled water and boiled for 15 min. After centrifugation, the resulting supernatants were collected as the vacuolar fraction. The amino acid compositions of the fractions were measured with an automatic amino acid analyzer (Hitachi L-8800, Hitachi, Tokyo, Japan). Table 1 shows the amino acid compositions in the who...

Vacuolar amino acid transporters upregulated by exogenous proline and involved in cellular localization of proline in <i>Saccharomyces cerevisiae</i>

“…5 and 6), indicating a preference of Avt3 and Avt4 for amino acid substrates. Since the in vitro ATP-dependent activity of 14 C-labelled proline efflux at the vacuolar membrane has been already detected (Ishimoto et al, 2012), it should be further determined whether Avt3 is fully responsible for this activity. Our result does not conflict with a finding in Schizosaccharomyces pombe that the SpAvt3 protein, an ortholog of Avt3 and Avt4, mediates the vacuolar export of proline and other various amino acids (Chardwiriyapreecha et al, 2015).…”

“…More recently, Avt3, Avt4, and Avt7 were shown to mediate the efflux of proline under nitrogen starvation conditions (Sekito et al, 2014;Tone et al, 2015a). Although the proline import activity of the purified vacuolar membrane has not been detected (Ohsumi and Anraku, 1981;Sato et al, 1984;Tone et al, 2015b), the in vitro export of proline from the vacuole occurs in an ATP-dependent manner (Ishimoto et al, 2012). Atg22 mediates the efflux of leucine and other amino acids resulting from autophagic degradation, and also redundantly functions with Avt3 and Avt4 (Yang et al, 2006).…”

Functional Expression ofSchizosaccharomyces pombeVba2p in the Vacuolar Membrane ofSaccharomyces cerevisiae

“…The vacuolar fractions of the S. cerevisiae cells were prepared by the cupric ion treatment method 6) as described previously. 7) Cu 2þ treatment causes selective lesions of the permeability barrier of the plasma membrane, but does not affect that of the vacuolar membrane. Amino acid compositions were measured by means of an automatic amino acid analyzer (L-8900, Hitachi, Tokyo).…”

“…In S. cerevisiae cells, basic amino acids such as lysine, histidine, and arginine are accumulated in large quantities in the vacuole. 7) The vacuolar levels of these amino acids were slightly decreased in strain y To whom correspondence should be addressed. Tel: +81-89-946-9968; Fax: +81-89-946-9757; E-mail: mikawa@agr.ehime-u.ac.jp Abbreviations: CCCP, carbonylcyanide m-chlorophenylhydrazone; DIC, differential interference contrast; DMSO, dimethyl sulfoxide; GFP, green fluorescent protein; MES, 2-morpholinoethanesulfonic acid; MFS, major facilitator superfamily; NQO, 4-nitroquinoline N-oxide; ORF, open reading frame; PCR, polymerase chain reaction; V-ATPase, vacuolar type H þ -ATPase STY4109/p416GPD.…”

“…Amino acid compositions were measured by means of an automatic amino acid analyzer (L-8900, Hitachi, Tokyo). 7) Figure 2A shows the contents of lysine, histidine, and arginine in the vacuolar fraction of wild-type strain STY3807 transformed with p416GPD (STY3807/p416GPD), strain STY4109 transformed with p416GPD (STY4109/p416GPD), and strain STY4109 transformed with pGFP-VBA2 (STY4109/ pGFP-VBA2). In S. cerevisiae cells, basic amino acids such as lysine, histidine, and arginine are accumulated in large quantities in the vacuole.…”