Figure 6. The Ex6 and Pro-Rich domains... | Rockefeller University Press

Figure 6.

$Figure 6. The Ex6 and Pro-Rich domains of Pyd mediate the interaction with Su(dx). (A) Schematic representation of the E3 ubiquitin ligase Su(dx). C2 phospholipid-binding domain, WW protein interaction domains, and the catalytic HECT domain are indicated. (B) Schematic representation of Pyd/ZO-1 indicating candidate WW domain–interacting motifs. (C) Fragments of Pyd, as indicated, were tested for binding to GST fusion proteins containing the WW domains of Su(dx). Autoradiograph of 35S-labeled input proteins (input, left) and of eluted bound fractions from the indicated combinations. Only fragments containing both Ex6 and Pro-Rich domains were retained by GST-Su(dx)-WW. Weak binding was detected with the Pro-Rich domain alone, and no specific binding was detected with other domains (PDZ2, PDZ3, SH3, and GUK; not depicted). (D) Pyd domains required for Su(dx) recruitment. Su(dx) (HA-tag: purple, middle; white, bottom) is recruited by full-length and, to a lesser extent, by ΔPro-Rich or by ΔEx6 Pyd variants but not by ΔEx6ΔPro-Rich (GFP tags: green, middle). (top) S2 cells transfected with the Pyd constructs only and stained for GFP (white). Percentages in the bottom images indicate percentage of transfected cells with some HA-Su(dx) detected at the cell cortex as the mean of more than three experiments. Bar, 7 µm. (E and F) Tryptophan fluorescence change upon titration of Su(dx) WW domains with synthetic Pyd-derived peptides. Single (WW1 and WW2) and paired (WW1–2 and WW3–4) WW domains were tested with peptide sequences EGLPPPYTV (E) and APPPQSYPQ (F). F′ are normalized fluorescence values. The data were fitted to a single-site binding model, as described in Materials and methods, to obtain Ka (association constant) and converted to Kd (dissociation constant). Errors of fit are smaller than SDs from multiple experiments; SD and mean Kd values are reported in Table S1 for each WW domain–peptide combination.$

The Ex6 and Pro-Rich domains of Pyd mediate the interaction with Su(dx). (A) Schematic representation of the E3 ubiquitin ligase Su(dx). C2 phospholipid-binding domain, WW protein interaction domains, and the catalytic HECT domain are indicated. (B) Schematic representation of Pyd/ZO-1 indicating candidate WW domain–interacting motifs. (C) Fragments of Pyd, as indicated, were tested for binding to GST fusion proteins containing the WW domains of Su(dx). Autoradiograph of ³⁵S-labeled input proteins (input, left) and of eluted bound fractions from the indicated combinations. Only fragments containing both Ex6 and Pro-Rich domains were retained by GST-Su(dx)-WW. Weak binding was detected with the Pro-Rich domain alone, and no specific binding was detected with other domains (PDZ2, PDZ3, SH3, and GUK; not depicted). (D) Pyd domains required for Su(dx) recruitment. Su(dx) (HA-tag: purple, middle; white, bottom) is recruited by full-length and, to a lesser extent, by ΔPro-Rich or by ΔEx6 Pyd variants but not by ΔEx6ΔPro-Rich (GFP tags: green, middle). (top) S2 cells transfected with the Pyd constructs only and stained for GFP (white). Percentages in the bottom images indicate percentage of transfected cells with some HA-Su(dx) detected at the cell cortex as the mean of more than three experiments. Bar, 7 µm. (E and F) Tryptophan fluorescence change upon titration of Su(dx) WW domains with synthetic Pyd-derived peptides. Single (WW¹ and WW²) and paired (WW^1–2 and WW^3–4) WW domains were tested with peptide sequences EGLPPPYTV (E) and APPPQSYPQ (F). F′ are normalized fluorescence values. The data were fitted to a single-site binding model, as described in Materials and methods, to obtain K_a (association constant) and converted to K_d (dissociation constant). Errors of fit are smaller than SDs from multiple experiments; SD and mean K_d values are reported in Table S1 for each WW domain–peptide combination.