Fig. 6. Interaction of Sla1p at the cell cortex with the endocytic machinery. (A) The effect of deletion of the C-terminal repeat region on Sla1p localisation. Cells expressing full-length 9xmyc-tagged Sla1p, KAY303 (left) or mutant sla1Ct-9xmyc, KAY363 (right) were grown in rich media to log phase and then processed for immunofluorescence as described in the Materials and Methods. Note the lack of cortical localisation of Sla1p in the absence of the C-terminal repeat region. Bar, 10 µM. (B) Localisation of Sla1-GFP in END3 and end3-1 cells following a temperature shift. KAY397 (END3) and KAY462 (end3-1) cells were grown overnight at 30°C in rich medium to log phase. Half of each culture was then shifted to 37°C, the non-permissive temperature for the end3-1 mutation. Cells were incubated at 37°C for 2 hours. Cells were then mounted on slides and images recorded as described in the Materials and Methods. (C) Samples of cells were also taken at this time, fixed and processed for rhodamine-phalloidin staining. Cells showing a complete deletion of END3 were grown at 30°C and analysed by rhodamine-phalloidin staining to observe their actin phenotype (D) and to assess localisation of Sla1GFP (E). Bar, 10 µM.

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