Figure 4. Mutations in sso2 inhibit cell growth and secretion of Bgl2 and invertase.

(A) Sequences of three sso2 mutants generated by site-directed mutagenesis. Residues in the first and/or the second NPY motif that were mutated to Ala (A) are shown in bold italics. (B) Sso2 mutants in an sso1Δ background partially inhibit cells growth at 37°C. Control sso1⊗ SSO2 or sso1Δ sso2 mutant cells were grown overnight in YPD medium. An aliquot (0.2 OD₆₀₀ units) of cells from each strain was collected, serially diluted by fivefold and spotted onto YPD plates. Plates were incubated at 25^oC, 34^oC, or 37^oC for 2 days. (C–D) The sso1Δ sso2 mutants were grown at 25^oC in YPD medium overnight to early log phase and shifted to 37^oC for 90 min. The internal and external fractions were prepared as described in Materials and methods. (C) The internal or external pools of Bgl2 were detected by western blotting. Several mutations in SSO2 caused inhibition of Bgl2 secretion. (D) Quantitation of internal Bgl2 was determined by ImageJ. Results were analyzed based on seven independent experiments. Error bar represents SD, n=7. *p<0.005, **p<2e5. (E) Strains were grown at 25°C in YP containing 5% (v/v) glucose medium overnight to early log phase. 1 OD₆₀₀ unit of cells was collected from each strain and shifted to YP containing 0.1% (v/v) glucose medium, and incubated at 37^oC for 45 min. Four independent experiments were performed. Error bars represent SD, n=4. *p<0.005.

Figure 4—figure supplement 1. Growth effect and influence on Bgl2 and invertase secretion of sso2 mutants of its first NPY motif.

(A) The sequence of sso2 mutants generated by site-directed mutagenesis. Residues in the first NPY motif that were mutated to Ala (A) in all mutants (i.e. M1-M5) are shown in bold italics. (B) The sso2 mutations in an sso1Δ background partially inhibit cells growth at 37oC. Control sso1Δ SSO2 or sso1Δ sso2 mutant cells were grown overnight in YPD medium. An aliquot (0.2 OD600 units) of cells from each strain was collected, serially diluted by 5 fold and spotted onto YPD plates. Plates were incubated at 25oC, 34 oC or 37oC for 2 days. (C) Control sso1Δ SSO2 strain and sso1Δ sso2 mutants were grown as described in Figure 3. The external and internal Bgl2 pools were detected by western blotting. Several mutations in SSO2 caused inhibition of Bgl2 secretion. (D) Quantitation of internal Bgl2. Results were analyzed based on four independent experiments. Error bar represents SD, n=5. *p<0.05. (E) Invertase secretion was determined in sso2 mutants. Strains were grown as in Figure 3. Samples were processed and quantified as described in Materials and Methods. Three independent experiments were performed. Error bar represents SD, n=3. *p < 0.05. Invertase accumulation was calculated by the formula of ${\displaystyle (Ex{t}_{45m}-Ex{t}_{0m})/[(Ex{t}_{45m}-Ex{t}_{0m})+(In{t}_{45m}-In{t}_{0m})]}$.