FIG 6.

Lack of negative regulation of Akl1 by Fpk1 impedes endocytosis. (A) Cultures of an akl1Δ strain (YFR479) containing either an empty vector (V; YCpUG) or expressing from the GAL1 promoter in the same vector either GFP-Akl1^AA (pFR303) or a catalytically inactive (kinase-dead) derivative, GFP-Akl1^AA(D181A) (pKL31), were induced on galactose medium for 2.5 h, incubated with 4 mg/ml of Lucifer yellow CH (LY) for 30 min at 24°C, and then viewed directly by fluorescence microscopy. (B) Cells (YFR515) expressing both Akl1-mCherry and Sla1-GFP from their endogenous promoters at their normal chromosomal loci were grown on YPD and viewed by fluorescence microscopy. (C) Strains expressing Akl1 Sla1-GFP Abp1-RFP (WT, YFR507) and Akl1^AA Sla1-GFP Abp1-RFP (YFR508) were examined by fluorescence video microscopy (see Movies S1 and S2 in the supplemental material), and kymographs were plotted as described in Materials and Methods. (D) The mean Sla1-GFP lifetime at the cell cortex was measured from multiple kymographs as for panel C. (E) The total number of Sla1-GFP patches per cell subsequently joined by Abp1-RFP and moved toward the cell center, a hallmark of actin-driven internalization (40), was determined in cells as in panel C. n, total number cells examined; ns, not significant.