Figure 7.

Lysine-less Ist1 is defective in endosomal recycling. (A) Immunoblot with indicated antibodies of lysates generated from ist1∆ cells transformed with plasmids expressing Ist1^WT-HA or Ist1^KR-HA from endogenous promoter, alongside WT cells. (B) Cycloheximide chase experiments using ist1∆ pdr5∆ cells expressing Ist1^WT-HA or Ist1^KR-HA. Cells grown to mid-log phase were then exposed to 25 mg/liter cycloheximide for the denoted time before harvesting and immunoblot with ⍺-Ist1 and ⍺-GAPDH antibodies. (C) Graph of Ist1 stability following cycloheximide chase at indicated times of Ist1^WT (gray) and Ist1^KR (green), with SD (n = 3) indicated with respective shaded regions. (D) Airyscan images of ist1∆ cells co-expressing Ist1^KR-GFP and Sec7-mCherry (upper) or Vps4-mCherry (lower) grown to mid-log phase. (E) Associated jitter plots from D showing Pearson’s correlation coefficient. *, P < 0.0001 from unpaired t test, n = 56 cells per condition. (F) Airyscan microscopy of ist1∆ cells coexpressing Mup1-GFP, Vps4-mCherry with either Ist1^WT-HA (upper) or Ist1^KR-HA (lower). (G) FM4-64 efflux measurements from WT cells (dark gray) and ist1∆ mutants expressing plasmid borne copies of Ist1^WT-HA (light gray), Ist1^KR-HA (green) or transformed with an empty vector (pink). (H and I) Plasmids of His₆-Ist1^WT and His₆-Ist1^KR were expressed in BL21 DE3 codon optimized E. coli strain using 0.5 mM IPTG at 15°C overnight. Lysates were generated by sonication and bound to 600 μl Ni²⁺-NTA bed volume, followed by washing in 20 mM imidazole and elution in 500 mM imidazole. Samples were analyzed by SDS-PAGE followed by Coomassie staining, showing protein levels in lysate pre- and postbinding to beads, the material lost during washes, and the final eluted products. (J–M) Intrinsic fluorescence at 350 nm (J and K) and unfolding induced aggregation via scattering in milli-absorbance units (mAU; L and M) was measured by nanoDSF for purified His₆-Ist1^WT and His₆-Ist1^KR samples exposed to a 1°C/min heat ramp. Scale bar, 5 μm. Source data are available for this figure: SourceData F7.