Basic amino acids (lysine, histidine and arginine) accumulated in Saccharomyces cerevisiae vacuoles should be mobilized to cytosolic nitrogen metabolism under starvation. We found that the decrease of vacuolar basic amino acids in response to nitrogen starvation was impaired by the deletion of AVT4 gene encoding a vacuolar transporter. In addition, overexpression of AVT4 reduced the accumulation of basic amino acids in vacuoles under nutrient-rich condition. In contrast to AVT4, the deletion and overexpression of AVT3, which encodes the closest homologue of Avt4p, did not affect the contents of vacuolar basic amino acids. Consistent with these, arginine uptake into vacuolar membrane vesicles was decreased by Avt4p-, but not by Avt3p-overproduction, whereas various neutral amino acids were excreted from vacuolar membrane vesicles in a manner dependent on either Avt4p or Avt3p. These results suggest that Avt4p is a vacuolar amino acid exporter involving in the recycling of basic amino acids.

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...

-Tributyltin (TBT) has long been recognized as a major environmental pollutant that can cause significant damage to the cellular functions as well as disruption of endocrine homeostasis. TBT induces apoptosis accompanied by production of reactive oxygen species (ROS) in mammalian and yeast cells. We observed that the budding yeast cells exposed to this compound at low concentrations exhibited cell growth arrest, but not cell death. Flow cytometric analysis of yeast cells without synchronization and morphological assessment of cells synchronized at M phase by nocodazole treatment indicated that TBT-exposed Saccharomyces cerevisiae cells were arrested at G1 phase of the cell cycle. This arrest was recovered by the addition of N-acetylcysteine, suggesting the involvement of ROS production by TBT. This is the first study to evaluate the action of TBT on cell cycle events.

This study investigated the process that contributes to the decay of short-term motor memory regarding force reproduction. Participants performed tonic flexion of the right index finger with the target force feedback (criterion phase) and reproduced this force level without feedback 3, 10, 30, or 60 s after the end of the criterion phase (recall phase). The constant error for force reproduction was significantly greater than zero, indicating that information about the somatosensation and/or motor command in the criterion phase is positively biased. Constant and absolute errors were not influenced by the retention interval, indicating that neither bias nor error represents the decay of short-term motor memory over time. Variable error, defined as SD of bias (force in the recall phase minus that in the criterion phase), increased as the retention interval increased. This indicates that the decay of short-term motor memory is represented by the increase in inconsistency of memory bias among the trials. The correlation coefficient of the force between the criterion and recall phases with 3-s retention interval was greater than that with longer intervals. This is explained by the view that the contribution of the information of the practiced force to the force reproduction process is great within 3 s after the end of the practice, but the additional contribution of the noise information becomes greater after this time, causing lesser relative contribution of the information of the practiced force to the force reproduction process.

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