A Novel Mammalian Retromer Component, Vps26B

Abstract

The mammalian retromer protein complex, which consists of three proteins -Vps26, Vps29, and Vps35 -in association with members of the sorting nexin family of proteins, has been implicated in the trafficking of receptors and their ligands within the endosomal/lysosomal system of mammalian cells. A bioinformatic analysis of the mouse genome identified an additional transcribed paralog of the Vps26 retromer protein, which we termed Vps26B. No paralogs were identified for Vps29 and Vps35. Phylogenetic studies indicate that the two paralogs of Vps26 become evident after the evolution of the chordates. We propose that the chordate Vps26-like gene published previously be renamed Vps26A to differentiate it from Vps26B. As for Vps26A, biochemical characterization of Vps26B established that this novel 336 amino acid residue protein is a peripheral membrane protein. Vps26B co-precipitated with Vps35 from transfected cells and the direct interaction between these two proteins was confirmed by yeast 2-hybrid analysis, thereby establishing Vps26B as a subunit of the retromer complex. Within HeLa cells, Vps26B was found in the cytoplasm with low levels at the plasma membrane, while Vps26A was predominantly associated with endosomal membranes. Within A549 cells, both Vps26A and Vps26B co-localized with actin-rich lamellipodia at the cell surface. These structures also co-localized with Vps35. Total internal reflection fluorescence microscopy confirmed the association of Vps26B with the plasma membrane in a stable HEK293 cell line expressing cyan fluorescent protein (CFP)-Vps26B. Based on these observations, we propose that the mammalian retromer complex is located at both endosomes and the plasma membrane in some cell types.

Evolutionary reconstruction of the retromer complex and its function in Trypanosoma brucei

“…Comparative genomics revealed evidence of species-specific expansions for Vps26, Vps29 and Vps35, especially in Metazoa, Archaeplastida and T. vaginalis. Evidence for a tissue-specific function for Vps26 isoforms in mouse, as well as distinct intracellular locations (Kerr et al, 2005;Kim et al, 2008), suggests that these expansions could result in diversification of retromer function. The membrane-deforming subcomplex, composed of two sorting nexins, was also highly conserved, although there was more variability within sorting nexin orthologues than for the other retromer components.…”

Visualisation of macropinosome maturation by the recruitment of sorting nexins

“…Cell monolayers were fixed in 0.9% or 4% (v/v) PFA and analysed by indirect immunofluorescence as described previously (Kerr et al, 2005). 3D reconstructions were generated by assembling Z stacks using the LSM 510 META software.…”

“…Yeast two-hybrid assays using the full-length cDNAs for human SNX1 and SNX5 subcloned into pLexA and pYESTrp yeast expression vectors were performed as described by Kerr et al (Kerr et al, 2005). Immunoprecipitation of GFP-SNX5 from HEK-GFP-SNX5 cell monolayers and western immunoblotting were performed as previously described (Kerr et al, 2005).…”

“…Immunoprecipitation of GFP-SNX5 from HEK-GFP-SNX5 cell monolayers and western immunoblotting were performed as previously described (Kerr et al, 2005).…”

A defect in the retromer accessory protein, SNX27, manifests by infantile myoclonic epilepsy and neurodegeneration

“…Formed from the assembly of Vacuolar Protein Sorting 35 (VPS35) with VPS29 and VPS26 [4,5], retromer serves to sort transmembrane proteins away from lysosomal-mediated degradation for recycling back to the cell surface, the trans-Golgi network (TGN) or alternatively, to specialized endomembrane compartments [6-9, 5, 10]. Retromer mediates cargo sorting by coordinating two processes: the sequence-dependent capture of specific cargo and the remodeling of membrane to form isolated cargo-enriched transport carriers [3,11,12].…”