Figure 5. Progressivity of Shr3-Gap1... | Rockefeller University Press

Figure 5.

$Progressivity of Shr3-Gap1 chaperone-substrate interactions. (A) Schematic diagram of split ubiquitin constructs including the gap1-Cub-GST, hxt1-Cub-GST, and gal2-Cub-GST truncation constructs. (B) Strain FGY135 (gap1Δ shr3Δ) carrying pPL1262 (SHR3-NubA) and pIM1 (gap1-2TM-Cub-GST), pIM2 (gap1-4TM-Cub-GST), pIM3 (gap1-6TM-Cub-GST), pIM4 (gap1-8TM-Cub-GST), pIM5 (gap1-10TM-Cub-GST), or pIM16 (gap1-12TM-Cub-GST; left panel), or pIM34 (hxt1-2TM-Cub-GST), pIM35 (hxt1-4TM-Cub-GST), pIM36 (hxt1-6TM-Cub-GST), pIM37 (hxt1-8TM-Cub-GST), pIM38 (hxt1-10TM-Cub-GST), or pIM39 (hxt1-12TM-Cub-GST; center panel) or pIM40 (gal2-2TM-Cub-GST), pIM41 (gal2-4TM-Cub-GST), pIM42 (gal2-6TM-Cub-GST), pIM43 (gal2-8TM-Cub-GST), pIM44 (gal2-10TM-Cub-GST), or pIM45 (gal2-12TM-Cub-GST; right panel) were induced with 2% galactose for 1 h. Extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. (C) The signal intensities of the immunoreactive forms of uncleaved Cub constructs and cleaved interaction marker (GST-6xHA) were quantified; the mean values of the fraction of split ubiquitin cleavage are plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F5.$

Progressivity of Shr3-Gap1 chaperone-substrate interactions. (A) Schematic diagram of split ubiquitin constructs including the gap1-Cub-GST, hxt1-Cub-GST, and gal2-Cub-GST truncation constructs. (B) Strain FGY135 (gap1Δ shr3Δ) carrying pPL1262 (SHR3-NubA) and pIM1 (gap1-2TM-Cub-GST), pIM2 (gap1-4TM-Cub-GST), pIM3 (gap1-6TM-Cub-GST), pIM4 (gap1-8TM-Cub-GST), pIM5 (gap1-10TM-Cub-GST), or pIM16 (gap1-12TM-Cub-GST; left panel), or pIM34 (hxt1-2TM-Cub-GST), pIM35 (hxt1-4TM-Cub-GST), pIM36 (hxt1-6TM-Cub-GST), pIM37 (hxt1-8TM-Cub-GST), pIM38 (hxt1-10TM-Cub-GST), or pIM39 (hxt1-12TM-Cub-GST; center panel) or pIM40 (gal2-2TM-Cub-GST), pIM41 (gal2-4TM-Cub-GST), pIM42 (gal2-6TM-Cub-GST), pIM43 (gal2-8TM-Cub-GST), pIM44 (gal2-10TM-Cub-GST), or pIM45 (gal2-12TM-Cub-GST; right panel) were induced with 2% galactose for 1 h. Extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. (C) The signal intensities of the immunoreactive forms of uncleaved Cub constructs and cleaved interaction marker (GST-6xHA) were quantified; the mean values of the fraction of split ubiquitin cleavage are plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F5.

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