Structural basis of TRAPPIII-mediated Rab1 activation (2021)
Figure 5

Figure 5: The Rab1/Ypt1 HVD points to the membrane binding surface

Cryo-EM density is shown at a low threshold to highlight the almost continuous density connecting the Rab1/Ypt1 nucleotide-binding domain (NBD) to its HVD (hypervariable domain). Inset is a closeup of the density corresponding to the Rab1/Ypt1 HVD region. Density is colored by subunit as in Figure 2, Rab1/Ypt1 is yellow. Density is depicted as it is likely oriented at the surface of organelle membranes.

2D class averages showing extra density in this region in the presence of Rab1/Ypt1. Circle highlights the Rab1/Ypt1 NBD, and the arrow points to the HVD density.

Network diagram of cross-links observed between Trs31 and Rab1/Ypt1. Many cross-links are observed at the C-terminal region of Rab1/Ypt1. Cross-links involving residues present in the atomic model are colored black, while cross-links between unmodeled residues are colored pink.

As in C, but the cross-links are mapped onto the structure. Cross-links involving residues present in the atomic model are colored black, while cross-links between unmodeled residues are colored pink.

E. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, **** p < 0.0001.

F. Quantification of the colocalization of TRAPPIII with mitochondrially-anchored Rab substrates. The sequence of wild-type Rab1/Ypt1 HVD is in black, the substituted residues are colored to correspond with their location on the graph. The "CIM" motif is required for enzymatic prenylation of the Rab, but these chimeras are anchored to the plasma membrane independently of prenylation, via Fis1-protein fusion. Average Mander's Overlap Coefficient (MOC) is plotted with 95% confidence intervals. Each dot represents the MOC for a cropped image containing 2-9 cells. Samples are compared using an ordinary one-way ANOVA with Tukey's multiple comparisons test, but significance indicators are only present for each substitution mutant compared to "wild-type" Ypt31HVDYpt1: vs. 181TTQ p = 0.9924, vs. 184KKE p = 0.9997, vs. 187DKGN * p = 0.0145, vs. 191VNL, 194KGQS, 198LTNT **** p < 0.0001, ns = not significant.

Surface representation of the TRAPPIII-Rab1/Ypt1 complex colored by electrostatic potential. Top panel is oriented as in A, the bottom panel is rotated 90° to visualize the most positive surface which likely lies at the surface of the membrane.

Quantification of in vitro TRAPPIII complex membrane pelleting assays performed with various lipid compositions. The strongest binding was observed with the most negatively charged liposomes. Binding is compared to wild-type (WT) on "Golgi" liposomes using an unpaired, two-tailed Student's t-test with Welch's correction: Golgi vs. Golgi-chol p = 0.1067, vs Anionic ** p = 0.0063, vs. No PS/PA p = 0.0522, vs. 10% PI4P * p = 0.0207, vs. No PI ** p = 0.0035, ns = not significant. The value for each of n=3 independent experiments is depicted. Error bars represent 95% confidence intervals.