Fig. 6.

Functional analysis of the VV motif in Sec14. SEC14 genes carrying the indicated for V₁₅₄ and V₁₅₅ missense substitutions (Phe, Ala, Glu, Tyr) were integrated into the LEU2 locus of a sec14-1^ts strain. The mock condition documents the phenotype of an isogenic strain where a SEC14-less integration cassette was transplaced into the LEU2 locus. A: Integrants were interrogated for in vivo function by scoring rescue of sec14-1^ts growth defects at the restrictive temperature of 37°C (left panel) or complementation of the normally lethal sec14Δ allele (right panel). Left panel: The YPD plates upon which the cells were spotted were incubated at 37°C for 48 h and images taken. Right panel: A colony color-based plasmid shuffle assay was used for determining whether the mutant Sec14 proteins could fulfill all biological functions of Sec14. Each mutant SEC14 gene was integrated into the MET17 locus of an ade2 ade3 sec14Δ strain carrying a Yep (SEC14, LEU2, ADE3) plasmid [strain CTY558 (9)]. The mock condition documents the phenotype of an isogenic strain where a SEC14-less integration cassette was transplaced into the MET17 locus. Plates were incubated at 30°C for 48 h and images were taken. The ability of mutant Sec14 expression to rescue lethality of the sec14Δ allele is recognized by loss of the Yep (SEC14, LEU2) plasmid upon relief of nutritional selection for LEU2 selection by spotting cells onto YPD agar. Plasmid loss is recognized by appearance of white colony segregants from the background of red plasmid-bearing colonies. All mutant proteins retained biological activity in this assay. B: Integrants expressing the indicated mutant Sec14 proteins in sec14-1^ts genetic background were examined for NPPM 6748-481 sensitivity by dilution spotting on DMSO and drug-supplemented YPD agar. Plates were incubated at 30°C for 48 h and images taken. For both panels, the mock condition documents the phenotype of an isogenic strain where a SEC14-less integration cassette was transplaced into the LEU2 locus.