Figure 7. Progressive Shr3-Agp1 and... | Rockefeller University Press

Figure 7.

$Progressive Shr3-Agp1 and Shr3-Ssy1 chaperone-substrate interactions. (A) Schematic diagram of agp1-Cub-GST truncation constructs. Strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262) or SHR3Δ94-NubA (pAR76) and carrying pIM9 (agp1-2TM-Cub-GST), pIM10 (agp1-4TM-Cub-GST), pIM11 (agp1-6TM-Cub-GST), pIM12 (agp1-8TM-Cub-GST), pIM13 (agp1-10TM-Cub-GST), or pIM26 (agp1-12TM-Cub-GST) were induced with 2% galactose for 1 h. Extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. The signal intensities of the immunoreactive forms of uncleaved Cub constructs and cleaved interaction marker (GST-6xHA) were quantified; the mean values of the fraction of split ubiquitin cleavage are plotted with error bars showing standard deviation (n = 3; biological replicates). (B) Schematic diagram of ssy1-Cub-GST truncation constructs. Strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262) or SHR3Δ94-NubA (pAR76) and carrying pIM20 (ssy1-2TM-Cub-GST), pIM21 (ssy1-4TM-Cub-GST), pIM22 (ssy1-6TM-Cub-GST), pIM23 (ssy1-8TM-Cub-GST), pIM24 (ssy1-10TM-Cub-GST), or pIM25 (ssy1-12TM-Cub-GST) were induced with 2% galactose for 1 h. Extracts were prepared and analyzed as in A, and the mean values of the fraction of split ubiquitin cleavage are plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F7.$

Progressive Shr3-Agp1 and Shr3-Ssy1 chaperone-substrate interactions. (A) Schematic diagram of agp1-Cub-GST truncation constructs. Strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262) or SHR3Δ94-NubA (pAR76) and carrying pIM9 (agp1-2TM-Cub-GST), pIM10 (agp1-4TM-Cub-GST), pIM11 (agp1-6TM-Cub-GST), pIM12 (agp1-8TM-Cub-GST), pIM13 (agp1-10TM-Cub-GST), or pIM26 (agp1-12TM-Cub-GST) were induced with 2% galactose for 1 h. Extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. 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). (B) Schematic diagram of ssy1-Cub-GST truncation constructs. Strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262) or SHR3Δ94-NubA (pAR76) and carrying pIM20 (ssy1-2TM-Cub-GST), pIM21 (ssy1-4TM-Cub-GST), pIM22 (ssy1-6TM-Cub-GST), pIM23 (ssy1-8TM-Cub-GST), pIM24 (ssy1-10TM-Cub-GST), or pIM25 (ssy1-12TM-Cub-GST) were induced with 2% galactose for 1 h. Extracts were prepared and analyzed as in A, and 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 F7.

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