Figure 6.

Arf1p and Pab1p are required for ASH1 mRNA localization to the bud tip. (A) Overview of scored phenotypes. (B) List of mutants used in the analysis in C. (C) The defect in mRNA localization is allele specific. Different mutants in ARF1 were grown to early log phase at 23°C and then shifted for 1 h to 37°C. ASH1 mRNA in the cells was visualized by FISH. At least 100 cells/strain were scored. (D) ASH1 mRNA levels are not sensitive to shift to the restrictive temperature. Different mutants in ARF1 were grown to early log phase at 23°C and then shifted for 1 h to 37°C; the deletion mutants in ARF1 and PAB1 SPB8 as well as a wild-type strain were grown at 30°C. Total RNA was extracted from the different strains and analyzed by Northern blot. The blot was incubated with probes for ASH1 mRNA and ADH1 mRNA. On temperature shift, no significant changes in mRNA levels were observed. (E) Ash1p levels are not altered in different mutants. The strains were transformed with a CEN plasmid encoding C-terminally myc₉-tagged Ash1p and grown as described in D. Soluble extracts were prepared and analyzed by immunoblot for the presence of Ash1p-myc and Arf1p. The translation of ASH1 mRNA seemed to be unaffected by shift to restrictive temperature. The Arf-signal in the Δarf1 mutant corresponds to Arf2p, which is up-regulated in Δarf1 strains. Arf2p is 10 times less than abundant than Arf1p in wild-type strains.