Fig. 2: EMC interaction with a TMD depends on its molecular composition.
a The minimal consensus membrane protein “ConMem” (left) is a model protein as described in ref. 32. Systematically changing its inert TMD sequence allows analyses of intramembrane recognition processes as depicted in the schematic on the right. For a first set of EMC binding experiments (shown in b), the central valine at position 13 (underlined) was exchanged. b Systematic analysis of the interaction of ConMem variants with the endogenous EMC in HEK 293T cells. The central valine at position 13 was replaced with all other 19 amino acids, and binding to endogenous EMC was assessed by co-IP against the HA-tag on ConMem. Residues analyzed are ordered according to the Kyte–Doolittle scale for hydrophobicity61 and color-coded according to physicochemical properties (gray: hydrophobic/aromatic, yellow: polar, red: negatively charged, blue: positively charged). Binding was assessed in five independent replicates and normalized to the original consensus sequence (V at position 13) (mean ± SD, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001, two-tailed Student’s t tests). c Positional binding dependency of the EMC to ConMem variants with substitutions at indicated positions throughout the ConMem TMD. Binding was assessed in at least three independent replicates by co-IP against the HA-tag on ConMem and normalized to the original consensus sequence (V at position 13) (mean ± SD, *P-value < 0.05, **P-value < 0.01, ***P-value < 0.001, ****P-value < 0.0001, two-tailed Student’s t tests).
