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. 2012 Mar 20;7(3):e33270. doi: 10.1371/journal.pone.0033270

Figure 4. MoTSC13 is functionally equivalent to S. cerevisiae TSC13, while MoVAC8 only partially substitutes for VAC8.

Figure 4

(A) MoTSC13 complemented the temperature-sensitive lethality of the yeast strain TDY2058 (double mutant tsc13-1 Δelo). Yeast tsc13-1 Δelo mutants were transformed either with empty vector or a plasmid expressing MoTSC13 cDNA under control of the galactose-inducible GAL1 promoter. Cells were grown in YPDA overnight, normalized to and subjected to 10-fold serial dilutions, spotted onto SD+Gal plates and incubated at 26°C, 30°C or 37°C for 3 days prior to photographing. The experiments were carried out in triplicate, examining two independent yeast transformants. (B) MoVAC8 complemented the caffeine sensitivity of yeast strain BY4741 vac8Δ::KANMX4. Yeast vac8Δ::KANMX4 mutants were transformed with empty vector or a plasmid expressing MoVAC8 cDNA under the control of the GAL1 promoter. Cells were grown in YPDA overnight, normalized and subjected to 10-fold serial dilutions, spotted onto SD+Gal plates containing either 0.05% or 0.1% caffeine, and incubated at 26°C for the indicated period of time. The experiments were carried out in triplicate, examining two independent yeast transformants. (C) MoVAC8 does not complement vacuole morphology and vacuole inheritance defects of yeast mutant vac8Δ::KANMX4. FM4–64 was used to stain cells of S. cerevisiae, vac8Δ::KANMX4 and vac8Δ::KANMX4 strain, expressing MoVAC8 by pulse-chase labelling. Arrows indicates presence (b) or absence (d, f, h, j) of segregating vacuoles. (D) MoVAC8 failed to complement vacuole inheritance defects of yeast mutant vac8Δ::KANMX4 during budding. Pulse-chase labelled S. cerevisiae cells by FM4–64 were counted for the proportion of daughter cells carrying vacuoles inherited from the mother cell during budding. (N indicates the total number of cells counted). (E) MoTSC13p localises to the perinuclear and peripheral ER membrane in S. cerevisiae. yEGFP tagged MoTSC13p was expressed in yeast strain TDY2058 under control of the GAL1 promoter in plasmid pYES2. Cells were stained with DAPI to visualise nuclei. (F) MoVAC8p failed to accumulate at the vacuolar membrane in S. cerevisiae. yEGFP tagged MoVAC8p was expressed in yeast strain vac8Δ::KANMX4 under control of GAL1p promoter in plasmid pYES2.