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. 2011 Nov;77(21):7517–7525. doi: 10.1128/AEM.00404-11

Fig. 4.

Fig. 4.

A high copy number of ALY2 reduces the cold-induced downregulation of the tryptophan transporter Tat2p, which is important for cold growth. (A) YEplac181 (control, LEU2), YEp181ALY2 (ALY2), and YEp181UBP13 (UBP13) transformants of tat1Δ and tat2Δ mutants of the S. cerevisiae CEN.PK2-1C background were examined for growth at 30 or 12°C. YEplac181 transformants (control) of the wild-type (wt) strain were also spotted as a reference. Cells were pregrown in liquid SCD-Leu, and then the cultures were diluted and spotted onto solid medium as described for Fig. 1. (B) Cell lysates were subjected to SDS-PAGE and immunoblotted with anti-HA or anti-HxK2 antibody. Analysis was performed of CEN.PK2-1C derivative strains cotransformed with plasmids 3HA-TAT2c (URA3) and YEplac181 (control, LEU2), YEp181ALY2 (ALY2), or YEp181UBP13 (UBP13). Cells were pregrown at 30°C and protein extracts were treated as described for Fig. 3C. (C) The levels of the general amino acid permease Gap1p were analyzed in cells cotransformed with plasmid YCpJ25, which contains the GAL1-promoter dependent GFP-tagged GAP1 gene (18), and YEplac181 (Control, LEU2), YEp181ALY2 (ALY2), or YEp181UBP13 (UBP13). Cells were grown as described for Fig. 3A, except that the cold treatment was extended to a 24-h period. Preparation of protein extracts, SDS-PAGE separation, and visualization of Gap1-GFP and Hxk2p (loading control) were performed as described in Materials and Methods. In all cases, a representative experiment is shown.