Fig. 3.

The TMD region of G1IP mediates interaction with AtGET1, and its cytosolic N terminus can interfere with the mammalian insertion system. (A) Schematic of the 2in1 rBiFC constructs used in B. G1IP and G1IPcyt were tagged C-terminally to avoid masking the N-terminal motif, marked with an asterisk. (B) rBiFC analysis using full-length and truncated versions of G1IP to test for interaction with AtGET1. Exemplary CLSM images of transiently transfected N. benthamiana leaves are depicted. Mean fluorescence of at least 25 areas was measured in YFP and RFP channels, ratioed, and plotted to show YFP complementation. Center lines of boxes represent the median, with outer limits at the 25th and 75th percentiles. Tukey whiskers extend to 1.5 times the IQR. All values are depicted as black dots. (C) Schematic of the 2in1 FRET constructs used for co-IP in D. (D) Co-IP of full-length and truncated G1IP with AtGET1, transiently expressed in N. benthamiana leaves. Protein extracts were immunoprecipitated with anti-RFP beads, and protein–protein interaction was detected by immunoblotting using anti-RFP and anti-GFP antibodies. IN, input; FT, flow-through; IP, immunoprecipitate. (E) Schematic representation of full-length, truncated, and mutated G1IP. The small alignment highlights a conserved cluster of positively charged amino acids and its charge-reversal mutation in the G1IP^4Ecyt mutant, respectively. (F and G) Insertion assays into microsomal membranes. Stx5-op was translated in vitro in rabbit reticulocyte lysate and incubated with recombinant cytosolic fragments and pancreatic rough microsomes. Protein extracts were immunoblotted with anti-Stx5 antibody, and ER insertion was monitored via band shift reporting glycosylation. Boxplots show quantification of the immunoblots from four independent experiments. The center lines of boxes represent the median, with outer limits at the 25th and 75th percentiles. Tukey whiskers extend to 1.5 times the IQR. Outliers are depicted as black dots. ***P < 0.001, Student’s t test.