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

Abstract

In the budding yeast Saccharomyces cerevisiae, the AVT genes (AVT1-7), which encode vacuolar amino acid transporters belonging to the amino acid vacuolar transport (AVT)-family, were significantly upregulated in response to exogenous proline. To reveal a novel role of the Avt proteins in proline homeostasis, we analyzed the effects of deletion or overexpression of the AVT genes on the subcellular distribution of amino acids after the addition of proline to the cells grown in minimal medium. Among seven AVT gene disruptants, avt1D and avt7D showed the lowest ratios of vacuolar proline. Consistently, overexpression of the AVT1 gene specifically enhanced the vacuolar localization of proline. Since double disruption of the AVT1 and AVT7 genes did not completely abrogate vacuolar accumulation of proline, it is presumed that Avt1 has a dominant role, and Avt7 and other Avt proteins have redundant functions, in the localization of proline into the vacuolar lumen. In contrast, deletion of the AVT3 gene increased vacuolar proline, although the highly expressed AVT3 gene interfered with the accumulation of proline in the vacuole. Based on these results, it appears that Avt3 is the major protein involved in the export of proline from the vacuole. We also observed vacuolar membrane localization of GFP-fused Avt1, Avt3, and Avt7 proteins. Taken together, our data suggest that the AVT genes induced by exogenous proline are involved in the bidirectional transport of proline across the vacuolar membrane.