VPS29 Is Not an Active Metallo-Phosphatase but Is a Rigid Scaffold Required for Retromer Interaction with Accessory Proteins (original) (raw)
Figure 8
VPS29 binds specifically to SNX1 but with low affinity in vitro.
(A) Immunoprecipitations from HeLa cells do not detect association of retromer with SNX1 even in the presence of increased levels of VPS29 expression. Cells expressing GFP-VPS29 or GFP-VPS29(L152E) mutant were subjected to immunoprecipitation with either VPS26 or SNX1 antibodies. VPS26 (and thus VPS29-containing retromer) associates readily with the effector complex containing strumpellin [32], confirming that known binding partners can be detected in the immuno-isolates. However, no SNX1 is detected, and furthermore, in reverse experiments SNX1 does not precipitate retromer indicating that their association in vivo is relatively weak or transient. (B) Titration of VPS29 with SNX1 in NMR experiments reveals specific but weak association in vitro. A selected region is shown for the 15N-HSQC spectra of VPS29 in the presence of increasing concentrations of SNX1. (C) Chemical shift perturbations are shown for VPS29 in the presence of SNX1. Inset shows a plot of the chemical shift perturbation for Leu26 NH as a function of SNX1 concentration. (D) SNX1 binds to VPS29 via the conserved hydrophobic surface on the opposite face to the metal-binding pocket and VPS35 binding interface. Residues that show the largest perturbations on SNX1 binding (>2 standard deviations) are mapped on the VPS29 structure in blue. The structure of VPS29 (surface, and green ribbons) is shown in complex with VPS35(476–780) (red ribbons) [24]. The side-chains of the VPS29 hydrophobic surface are indicated. (E) Mutation of the hydrophobic surface of VPS29 (L152E) prevents VPS29-SNX1 association. The [1H,15N]-HSQC spectra for VPS29(L152E) in the absence (black) and presence (red) of SNX1 indicates no significant association is occurring.