Fig 1: Model and evolution of the VPS29/VARP/TBC1D5 interactions.a Model depicting possible assemblage of VARP, TBC1D5 and their functional partners onto endosomal membrane-attached retromer arches. b–d Hypothesized cellular configuration of VARP, TBC1D5, and VPS29 and associated endosomal partners (coloured as in key) in three reconstructed ancestral nodes. Reading anticlockwise, in the LECA (b), Retromer interacts with Rab7 mediated by TBC1D5, while Varp interacts with Rab21 at the endosome and Plasma membrane. In the opisthokont ancestor (c), the addition of the ankyrin repeats allows for possible interaction of Varp with Vamp7. By the time of the holozoan ancestor (d), all of the relevant domains (including the two Zn-fingernails) had been added to VARP to compete for Vps29 with TBC1D5 using the same conserved motif providing for direct interaction with retromer. e Proposed origin timing of the VPS29 Leu26, Leu152, and Tyr165 residues, the VARP Zn-fingernail, and the TBC1D5 AsnProLeu motif in eukaryotes. This schematic of eukaryotic relationships, with emphasis on the lineages leading to metazoan shows the acquisition points of proteins (bold) and motifs/residues at relevant nodes. See Supplementary Fig. 4 for supporting alignments.
Fig 2: Identification of the VPS35 interactome using a quantitative SILAC proteomic approach reveals novel endosomally localized retromer interactors. (A) Lysates from control RPE1 cells and RPE1 cells with stable suppression of endogenous VPS35 (using an shRNA lentivirus targeting the 3′ UTR), prior to the stable re-expression of GFP–VPS35, were immunoblotted with anti-VPS35 and anti-tubulin antibodies. (B) Gene Ontology annotations revealed a preponderance of proteins involved in ‘establishment of localization’, ‘localization’ and ‘transport’ in the VPS35 interactome. DAVID was used to assign Gene Ontology annotations to proteins identified in the VPS35 SILAC proteomics with a >2.5-fold enrichment and with a minimum of two peptides. The larger the red node, the greater the number of proteins classified in that category; the thicker the edge between nodes, the greater the overlap of proteins within those classifications. (C) The majority of known retromer interactors were found in the VPS35 interactome. Network analysis of VPS35 interactome components identified in the SILAC proteomics was performed using the STRING database. Colours represent protein–protein interactions or protein complexes known to associate. (D) Novel interactions were confirmed by western blotting. Cell extracts derived from RPE1 cells lentivirally transduced with GFP or GFP–VPS35 were subjected to a GFP-nanotrap (GFP-IP) and subsequently analyzed for binding to the indicated proteins. The number of peptides and fold enrichment of the indicated proteins in the VPS35 SILAC proteomics are also indicated. (E) VARP, SDCCAG3 and RME-8 colocalize with VPS35. HeLa cells transiently transfected with VARP–Myc and untransfected HeLa cells were fixed and stained with antibodies raised against Myc, RME-8 or SDCCAG3 and co-stained with an antibody against endogenous VPS35 (red). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm.
Fig 3: Suppression of the novel interactors identified in the VPS35 SILAC proteomics screen affects retromer-mediated endosome-to-plasma-membrane transport. (A) Western blot analysis of lysates derived from HeLa cells that were transfected with siRNAs against the indicated targets. Tubulin is shown as a loading control. (B,C) VPS35, ANKRD50 and FAM21 suppression leads to an increase in the lysosomal accumulation of GLUT1. Immunofluorescent staining of endogenous GLUT1 and the lysosomal marker LAMP1 in HeLa cells deficient for VPS35, FAM21, RME-8, SDCCAG3, VARP or ANKRD50 (B). Boxed areas are shown at higher magnification to the right. Scale bars: 10 µm. (C) The data show the mean±s.e.m. (150 cells acquired in three independent experiments, n = 3); *P<0.05 (unpaired Student's t-test). (D,E) Surface levels of MCT1 and GLUT1 are decreased upon suppression of certain retromer interactors. HeLa cells were transfected with the indicated siRNAs and the surface abundance of GLUT1 and MCT1 was determined by quantitative western blotting (D). The abundance of GLUT1 in total cell lysates is also shown. (E) Graphical representation of the loss of GLUT1 and MCT1 from the surface of HeLa cells transfected with the indicated siRNAs. Data show the mean±s.e.m. [three (MCT1) and six (GLUT1) independent experiments]; *P<0.05 (unpaired Student's t-test).
Fig 4: Analysis of VARP residues 692-746:VPS29 interface.a View of VARP:VPS29 complex rotated from view in Fig. 2 to better show binding surface. b Cut away surface rendering of VPS29 to highlight the hydrophobic pocket in which HisProLeu residues of VARP bind. Key side chains in the interaction are shown and labelled pink (VPS29) or cyan (VARP). c Schematic representation of His, Pro, Leu triplet binding to VPS29. d KDs determined by SPR for mutants in key residues of the VPS29:VARP interface. e Same view as (a) but shown as surface representation with residues whose mutation abolish binding to residues 692-746 of VARP highlighted in red forming a single shallow cavity on the surface of VPS29.
Fig 5: TRAILR1 surface levels are perturbed by the suppression of VPS35 interactors. (A,B) Flow cytometric analysis of surface-resident TRAILR1 in cells deficient for FAM21, RME-8, SDCCAG3, ANKRD50, VARP and VPS35. Data in B show the mean±s.e.m.; *P<0.025 (unpaired Student's t-test). (C) HeLa cells transfected with siRNA against the indicated targets were fixed and stained to examine the distribution of endogenous TRAILR1 and its colocalization with LAMP1-decorated late endosomes and lysosomes. Boxed areas are shown at higher magnification above the main image. Scale bars: 10 µm.
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