Figure 2.
GSK3/AtSK sites in TRAPPII and binary interactions. Yeast two-hybrid assays were carried out by pairwise one-on-one mating in four independent replicate experiments, of which three (B) or four (F) are shown. As a negative control, the respective AD constructs were tested with the empty DB vector. The panels are from different plates (in B, F). (A) TRAPPII-specific truncations for yeast two-hybrid were based on phylogenetic analysis (Steiner et al., 2016). Conserved sequences are depicted in red, intermediate degrees of conservation in orange, and plant-specific sequences are in green. All three GSK3 sites found to be phosphorylated in vivo reside in the plant-specific moiety of TRS120-T2 (green; a blue rectangle delineates the region of interest). (B) Yeast two-hybrid assays of interactions between BIN2 and TRAPPII subunits. BIN2 was fused to the GAL4 DNA-binding domain (DB) and TRAPPII subunits and truncations thereof (CLUB-C1, -C2, -C3 and TRS120-T1, -T2, -T3) to the GAL4 activation domain (AD). The results show interactions between BIN2 and AtTRS120-T2 wild type. BIN2 bound specifically to the TRAPPII subunit TRS120 and not with other TRAPPII subunits or truncations. In a total of >2,400 pairwise tests, BIN2 was the only kinase we found that interacted with a TRAPP subunit. The Y2H did not detect CLUB/AtTRS130-AtSK interaction shown in Fig. 1 B, which shows copurified proteins that can include indirect—as opposed to binary—interactors. (C) GSK3 sites in AtTRS120-T2. The α-phosphosite of AtTRS120 spans amino acid positions S922 and S923, the β-phosphosite amino acids S971-S975 and the γ-phosphosite amino acid residue S1165. The canonical GSK3 consensus sequence is: (pS/pT)XXX(S/T), and the β-phosphosite fits this definition (see underlined SXXXS). Even though they are annotated as GSK3 sites in the PPSP (https://www.phosphosite.org) database, the α-phosphosite deviates somewhat with SXXXXS, and the γ-phosphosite deviates completely. Amino acids in red were mutated to A or D via site-directed mutagenesis. Amino acids highlighted in yellow were found to be phosphorylated in vivo (see Fig. S2) and these were all serines. (D) The Arabidopsis TRAPPII complex consists of seven shared core subunits (TCA17, TRS33, BET3, BET5, TRS23, TRS31, TRS20; light blue) and three TRAPPII-specific subunits (CLUB/AtTRS130, AtTRS120, and the plant-specific subunit TRIPP), and forms a dimer with plant-specific domains (green) at the predicted dimer interface (Kalde et al., 2019; Garcia et al., 2020). This model is based on extensive pair-wise yeast two-hybrid analysis between TRAPPII subunits in Arabidopsis (Kalde et al., 2019; Garcia et al., 2020). (E) Mapping of GSK3/AtSK sites on an AlphaFold structural prediction (Jumper et al., 2021; Varadi et al., 2022) of AtTRS120. A lateral view and a frontal perspective are shown. Note that all three GSK3/AtSK sites (magenta sticks) reside in unstructured, flexible, and accessible regions of the protein. (F) Interactions between BIN2 and TRAPPII complex subunits (CLUB-C2, CLUB-C3, TRS120-T1, TRS120-T3 truncations), as positive controls, fused to the GAL4 DNA-binding domain (DB) and TRS120-T2 truncation and its phosphomutants TRS120-T2 SαD, SβD, SγD, SαβD, and SαβγD fused to the GAL4 activation domain (AD). Note the positive interactions between BIN2 and AtTRS120-T2 wild type and phosphovariants; there was, however, no reproducible interaction if all three target sites were phosphomimetic (TRS120-T2 SαβγD). Related to Figs. S2, S3, and S4.

GSK3/AtSK sites in TRAPPII and binary interactions. Yeast two-hybrid assays were carried out by pairwise one-on-one mating in four independent replicate experiments, of which three (B) or four (F) are shown. As a negative control, the respective AD constructs were tested with the empty DB vector. The panels are from different plates (in B, F). (A) TRAPPII-specific truncations for yeast two-hybrid were based on phylogenetic analysis (Steiner et al., 2016). Conserved sequences are depicted in red, intermediate degrees of conservation in orange, and plant-specific sequences are in green. All three GSK3 sites found to be phosphorylated in vivo reside in the plant-specific moiety of TRS120-T2 (green; a blue rectangle delineates the region of interest). (B) Yeast two-hybrid assays of interactions between BIN2 and TRAPPII subunits. BIN2 was fused to the GAL4 DNA-binding domain (DB) and TRAPPII subunits and truncations thereof (CLUB-C1, -C2, -C3 and TRS120-T1, -T2, -T3) to the GAL4 activation domain (AD). The results show interactions between BIN2 and AtTRS120-T2 wild type. BIN2 bound specifically to the TRAPPII subunit TRS120 and not with other TRAPPII subunits or truncations. In a total of >2,400 pairwise tests, BIN2 was the only kinase we found that interacted with a TRAPP subunit. The Y2H did not detect CLUB/AtTRS130-AtSK interaction shown in Fig. 1 B, which shows copurified proteins that can include indirect—as opposed to binary—interactors. (C) GSK3 sites in AtTRS120-T2. The α-phosphosite of AtTRS120 spans amino acid positions S922 and S923, the β-phosphosite amino acids S971-S975 and the γ-phosphosite amino acid residue S1165. The canonical GSK3 consensus sequence is: (pS/pT)XXX(S/T), and the β-phosphosite fits this definition (see underlined SXXXS). Even though they are annotated as GSK3 sites in the PPSP (https://www.phosphosite.org) database, the α-phosphosite deviates somewhat with SXXXXS, and the γ-phosphosite deviates completely. Amino acids in red were mutated to A or D via site-directed mutagenesis. Amino acids highlighted in yellow were found to be phosphorylated in vivo (see Fig. S2) and these were all serines. (D) The Arabidopsis TRAPPII complex consists of seven shared core subunits (TCA17, TRS33, BET3, BET5, TRS23, TRS31, TRS20; light blue) and three TRAPPII-specific subunits (CLUB/AtTRS130, AtTRS120, and the plant-specific subunit TRIPP), and forms a dimer with plant-specific domains (green) at the predicted dimer interface (Kalde et al., 2019; Garcia et al., 2020). This model is based on extensive pair-wise yeast two-hybrid analysis between TRAPPII subunits in Arabidopsis (Kalde et al., 2019; Garcia et al., 2020). (E) Mapping of GSK3/AtSK sites on an AlphaFold structural prediction (Jumper et al., 2021; Varadi et al., 2022) of AtTRS120. A lateral view and a frontal perspective are shown. Note that all three GSK3/AtSK sites (magenta sticks) reside in unstructured, flexible, and accessible regions of the protein. (F) Interactions between BIN2 and TRAPPII complex subunits (CLUB-C2, CLUB-C3, TRS120-T1, TRS120-T3 truncations), as positive controls, fused to the GAL4 DNA-binding domain (DB) and TRS120-T2 truncation and its phosphomutants TRS120-T2 SαD, SβD, SγD, SαβD, and SαβγD fused to the GAL4 activation domain (AD). Note the positive interactions between BIN2 and AtTRS120-T2 wild type and phosphovariants; there was, however, no reproducible interaction if all three target sites were phosphomimetic (TRS120-T2 SαβγD). Related to Figs. S2, S3, and S4.

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