Figure 9.
Reversal of Ist1 ubiquitination inhibits endosomal recycling. (A) Schematic illustrating the strategy to fuse Ist1-GFP to an active DUb and an inactive catalytically dead (dubC>S) mutant enzyme. (B–D) Airyscan microscopy of indicated fluorescent proteins expressed in WT cells grown to exponential phase prior to imaging. Insets (D) show of colocalization and distinct foci in yellow and blue, respectively. (E) Jitter plots showing Pearson’s correlation coefficient (PCC) from imaging shown in B–D. *, P < 0.0001 from unpaired t test, n = 55–65 cells per condition. (F and G) FM4-64 efflux measurements from dye loaded to cells expressing active Ist1-GFP-DUb (F) and inactive Ist1-GFP-DUbC>S (G) fusion proteins, with profiles from WT and ist1∆ mutants included for reference. Scale bar, 5 μm (white); 0.5 μm (yellow).

Reversal of Ist1 ubiquitination inhibits endosomal recycling. (A) Schematic illustrating the strategy to fuse Ist1-GFP to an active DUb and an inactive catalytically dead (dubC>S) mutant enzyme. (B–D) Airyscan microscopy of indicated fluorescent proteins expressed in WT cells grown to exponential phase prior to imaging. Insets (D) show of colocalization and distinct foci in yellow and blue, respectively. (E) Jitter plots showing Pearson’s correlation coefficient (PCC) from imaging shown in B–D. *, P < 0.0001 from unpaired t test, n = 55–65 cells per condition. (F and G) FM4-64 efflux measurements from dye loaded to cells expressing active Ist1-GFP-DUb (F) and inactive Ist1-GFP-DUbC>S (G) fusion proteins, with profiles from WT and ist1∆ mutants included for reference. Scale bar, 5 μm (white); 0.5 μm (yellow).

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