Figure 6.

Tolerance of rho2Δ, pck2Δ, and pck1Δ strains to mok1⁺ overexpression. (A) Strains SKP103, TMC115, SKP170, and TMC116 (corresponding to wild-type, rho2Δ, pck2Δ, and pck1Δ cells carrying the integrated Pnmt1–mok1⁺ gene, respectively) were streaked on minimal medium in the presence (bottom plate) or absence (top plate) of thiamine and incubated at 28° for 3 d. (B) Mok1p levels in SKP103 (1, 4), TMC115 (2,5), and SKP170 (3,6) cells. Total cell extracts (5 μg) were prepared from cells grown for 14 h in medium with (1–3) or without (4—6) thiamine, run on 7.5% SDS-PAGE, and immunoblotted with anti-Mok1p antiserum and TAT1 anti-tubulin monoclonal antibody. (C) Cell morphologies and Calcofluor staining of the same S. pombe strains grown in EMM without thiamine for 14 h. Bar, 8 μm. (D) Morphologies of SKP103, TMC115, SKP170, and TMC116 cells grown in EMM with 5 μM thiamine for 48 h. Bar, 10 μm. (E) Cell wall composition of different S. pombe strains analyzed for ¹⁴C-glucose radioactivity incorporated into each cell wall polysaccharide. Strains HM123, SKP103, TMC115, and SKP170 were grown in the presence or absence of thiamine for 18 h, and ¹⁴C-glucose was added 2 h before the cells were harvested. Values are the means of three independent experiments with duplicated samples. SDs for the total carbohydrate values are shown.