Figure 4.

The Sla2p-PtdIns(4,5)P₂ interaction is important for cell growth and the endocytic internalization. (A) Extracts of cells grown at 25°C or after 2 h at 37°C were prepared and analyzed by Western blotting with an anti-Sla2p antibody or an anti-actin antibody. (B) The ANTH domain is essential for cell growth at elevated temperatures. Dilution series of wild-type, sla2 4K-A, sla2 ANTHΔ, and sla2Δ cells were plated on YPD medium and incubated for 2 d at 25, 34, or 37°C, respectively. (C and D) Receptor-mediated internalization of [³⁵S]methionine-labeled α-factor in sla2 mutants after a 5-min preincubation at either 25 or 37°C. Each assay was performed three times, and similar results were obtained each time. (E) Deletion of SJL1 suppressed the temperature-sensitive growth defect of the sla2 4K-A mutant. Dilution series of wild-type, sla2 4K-A, sjl1Δ, and sla2 4K-A sjl1Δ cells were plated on YPD medium and incubated for 2 d at either 25 or 37°C. (F) Deletion of SJL1 suppressed the endocytic internalization defect of the sla2 4K-A mutant. Endocytosis of [³⁵S]methionine-labeled α-factor was measured after a 5-min preincubation at 37°C. This assay was performed three times, and similar results were obtained each time.