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Figure 7 SAR1A is a direct ubiquitylation target of the KLHL40-CUL3 complex and is differently ubiquitylated by a disease-causing mutation in KLHL40. (A) Coimmunoprecipitation in C2C12 cells showing KLHL40 directly interacts with SAR1A. (B) Co-overexpression of decreasing KLHL40-FLAG and constant SAR1A-V5 in C2C12 myoblasts demonstrates that KLHL40 is a regulator of Sar1A protein. (C) Co-overexpression of decreasing amounts of KLHL40-FLAG and constant amount of SAR1A-V5 in C2C12 myoblasts in the presence of UPS inhibitor MG132 increases the SAR1A protein levels in comparison to MG132- condition. (D) Alignment of the amino acid sequence of the SAR1A ubiquitylation site demonstrates high conservation in vertebrates (K182 in all species, marked by the asterisk). (E) Localization of different disease-causing variants in KLHL40 in the protein domains. (F) In vitro ubiquitylation of human SAR1A by CUL3 protein complex in the presence of wild-type and disease-causing KLHL40 proteins. (G) Quantifying the relative human SAR1A ubiquitylation by wild-type and disease-causing KLHL40-CUL3 complex. (H) Ubiquitylation of overexpressed SAR1A in the presence of KLHL40 in C2C12 myoblasts. Data are mean ± S.E.M; with one-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test and Brown-Forsythe test (****p<0.001; n.s. non significant) n=3.