A Family of Basic Amino Acid Transporters of the Vacuolar Membrane from Saccharomyces cerevisiae

Koichi Akiyama,Takayuki Sekito,Yoshimi Kakinuma,Masamitsu Shimazu,Yoshinori Ohsumi

doi:10.1074/jbc.m412617200

Abstract

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine. ATP-dependent uptake of histidine and lysine by isolated vacuolar membrane vesicles was impaired in YMR088c, a vacuolar basic amino acid transporter 1 (VBA1)-deleted strain, whereas uptake of tyrosine or calcium was little affected. This defect in histidine and lysine uptake was complemented fully by introducing the VBA1 gene and partially by a gene encoding Vba1p fused with green fluorescent protein, which was determined to localize exclusively to the vacuolar membrane. A defect in the uptake of histidine, lysine, or arginine was also observed in the vacuolar membrane vesicles of mutants YBR293w (VBA2) and YCL069w (VBA3). These three VBA genes are closely related phylogenetically and constitute a new family of basic amino acid transporters in the yeast vacuole.

Highlights

Among the members of the major facilitator superfamily of Saccharomyces cerevisiae, we identified genes involved in the transport into vacuoles of the basic amino acids histidine, lysine, and arginine
Amino Acid Uptake by S. cerevisiae Mutants—Based on the complete genome sequence of S. cerevisiae [16, 17], a computeraided analysis suggested the presence of the major facilitator superfamily (MFS) comprising permeases that typically contain 12 transmembrane-spanning domains
Active transport systems for amino acids operate in yeast vacuoles

Summary

EXPERIMENTAL PROCEDURES

Media, and Gene Manipulation—S. cerevisiae strains used in this study were YPH499 (MATa ura lys 801 ade101 trp1-⌬63 his3-⌬200 leu2-⌬1), its VMA1-deleted mutant RH104 (⌬vma1::TRP1) [8], ATCC 201388 (BY4741) (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0), and its major facilitator gene-deleted strains such as ATCC 4006223 (ymr088c::KanMX4), ATCC 4015741 (ybr293w::KanMX4), or ATCC 4003476 (ycl069w::KanMX4), all purchased from the American Type Culture Collection (ATCC, Manassas, VA). Homologous recombination in the identical promoter and terminator sequences between KanMX4 and NatMX4 results in nourseothricin-resistant but kanamycin-sensitive transformants (⌬ybr293w::NatMX4 and ⌬ycl069w::NatMX4), which were further transformed KanMX4 disruption cassette amplified by PCR for the second disruption as follows. The ⌬ybr293w::NatMX4 strain was the transformed with ⌬ymr088c::KanMX4 cassette amplified by PCR from genomic DNA of the ATCC 4006223 (⌬ymr088c::KanMX4) strain to obtain the ⌬ymr088c::KanMX4 ⌬ybr293w::NatMX4 strain. ⌬ymr088c::KanMX4 ⌬ycl069w::NatMX4 and ⌬ybr293w::KanMX4 ⌬ycl069w::NatMX4 strains were constructed by integrating ⌬ymr088c::KanMX4 and ⌬ybr293w::KanMX4 cassettes, respectively, into the ⌬ycl069w::NatMX4 strain. For triple null disruptant ⌬ymr088c::URA3MX4 ⌬ybr293w::KanMX4 ⌬ycl069w::NatMX4, the third gene, YBR293W, was disrupted by transforming the ⌬ybr293w::KanMX4 disruption cassette into the ⌬ymr088c::URA3MX4 ⌬yclo69w::NatMX4 strain constructed by a replacement KanMX4 with URA3MX4 in the ⌬ymr088c::KanMX4 ⌬yclo69w::NatMX4 strain by homologous recombination as described above.

A Family of Basic Amino Acid Transporters of Yeast Vacuole

RESULTS

DISCUSSION