Fig. 4. Conditional yif1 mutants are blocked in ER-to-Golgi transport. (A) Total cellular proteins of wild-type cells (wt), of various yif1 mutant cells, and of wt cells expressing YIF1 from the multicopy vector pRS326 were probed for Yif1p when grown at 25°C or after a shift to 37°C for 60 min. Note that the mutant proteins are unstable at non-permissive temperature and that some of them have an electrophoretic behaviour different from wild-type Yif1p. (B) Pulse–chase experiments performed with wild-type (wt) and yif1 mutant cells at either 25 or 37°C (15 min pulse with ³⁵S-labelled amino acids, 30 min chase time). Note the accumulation of the ER forms of vacuolar CPY and ALP at 25°C (partial) and 37°C (complete). (C) Defects in invertase glycosylation and secretion. Invertase synthesis was induced for 60 min at 25 or 37°C in wild-type (wt) and mutant cells. Highly glycosylated (S) and ER core-glycosylated enzyme (ER) in intracellular (I) and extracellular fractions (E) as well as cytoplasmic invertase (C) were identified by an activity stain in non-denaturing gels.