Figure 5.

Gyp7 is activated by a distinct membrane environment. (A) Membrane association of Gyp7 with DOGS-NTA containing liposomes. 715 nM Gyp7 was incubated with 715 μM liposomes (VML + DOGS-NTA, PC/PE + DOGS-NTA, PC/PE) for 10 min. Membranes were separated from supernatant by centrifugation at 100,000 g and both fractions were analyzed by SDS-PAGE and Coomassie staining. Control reaction contained no liposomes. (B) Quantification of the relative Gyp7 amount in the pellet in A. Band intensity of Gyp7 signal in the pellet was measured in Fiji and compared with Gyp7 signal in the supernatant. Bar graphs represent the averages from three independent experiments and puncta represent the mean of each experiment. P value ns, **<0.01, ***<0.001, using ANOVA one-way test. (C) Comparison of Gyp7 activity on DOGS-NTA containing liposomes. 250 μM liposomes were preloaded with 0.6 μM Ypt7:GDI complex in the presence of 3.75 mM EDTA and 125 μM GTP. Nucleotide binding was stabilized by addition of 7.5 mM MgCl₂. Reactions were incubated with 3.75 μM Gyp7 for 10 min. Liposomes were floated in a sucrose gradient. Control reactions contained no Gyp7. 40% of the float was analyzed together with 3% input by western blotting using an anti-Ypt7 antibody. (D) Quantification of bound Ypt7 to liposomes in C. Band intensity of Ypt7 signal in float was measured in Fiji and compared to input. Reactions containing Gyp7 were normalized to the average value of the respective control reaction. Bar graphs represent the averages from three independent experiments and puncta represent the mean of each experiment. P value ns, ***<0.001, using ANOVA one-way test. (E) AlphaFold2 structure prediction of Gyp7. The N-terminal PH domain is colored blue and the C-terminal TBC domain is colored cyan with the catalytic Arg (R458) and Glu (Q531) residues shown red in stick representation. A middle domain, which is modeled with low predicted local distance difference test (pLDDT) confidence scores (Fig. S3, C and D), is colored green. (F) Membrane association of the TBC domain compared to full-length Gyp7. Gyp7 and the TBC domain were incubated with liposomes of VML composition as in A. Control reactions contained no liposomes. (G) Quantification of the relative amount of Gyp7 in the pellet in F. Quantification performed as in B. P value *<0.05, using ANOVA one-way test. (H) Comparison of Gyp7 and TBC domain activities on liposomes with VML composition. Assay was performed as in C. Pre-loaded liposomes were incubated with different amounts of Gyp7 or the TBC domain for 10 min. (I) Quantification of bound Ypt7 to liposomes in H. Quantification was performed as in D. Reactions containing GAP were normalized to the average value of the control reaction. P value ns, *<0.05, using ANOVA one-way test. (J) Comparison of Gyp7 activity toward soluble Ypt7-GTP in solution and on membranes. 5 μM Ypt7 was incubated with 5 μM GAP and 50 μM GTP in the presence of 1 mM DTT, 20 mM EDTA, and 5 mM MgCl₂. Where indicated, reactions contained 1 mM liposomes with VML composition or PC/PE liposomes. Control reactions contained no Ypt7, no GAP, or neither Ypt7 nor GAP (see Fig. S3 I). Reactions were stopped after 0, 10, 60, 180, and 300 min by snap-freezing and boiling at 95°C. Samples were applied to a HPLC system and the absorbance of GDP and GTP was monitored at 254 nm. Peaks were analyzed with OpenChrom and for each time point the percentage of GDP and GTP in the samples was determined. The percentage of GTP left at each time point was normalized to the respective percentage of GTP at t = 0 min. Normalized % GTP left plotted against the time in min. Bar graphs represent the averages and error bars the SD from three independent experiments. P value **<0.01, ***<0.001, using ANOVA one-way test. Source data are available for this figure: SourceData F5.