Fig. 7.

Eisosomes are required for efficient recovery following glucose starvation. (A) Mid-log cultures of the indicated yeast strains were incubated in glucose (replete) or raffinose media for 2 h, before plating on YPD or SC plates for two days at 30°C. (B) Colocalisation of Pil1–mCherry and Fur4–mNG, by Airyscan confocal microscopy focussed at the centre and top, in cells grown to mid-log phase before imaging in minimal medium. (C) Growth of wild-type and fur4Δ cells grown in rich medium prior to serial dilution and growth on YPD and SC (minimal) solid media for 2 days at 30°C. (D) Levels of Pil1–GFP expressed from a plasmid in the indicated strains were assessed by immunoblot of lysates generated from mid-log phase cells using anti-GFP antibodies, with anti-Rsp5 antibodies used as a loading control. (E) Pil1–GFP localisation of transformants from D grown to log phase was recorded by confocal microscopy at centre- and top-focussed planes. Images on the right show cells coloured using a lookup table of GFP intensity. (F) The indicated strains were transformed with a plasmid expressing Fur4–mNG from its endogenous promoter and imaged by confocal microscopy. Yellow arrows on centre-focussed images indicate pronounced eisosomal concentrations. (G) The numbers of eisosomally localised Fur4–mNG puncta per cell were quantified from the indicated yeast strains. Mean±s.d. (H) Levels of Fur4–GFP sorted to the vacuole (left) and full-length Mup1–GFP (right) were assessed from indicated strains and conditions by anti-GFP immunoblotting, using GAPDH as a loading control. (I) Indicated strains were grown to mid-log phase in SC complete medium before harvesting and growth in raffinose medium for two hours, before average recovery growth was assessed by OD₆₀₀ measurements from cells incubated in SC medium. (J–M) Average recovery from assay described in I was quantified relative to the wild-type control and plotted over time for the indicated mutants at either 20 mg/l uracil (J,L,M) or 2 mg/l uracil (K). Mean±s.d. from three biological replicates. Black asterisks indicate Student's t-test comparisons with wild-type samples, red asterisks indicate comparisons between strains; *P<0.05. Scale bars: 5 µm.