Figure 4.
Assessing Shr3-Gap1 interactions using split ubiquitin. (A) Schematic diagram of the split ubiquitin constructs used to evaluate Shr3-AAP, Shr3-Ssy1, and Shr3-HXT interactions. (B) Overview of the split-ubiquitin assay and expected outcomes with Shr3-AAP interactions. (C) Left panels: Serial dilutions of cell suspensions from strain JKY2 (shr3Δ) carrying pRS316 (VC), pPL210 (SHR3), pPL1262 (SHR3-NubA), pAR67 (shr3-35-NubA), or pAR76 (SHR3Δ94-NubA) spotted on YPD and YPD+MM plates. Plates were incubated at 30°C for 2 d and photographed. Right panel: Serial dilutions of cell suspensions from strain FGY15 (gap1Δ) carrying pRS317 (VC), pJK92 (GAP1), pPL1257 (GAP1-Cub-GST), or pIM28 (gap1-ERXAAA-Cub-GST) were spotted on minimal medium with 2% galactose as carbon source and 1 mM L-citrulline as sole nitrogen source. Plates were incubated for 7 d and photographed. (D) Shr3-Gap1 interactions; strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262), shr3-35-NubA (pAR67), or SHR3Δ94-NubA (pAR76) and carrying pPL1257 (GAP1-Cub-GST) were induced with 2% galactose for 1 h. Protein extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. The signal intensities of the immunoreactive forms of full-length and cleaved Gap1 were quantified. The fraction of split-ubiquitin cleavage was determined; the mean values were plotted with error bars showing standard deviation (n = 3). (E) Shr3-Agp1, Shr3-Gnp1, and Shr3-Bap2 interactions; FGY135 strains as in D carrying pIM6 (AGP1-Cub-GST), pIM17 (GNP1-Cub-GST), or pIM7 (BAP2-Cub-GST). (F) Shr3-Ssy1 interactions; FGY135 strains as in D carrying pIM19 (SSY1-Cub-GST). (G) Shr3-Can1 and Shr3-Lyp1 interactions; FGY135 strains as in D carrying pIM8 (CAN1-Cub-GST) or pIM18 (LYP1-Cub-GST). (H) Shr3-Hxt1 and Shr3-Gal2 interactions; FGY135 strains as in D carrying pIM32 (HXT1-Cub-GST) or pIM33 (GAL2-Cub-GST). Strains in E–H were induced with 2% galactose for 1 h. Protein extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. The signal intensities of the immunoreactive forms of full-length and cleaved Agp1, Gnp1, Bap2, Ssy1, Can1, Lyp1, Hxt1, and Gal2 constructs were quantified. The fraction of split-ubiquitin cleavage was determined; the mean values were plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F4.

Assessing Shr3-Gap1 interactions using split ubiquitin. (A) Schematic diagram of the split ubiquitin constructs used to evaluate Shr3-AAP, Shr3-Ssy1, and Shr3-HXT interactions. (B) Overview of the split-ubiquitin assay and expected outcomes with Shr3-AAP interactions. (C) Left panels: Serial dilutions of cell suspensions from strain JKY2 (shr3Δ) carrying pRS316 (VC), pPL210 (SHR3), pPL1262 (SHR3-NubA), pAR67 (shr3-35-NubA), or pAR76 (SHR3Δ94-NubA) spotted on YPD and YPD+MM plates. Plates were incubated at 30°C for 2 d and photographed. Right panel: Serial dilutions of cell suspensions from strain FGY15 (gap1Δ) carrying pRS317 (VC), pJK92 (GAP1), pPL1257 (GAP1-Cub-GST), or pIM28 (gap1-ERXAAA-Cub-GST) were spotted on minimal medium with 2% galactose as carbon source and 1 mM L-citrulline as sole nitrogen source. Plates were incubated for 7 d and photographed. (D) Shr3-Gap1 interactions; strain FGY135 (gap1Δ shr3Δ) expressing SHR3-NubA (pPL1262), shr3-35-NubA (pAR67), or SHR3Δ94-NubA (pAR76) and carrying pPL1257 (GAP1-Cub-GST) were induced with 2% galactose for 1 h. Protein extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. The signal intensities of the immunoreactive forms of full-length and cleaved Gap1 were quantified. The fraction of split-ubiquitin cleavage was determined; the mean values were plotted with error bars showing standard deviation (n = 3). (E) Shr3-Agp1, Shr3-Gnp1, and Shr3-Bap2 interactions; FGY135 strains as in D carrying pIM6 (AGP1-Cub-GST), pIM17 (GNP1-Cub-GST), or pIM7 (BAP2-Cub-GST). (F) Shr3-Ssy1 interactions; FGY135 strains as in D carrying pIM19 (SSY1-Cub-GST). (G) Shr3-Can1 and Shr3-Lyp1 interactions; FGY135 strains as in D carrying pIM8 (CAN1-Cub-GST) or pIM18 (LYP1-Cub-GST). (H) Shr3-Hxt1 and Shr3-Gal2 interactions; FGY135 strains as in D carrying pIM32 (HXT1-Cub-GST) or pIM33 (GAL2-Cub-GST). Strains in E–H were induced with 2% galactose for 1 h. Protein extracts were prepared, separated by SDS-PAGE, and analyzed by immunoblotting using α-HA antibody. The signal intensities of the immunoreactive forms of full-length and cleaved Agp1, Gnp1, Bap2, Ssy1, Can1, Lyp1, Hxt1, and Gal2 constructs were quantified. The fraction of split-ubiquitin cleavage was determined; the mean values were plotted with error bars showing standard deviation (n = 3; biological replicates). Source data are available for this figure: SourceData F4.

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