The mammalian retromer regulates transcytosis of the polymeric immunoglobulin receptor

Epithelial cells have separate apical and basolateral plasma membrane domains with distinct compositions. After delivery to one surface, proteins can be endocytosed and then recycled, degraded or transcytosed to the opposite surface. Proper sorting into the transcytotic pathway is essential for maintaining polarity, as most proteins are endocytosed many times during their lifespan. The polymeric immunoglobulin receptor (pIgR) transcytoses polymeric IgA (pIgA) from the basolateral to the apical surface of epithelial cells and hepatocytes. However, the molecular machinery that controls polarized sorting of pIgR-pIgA and other receptors is only partially understood. The retromer is a multimeric protein complex, originally described in yeast, which mediates intracellular sorting of Vps10p, a receptor that transports vacuolar enzymes. The yeast retromer contains two sub-complexes. One includes the Vps5p and Vps17p subunits, which provide mechanical force for vesicle budding. The other is the Vps35p-Vps29p-Vps26p subcomplex, which provides cargo specificity. The mammalian retromer binds to the mannose 6-phosphate receptor, which sorts lysosomal enzymes from the trans-Golgi network to the lysosomal pathway. Here, we show a function for the mammalian Vps35-Vps29-Vps26 retromer subcomplex in promoting pIgR-pIgA transcytosis.     

Identification of a conserved motif required for Vps35p/Vps26p interaction and assembly of the retromer complex

The retromer complex is a conserved cytoplasmic coat complex that mediates the endosome-to-Golgi retrieval of vacuole/lysosome hydrolase receptors in yeast and mammals. The recognition of cargo proteins by the retromer is performed by the Vps35p/VPS35 (where Vps is vacuolar protein sorting) component, which together with Vps26p/VPS26 and Vps29p/VPS29, forms the cargo-selective subcomplex. In this report, we have identified a highly-conserved region of Vps35p/VPS35 that is essential for the interaction with Vps26p/VPS26 and for assembly of the retromer complex. Mutation of residues within the conserved region results in Vps35p/VPS35 mutants, which cannot bind to Vps26p/VPS26 and are not efficiently targeted to the endosomal membrane. These data implicate Vps26p/VPS26 in regulating Vps35p/VPS35 membrane association and therefore suggest a role for Vps26p/VPS26 in cargo recognition.     

Dominant-negative behavior of mammalian Vps35 in yeast requires a conserved PRLYL motif involved in retromer assembly

The retromer protein complex assists in recycling selected integral membrane proteins from endosomes to the trans Golgi network. One protein subcomplex (Vps35p, Vps26p and Vps29p) combines with a second (Vps17p and Vps5p) to form a coat involved in sorting and budding of endosomal vesicles. Yeast Vps35p (yVps35) exhibits similarity to human Vps35 (hVps35), especially in a completely conserved PRLYL motif contained within an amino-terminal domain. Companion studies indicate that an R(98)W mutation in yVps35 causes defective retromer assembly in Saccharomyces cerevisiae. Herein, we find that the expression of hVps35 in yeast confers dominant-negative vacuolar proenzyme secretion and defective secretory proprotein processing. The mutant phenotype appears to be driven by hVps35 competing with endogenous yVps35, becoming incorporated into defective retromer complexes and causing proteasomal degradation of endogenous Vps26 and Vps29. Increased expression of yVps35 displaces some hVps35 to a 100 000 x g supernatant and suppresses the dominant-negative phenotype. Remarkably, mutation of the conserved R(107)W of hVps35 displaces some of the protein to the 100 000 x g supernatant, slows protein turnover and restores stability of Vps26p and Vps29p and completely abrogates dominant-negative trafficking behavior. We show that hVps35 coprecipitates Vps26, whereas the R(107)W mutant does not. In pancreatic beta cells, the R(107)W mutant shifts hVps35 from peripheral endosomes to a juxtanuclear compartment, affecting both mannose phosphate receptors and insulin. These data underscore importance of the Vps35 PRLYL motif in retromer subcomplex interactions and function.     

The retromer subunit Vps26 has an arrestin fold and binds Vps35 through its C-terminal domain

The mammalian retromer complex consists of SNX1, SNX2, Vps26, Vps29 and Vps35, and retrieves lysosomal enzyme receptors from endosomes to the trans-Golgi network. The structure of human Vps26A at 2.1-A resolution reveals two curved beta-sandwich domains connected by a polar core and a flexible linker. Vps26 has an unpredicted structural relationship to arrestins. The Vps35-binding site on Vps26 maps to a mobile loop spanning residues 235-246, near the tip of the C-terminal domain. The loop is phylogenetically conserved and provides a mechanism for Vps26 integration into the complex that leaves the rest of the structure free for engagements with membranes and for conformational changes. Hydrophobic residues and a glycine in this loop are required for integration into the retromer complex and endosomal localization of human Vps26, and for the function of yeast Vps26 in carboxypeptidase Y sorting.     

Vps26p, a component of retromer, directs the interactions of Vps35p in endosome-to-Golgi retrieval

Endosome-to-Golgi retrieval of the carboxypeptidase Y receptor Vps10p is mediated by a recently discovered membrane coat complex termed retromer. Retromer comprises five conserved proteins: Vps35p, Vps29p, Vps5p, Vps17p, and Vps26p. Vps35p recognizes cargo molecules such as Vps10p and interacts strongly with Vps29p. Vps5p forms a subcomplex with Vps17p and has been proposed to play a structural role by self-assembling into large multimeric structures. The function of Vps26p is currently unknown. We have investigated the role that Vps26p plays in retromer-mediated endosome-to-Golgi transport by analyzing dominant negative alleles of Vps26p. These mutants have identified a crucial region of Vps26p that plays an important role in its function. Functional domains of Vps26p have been investigated by the creation of yeast-mouse hybrid molecules in which domains of Vps26p have been replaced by the similar domain in the protein encoded by the mouse VPS26 gene, Hbeta58. These domain swap experiments have shown that Vps26p promotes the interactions between the cargo-selective component Vps35p and the structural components Vps5p/Vps17p.     

Structure of Vps26B and mapping of its interaction with the retromer protein complex

Retromer is a heteromeric protein complex with important roles in endosomal membrane trafficking, most notably in the retrograde transport of lysosomal hydrolase receptors from endosomes to the Golgi. The core of retromer is composed of three subunits vacuolar protein sorting (Vps)35, Vps26 and Vps29, and in mammals, there are two paralogues of the medium subunit Vps26A and Vps26B. We find that both Vps26A and Vps26B bind to Vps35/Vps29 with nanomolar affinity and compete for a single-binding site to define distinct retromer complexes in vitro and in vivo. We have determined the crystal structure of mouse Vps26B and compare this structure with that of Vps26A. Vps26 proteins have a striking similarity to the arrestin family of proteins that regulate the signalling and endocytosis of G-protein-coupled receptors, although we observe that surface residues involved in arrestin function are not conserved in Vps26. Using structure-based mutagenesis, we show that both Vps26A and Vps26B are incorporated into retromer complexes through binding of Vps35 to a highly conserved surface patch within the C-terminal subdomain and that this interaction is required for endosomal recruitment of the proteins.     

Human homologues of yeast vacuolar protein sorting 29 and 35

In the yeast Saccharomyces cerevisiae, a membrane coat complex is required for endosome to Golgi retrograde transport. The vacuolar protein sorting proteins Vps29p, Vps35p, and Vps26p are required for pre-vacuolar/late endosome to Golgi retrieval of the vacuolar hydrolase receptor Vps10p. They form a cargo recognition and concentration subcomplex, termed the inner shell of the retromer coat, prior to vesicle formation by the addition of the membrane-deforming outer shell. We have cloned the human and murine homologues of yeast Vps29p and the human homologue of Vps35p. They encode 182 and 796 residue proteins, with 43 and 29% identity to their respective yeast. The 10.5 kb, 5 exon, VPS29 gene is located on chromosome 12q24 and the 29.6 kb, 17 exon, VPS35 gene is on chromosome 16. In humans, Vps29p, Vps35p, and Hbeta58, the homologue of Vps26p, may form an inner shell of the retromer coat similar to that found in yeast.     

Regulation of retromer recruitment to endosomes by sequential action of Rab5 and Rab7

The retromer complex mediates retrograde transport of transmembrane cargo from endosomes to the trans-Golgi network (TGN). Mammalian retromer is composed of a sorting nexin (SNX) dimer that binds to phosphatidylinositol 3-phosphate–enriched endosomal membranes and a vacuolar protein sorting (Vps) 26/29/35 trimer that participates in cargo recognition. The mammalian SNX dimer is necessary but not sufficient for recruitment of the Vps26/29/35 trimer to membranes. In this study, we demonstrate that the guanosine triphosphatase Rab7 contributes to this recruitment. The Vps26/29/35 trimer specifically binds to Rab7–guanosine triphosphate (GTP) and localizes to Rab7-containing endosomal domains. Interference with Rab7 function causes dissociation of the Vps26/29/35 trimer but not the SNX dimer from membranes. This blocks retrieval of mannose 6-phosphate receptors to the TGN and impairs cathepsin D sorting. Rab5-GTP does not bind to the Vps26/29/35 trimer, but perturbation of Rab5 function causes dissociation of both the SNX and Vps26/29/35 components from membranes through inhibition of a pathway involving phosphatidylinositol 3-kinase. These findings demonstrate that Rab5 and Rab7 act in concert to regulate retromer recruitment to endosomes.     

Phosphoinositide 3-kinase regulates the role of retromer in transcytosis of the polymeric immunoglobulin receptor

Retromer is a multimeric protein complex that mediates intracellular receptor sorting. One of the roles of retromer is to promote transcytosis of the polymeric immunoglobulin receptor (pIgR) and its ligand polymeric immunoglobulin A (pIgA) in polarized epithelial cells. In Madin-Darby Canine Kidney (MDCK) cells, overexpression of Vps35, the retromer subunit key for cargo recognition, restores transcytosis to a pIgR mutant that is normally degraded. Here we show that pIgA transcytosis was not restored in these cells when treated with the specific phosphoinositide 3-kinase (PI3K) inhibitor LY294002. Likewise, the decrease in pIgA transcytosis by wild-type pIgR seen upon PI3K inhibition was not reverted by Vps35 overexpression. PI3K inhibition reduced membrane association of sorting-nexins (SNX) 1 and 2, which constitute the retromer subcomplex involved in membrane deformation, while association of the Vps35-Vps26-Vps29 subcomplex, involved in cargo recognition, remained virtually unaffected. Colocalization between the two retromer subcomplexes was reduced upon the treatment. Whereas the interaction among the subunits of the Vps35-Vps26-Vps29 subcomplex remained unchanged, less Vps35 was found associated with pIgR upon PI3K inhibition. In addition, colocalization of internalized pIgA with subunits of both retromer subcomplexes throughout the transcytotic pathway was substantially reduced by LY294002 treatment. These data implicate PI3K in controlling retromer's role in pIgR-pIgA transcytosis.     

A novel mammalian retromer component, Vps26B

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.     

Rab GTPase regulation of retromer-mediated cargo export during endosome maturation

The retromer complex, composed of sorting nexin subunits and a Vps26/Vps29/Vps35 trimer, mediates sorting of retrograde cargo from the endosome to the trans-Golgi network. The retromer trimer subcomplex is an effector of Rab7 (Ypt7 in yeast). Whereas endosome targeting of human retromer has been shown to require Rab7-GTP, targeting of yeast retromer to the endosome is independent of Ypt7-GTP and requires the Vps5 and Vps17 retromer sorting nexin subunits. An evolutionarily conserved amino acid segment within Vps35 is required for Ypt7/Rab7 recognition in vivo by both yeast and human retromer, establishing that Rab recognition is a conserved feature of this subunit. Recognition of Ypt7 by retromer is required for its function in retrograde sorting, and in yeast cells lacking the guanine nucleotide exchange factor for Ypt7, retrograde cargo accumulates in endosomes that are decorated with retromer, revealing an additional role for Rab recognition at the cargo export stage of the retromer functional cycle. In addition, yeast retromer trimer antagonizes Ypt7-regulated organelle tethering and fusion of endosomes/vacuoles via recognition of Ypt7. Thus retromer has dual roles in retrograde cargo export and in controlling the fusion dynamics of the late endovacuolar system.     

Interchangeable but essential functions of SNX1 and SNX2 in the association of retromer with endosomes and the trafficking of mannose 6-phosphate receptors

The retromer is a cytosolic/peripheral membrane protein complex that mediates the retrieval of the cation-independent mannose 6-phosphate receptor from endosomes to the trans-Golgi network (TGN) in mammalian cells. Previous studies showed that the mammalian retromer comprises three proteins, named Vps26, Vps29, and Vps35, plus the sorting nexin, SNX1. There is conflicting evidence, however, as to whether a homologous sorting nexin, SNX2, is truly a component of the retromer. In addition, the nature of the subunit interactions and assembly of the mammalian retromer complex are poorly understood. We have addressed these issues by performing biochemical and functional analyses of endogenous retromers in the human cell line HeLa. We found that the mammalian retromer complex consists of two autonomously assembling subcomplexes, namely, a Vps26-Vps29-Vps35 obligate heterotrimer and a SNX1/2 alternative heterodimer or homodimer. The association of Vps26-Vps29-Vps35 with endosomes requires the presence of either SNX1 or SNX2, whereas SNX1/2 can be recruited to endosomes independently of Vps26-Vps29-Vps35. We also found that the presence of either SNX1 or SNX2 is essential for the retrieval of the cation-independent mannose 6-phosphate receptor to the TGN. These observations indicate that the mammalian retromer complex assembles by sequential association of SNX1/2 and Vps26-Vps29-Vps35 subcomplexes on endosomal membranes and that SNX1 and SNX2 play interchangeable but essential roles in retromer structure and function.     

Structural features of vps35p involved in interaction with other subunits of the retromer complex

The penta-subunit retromer complex of yeast mediates selective retrieval of membrane proteins from the prevacuolar endosome to the trans Golgi network. In this study, we set out to generate a panel of vps35 dominant-negative mutants that disrupt retromer-mediated cargo sorting. Mapping of the mutations revealed two types of alterations leading to dominant-negative behavior of the 944-amino acid protein: (i) mutations at or near the R(98) residue or (ii) C-terminal truncations exemplified by a nonsense mutation at codon 733. Both could be suppressed by overexpression of wild-type Vps35p, suggesting that these dominant-negative mutants compete for interactions with other retromer subunits. Interestingly, Vps35-R(98)W expression destabilized Vps26p while having no effect on Vps29p stability, while Vps35-Q(733)* expression affected Vps29p stability but had no effect on Vps26p. Measurement of Vps35/Vps26 and Vps35/Vps29 pairwise associations by coimmunoprecipitation in the presence or absence of other retromer subunits indicated that the R(98) residue, which is part of a conserved PRLYL motif, is critical for Vps35p binding to Vps26p, while both R(98) and residues 733-944 are needed for efficient binding to Vps29p.     

A Membrane Coat Complex Essential for Endosome-to-Golgi Retrograde Transport in Yeast

We have recently characterized three yeast gene products (Vps35p, Vps29p, and Vps30p) as candidate components of the sorting machinery required for the endosome-to-Golgi retrieval of the vacuolar protein sorting receptor Vps10p (Seaman, M.N.J., E.G. Marcusson, J.-L. Cereghino, and S.D. Emr. 1997. J. Cell Biol. 137:79–92). By genetic and biochemical means we now show that Vps35p and Vps29p interact and form part of a multimeric membrane-associated complex that also contains Vps26p, Vps17p, and Vps5p. This complex, designated here as the retromer complex, assembles from two distinct subcomplexes comprising (a) Vps35p, Vps29p, and Vps26p; and (b) Vps5p and Vps17p. Density gradient fractionation of Golgi/endosomal/vesicular membranes reveals that Vps35p cofractionates with Vps5p/Vps17p in a vesicle-enriched dense membrane fraction. Furthermore, gel filtration analysis indicates that Vps35p and Vps5p are present on a population of vesicles and tubules slightly larger than COPI/coatomer-coated vesicles. We also show by immunogold EM that Vps5p is localized to discrete regions at the rims of the prevacuolar endosome where vesicles appear to be budding. Size fractionation of cytosolic and recombinant Vps5p reveals that Vps5p can self-assemble in vitro, suggesting that Vps5p may provide the mechanical impetus to drive vesicle formation. Based on these findings we propose a model in which Vps35p/Vps29p/Vps26p function to select cargo for retrieval, and Vps5p/Vps17p assemble onto the membrane to promote vesicle formation. Conservation of the yeast retromer complex components in higher eukaryotes suggests an important general role for this complex in endosome-to-Golgi retrieval.     

Inhibition of retromer activity by herpesvirus saimiri tip leads to CD4 downregulation and efficient T cell transformation

The mammalian retromer is an evolutionally conserved protein complex composed of a vacuolar protein sorting trimer (Vps 26/29/35) that participates in cargo recognition and a sorting nexin (SNX) dimer that binds to endosomal membranes. The retromer plays an important role in efficient retrograde transport for endosome-to-Golgi retrieval of the cation-independent mannose-6-phosphate receptor (CI-MPR), a receptor for lysosomal hydrolases, and other endosomal proteins. This ultimately contributes to the control of cell growth, cell adhesion, and cell migration. The herpesvirus saimiri (HVS) tyrosine kinase-interacting protein (Tip), required for the immortalization of primary T lymphocytes, targets cellular signaling molecules, including Lck tyrosine kinases and the p80 endosomal trafficking protein. Despite the pronounced effects of HVS Tip on T cell signal transduction, the details of its activity on T cell immortalization remain elusive. Here, we report that the amino-terminal conserved, glutamate-rich sequence of Tip specifically interacts with the retromer subunit Vps35 and that this interaction not only causes the redistribution of Vps35 from the early endosome to the lysosome but also drastically inhibits retromer activity, as measured by decreased levels of CI-MPR and lower activities of cellular lysosomal hydrolases. Physiologically, the inhibition of intracellular retromer activity by Tip is ultimately linked to the downregulation of CD4 surface expression and to the efficient in vitro immortalization of primary human T cells to interleukin-2 (IL-2)-independent permanent growth. Therefore, HVS Tip uniquely targets the retromer complex to impair the intracellular trafficking functions of infected cells, ultimately contributing to efficient T cell transformation.     

The human Vps29 retromer component is a metallo-phosphoesterase for a cation-independent mannose 6-phosphate receptor substrate peptide

The retromer complex is involved in the retrograde transport of the CI-M6PR (cation-independent mannose 6-phosphate receptor) from endosomes to the Golgi. It is a hetero-trimeric complex composed of Vps26 (vacuolar sorting protein 26), Vps29 and Vps35 proteins, which are conserved in eukaryote evolution. Recently, elucidation of the crystal structure of Vps29 revealed that Vps29 contains a metallo-phosphoesterase fold [Wang, Guo, Liang, Fan, Zhu, Zang, Zhu, Li, Teng, Niu et al. (2005) J. Biol. Chem. 280, 22962-22967; Collins, Skinner, Watson, Seaman and Owen (2005) Nat. Struct. Mol. Biol. 12, 594-602]. We demonstrate that recombinant hVps29 (human Vps29) displays in vitro phosphatase activity towards a serine-phosphorylated peptide, containing the acidic-cluster dileucine motif of the cytoplasmatic tail of the CI-M6PR. Efficient dephosphorylation required the additional presence of recombinant hVps26 and hVps35 proteins, which interact with hVps29. Phosphatase activity of hVps29 was greatly decreased by alanine substitutions of active-site residues that are predicted to co-ordinate metal ions. Using inductively coupled plasma MS, we demonstrate that recombinant hVps29 binds zinc. Moreover, hVps29-dependent phosphatase activity is greatly reduced by non-specific and zinc-specific metal ion chelators, which can be completely restored by addition of excess ZnCl2. The binuclear Zn2+ centre and phosphate group were modelled into the hVps29 catalytic site and pKa calculations provided further insight into the molecular mechanisms of Vps29 phosphatase activity. We conclude that the retromer complex displays Vps29-dependent in vitro phosphatase activity towards a serinephosphorylated acidic-cluster dileucine motif that is involved in endosomal trafficking of the CI-M6PR. The potential significance of these findings with respect to regulation of transport of cycling trans-Golgi network proteins is discussed.     

The Vps35 D620N Mutation Linked to Parkinson's Disease Disrupts the Cargo Sorting Function of Retromer

The retromer is a trimeric cargo‐recognition protein complex composed of Vps26, Vps29 and Vps35 associated with protein trafficking within endosomes. Recently, a pathogenic point mutation within the Vps35 subunit (D620N) was linked to the manifestation of Parkinson's disease (PD). Here, we investigated details underlying the molecular mechanism by which the D620N mutation in Vps35 modulates retromer function, including examination of retromer's subcellular localization and its capacity to sort cargo. We show that expression of the PD‐linked Vps35 D620N mutant redistributes retromer‐positive endosomes to a perinuclear subcellular localization and that these endosomes are enlarged in both model cell lines and fibroblasts isolated from a PD patient. Vps35 D620N is correctly folded and binds Vps29 and Vps26A with the same affinity as wild‐type Vps35. While PD‐linked point mutant Vps35 D620N interacts with the cation‐independent mannose‐6‐phosphate receptor (CI‐M6PR), a known retromer cargo, we find that its expression disrupts the trafficking of cathepsin D, a CI‐M6PR ligand and protease responsible for degradation of α‐synuclein, a causative agent of PD. In summary, we find that the expression of Vps35 D620N leads to endosomal alterations and trafficking defects that may partly explain its action in PD.     

Molecular Insights into Rab7‐Mediated Endosomal Recruitment of Core Retromer: Deciphering the Role of Vps26 and Vps35

Retromer, a peripheral membrane protein complex, plays an instrumental role in host of cellular processes by its ability to recycle receptors from endosomes to the trans‐Golgi network. It consists of two distinct sub‐complexes, a membrane recognizing, sorting nexins (SNX) complex and a cargo recognition, vacuolar protein sorting (Vps) complex. Small GTPase, Rab7 is known to recruit retromer on endosomal membrane via interactions with the Vps sub‐complex. The molecular mechanism underlying the recruitment process including the role of individual Vps proteins is yet to be deciphered. In this study, we developed a FRET‐based assay in HeLa cells that demonstrated the interaction of Rab7 with Vps35 and Vps26 in vivo. Furthermore, we showed that Rab7 recruits retromer to late endosomes via direct interactions with N‐terminal conserved regions in Vps35. However, the single point mutation, which disrupts the interaction between Vps35 and Vps26, perturbed the Rab7‐mediated recruitment of retromer in HeLa cells. Using biophysical measurements, we demonstrate that the association of Vps26 with Vps35 resulted in high affinity binding between the Vps sub‐complex and the activated Rab7 suggesting for a possible allosteric role of Vps26. Thus, this study provides molecular insights into the essential role of Vps26 and Vps35 in Rab7‐mediated recruitment of the core retromer complex.      

Implication of mouse Vps26b-Vps29-Vps35 retromer complex in sortilin trafficking

The retromer complex, which mediates retrograde transport from endosomes to the trans-Golgi network, is a heteropentameric complex that contains a multifunctional cargo recognition heterotrimer consisted of the vacuolar protein sorting (Vps) subunits Vps26, Vps29, and Vps35. In mammals, there are two different isoforms of Vps26, Vps26a and Vps26b, that localize to the endosome, and to the plasma membrane, respectively. To elucidate the biological significance of the Vps26b isoform, we generated Vps26b knockout mice and studied their molecular, histological, and behavioral phenotypes. We found that the loss of Vps26b results in no significant defects in the behavior, body size, and health of the mice. Vps26b-deficient mice showed a severe reduction of Vps35 protein at cellular level and lacked the Vps26b-Vps29-Vps35 retromer complex, despite the normal presence of the Vps26a-Vps29-Vps35 retromer complex. Relatively, the amount of sortilin was increased approximately 20% in the Vps26b-deficient mice, whereas the sorLA was normal. These results suggest that mouse Vps26b-Vps29-Vps35 retromer complex is implicated in the transport of sortilin from endosomes to the trans-Golgi network (TGN).     

Vps26A and Vps26B subunits define distinct retromer complexes

The trimeric Vps29-Vps35-Vps26 sub-complex of retromer mediates retrograde transport of transmembrane proteins from endosomes to the trans-Golgi network. Our group has recently identified a Vps26 paralogue, Vps26B, which is able to suppress the expression of Vps26A when exogenously expressed in mammalian cells and defines a distinct retromer complex (Vps26B-retromer) in vivo and in vitro. In this study, we use HEK293 cells stably expressing either Vps26A-myc or Vps26B-myc to address the role of retromer cargo transport and subcellular localization of the two core retromer complexes as defined by the two mammalian-specific Vps26 paralogues. Vps26B-retromer, like Vps26A-retromer, associates with TBC1D5 and GOLPH3. In contrast, no interaction between Vps26B-retromer and cation-independent mannose 6-phosphate receptor (CI-M6PR) was detected, leading to a degradation of this receptor and an increase in cathepsin D secretion. Colocalization of Vps26 paralogues with different endosomally located Rab proteins shows prolonged association of Vps26B-retromer with maturing endosomes relative to Vps26A-retromer. Interestingly, the cycling of CI-M6PR is restored upon deletion of the variable Vps26B C-terminal region indicating that this region is directly responsible for the differential function of the two paralogues. In summary, we show that the two distinct retromer complexes defined by different Vps26 paralogues are not functionally equivalent and that the Vps26B C-terminal region can control cargo selection of the Vps26B-retromer.