FIGURE 6:

Sds23 and Ssp1 are required for elevated expression of Ght5 under low glucose. (A) Immunoblotting for Ght5-GFP in WT, Δsds23, and ssp1-837 (ssp1^ts) mutant strains. WT and mutant cells were cultivated in EMM2 containing either 111 or 4.4 mM glucose for 6 h at 33°C before sample preparation. Blots with anti-PSTAIR antibody are shown as the loading control. (B) Time course of ght5⁺ mRNA levels was examined in the Δsds23 and the ssp1^ts strains. Exponentially growing cells were transferred to fresh EMM2 containing 111 (filled rectangles) or 4.4 mM (open circles) glucose and cultivated at 33°C. mRNA levels of ght5⁺ relative to those of act1⁺ were measured by RT-qPCR at 2-h intervals. (C) Immunoblotting for Ght2- and Ght8-GFP in WT, Δsds23, and ssp1-837 (ssp1^ts) mutant strains. WT and mutant cells were cultivated in EMM2 containing either 111 or 4.4 mM glucose for 6 h at 33°C before sample preparation. Blots with anti-PSTAIR antibody are shown as the loading control. (D) Intracellular localization of Ght5 was examined in Δsds23 and ssp1^ts mutants, as well as in WT control. Cells harboring Ght5-GFP were cultivated at 33°C in a microfluidic perfusion chamber with a continuous supply of medium, and the glucose concentration was switched from 111 to 2.8 mM. GFP and brightfield (BF) microscopy images of the cells before medium switching and after 6-h cultivation in 2.8 mM glucose are shown. All GFP images here were processed under the same conditions. Bar, 10 μm. (E) Aliquots of 10⁴ cells of the WT, Δcbs2, and ts ssp1-837 mutant strains were serially diluted 10-fold, spotted onto YES medium plates containing the indicated concentrations of glucose, and incubated at 33°C for 3 d. See also Supplemental Figure S3.