Figure 3.

Mutations in either the Sec9 H1 or H2 helix result in loss of function that is suppressed by the expression of SNC2 with an R-to-Q substitution. (A) The sec9-Q468R mutant is lethal and the sec9-Q622R mutant is temperature-sensitive as the only source of SEC9. A diploid strain heterozygous for a deletion of SEC9 (a/α; sec9Δ::HIS3/sec9; ura3-52/ura3-52; leu2-3,112/leu2-3,112; his3-Δ200/his3-Δ200) was transformed with sec9-Q468R or sec9-Q622R CEN-LEU2 plasmids. Transformants were sporulated, and tetrads were picked for each mutant and allowed to grow for 3 d on YPD plates at 25°C. (B) Rescue of sec9-Q468R lethality by SNC2-R52Q. Diploids were cotransformed with either SNC2 (left panel) or SNC2-R52Q (right panel) carrying plasmids along with sec9-Q468R and analyzed as described in A. The left panel shows the resulting tetrads after cotransformation with SNC2 and sec9-Q468R plasmids. Subsequent analysis of the viable spores indicated that both plasmids were present but were unable to support growth in the spores containing the deleted sec9Δ::HIS3 allele. The right panel shows replica-plate analysis of four tetrads resulting from cotransformation with the SNC2-R52Q and sec9-Q468R plasmids. Rescue of the deleted SEC9 by the combination of SNC2-R52Q and sec9-Q468R plasmids was clearly evident by the presence of more than two viable spores and by the analysis of auxotrophic markers, which demonstrated that each of the viable spores contained either the wild-type SEC9 allele (his−) or sec9Δ::HIS3 disruption (his+) and both CEN-SNC2-R52Q (ura+) and CEN/sec9-Q468R (leu+). Rescued segregants show wild-type growth on YPD medium at both 25 and 37°C. (C) The SNC2-R52Q allele dominantly suppresses the temperature sensitivity of the sec9-Q622R mutant. The sec9-Q622R mutant sec9Δ::HIS3; CEN/sec9-Q622R (ts-) strain constructed in A was transformed with the SNC2 or SNC2-R52Q alleles on CEN/URA3 plasmids. Transformants were replica-plated onto YPD plates at 25 and 37°C.