Direct Binding of Retromer to Human Papillomavirus Type 16 Minor Capsid Protein L2 Mediates Endosome Exit during Viral Infection

Trafficking of human papillomaviruses to the Golgi apparatus during virus entry requires retromer, an endosomal coat protein complex that mediates the vesicular transport of cellular transmembrane proteins from the endosome to the Golgi apparatus or the plasma membrane. Here we show that the HPV16 L2 minor capsid protein is a retromer cargo, even though L2 is not a transmembrane protein. We show that direct binding of retromer to a conserved sequence in the carboxy-terminus of L2 is required for exit of L2 from the early endosome and delivery to the trans-Golgi network during virus entry. This binding site is different from known retromer binding motifs and can be replaced by a sorting signal from a cellular retromer cargo. Thus, HPV16 is an unconventional particulate retromer cargo, and retromer binding initiates retrograde transport of viral components from the endosome to the trans-Golgi network during virus entry. We propose that the carboxy-terminal segment of L2 protein protrudes through the endosomal membrane and is accessed by retromer in the cytoplasm.     

Two novel WD40 domain-containing proteins, Ere1 and Ere2, function in the retromer-mediated endosomal recycling pathway

Regulated secretion, nutrient uptake, and responses to extracellular signals depend on cell-surface proteins that are internalized and recycled back to the plasma membrane. However, the underlying mechanisms that govern membrane protein recycling to the cell surface are not fully known. Using a chemical-genetic screen in yeast, we show that the arginine transporter Can1 is recycled back to the cell surface via two independent pathways mediated by the sorting nexins Snx4/41/42 and the retromer complex, respectively. In addition, we identify two novel WD40-domain endosomal recycling proteins, Ere1 and Ere2, that function in the retromer pathway. Ere1 is required for Can1 recycling via the retromer-mediated pathway, but it is not required for the transport of other retromer cargoes, such as Vps10 and Ftr1. Biochemical studies reveal that Ere1 physically interacts with internalized Can1. Ere2 is present in a complex containing Ere1 on endosomes and functions as a regulator of Ere1. Taken together, our results suggest that Snx4/41/42 and the retromer comprise two independent pathways for the recycling of internalized cell-surface proteins. Moreover, a complex containing the two novel proteins Ere1 and Ere2 mediates cargo-specific recognition by the retromer pathway.     

Recruitment of the endosomal WASH complex is mediated by the extended 'tail' of Fam21 binding to the retromer protein Vps35

The retromer complex is a conserved endosomal protein sorting complex that sorts membrane proteins into nascent endosomal tubules. The recognition of membrane proteins is mediated by the cargo-selective retromer complex, a stable trimer of the Vps35 (vacuolar protein sorting 35), Vps29 and Vps26 proteins. We have recently reported that the cargo-selective retromer complex associates with the WASH (Wiskott-Aldrich syndrome homologue) complex, a multimeric protein complex that regulates tubule dynamics at endosomes. In the present study, we show that the retromer-WASH complex interaction occurs through the long unstructured 'tail' domain of the WASH complex-Fam21 protein binding to Vps35, an interaction that is necessary and sufficient to target the WASH complex to endosomes. The Fam21-tail also binds to FKBP15 (FK506-binding protein 15), a protein associated with ulcerative colitis, to mediate the membrane association of FKBP15. Elevated Fam21-tail expression inhibits the association of the WASH complex with retromer, resulting in increased cytoplasmic WASH complex. Additionally, overexpression of the Fam21-tail results in cell-spreading defects, implicating the activity of the WASH complex in regulating the mobilization of membrane into the endosome-to-cell surface pathway.     

Endosomal functions in plants

Plant endosomes are highly dynamic organelles that are involved in the constitutive recycling of plasma membrane cargo and the trafficking of polarized plasma membrane proteins such as auxin carriers. In addition, recent studies have shown that surface receptors such as the plant defense-related FLS2 receptor and the brassinosteroid receptor BRI1 appear to signal from endosomes upon ligand binding and internalization. In yeast and mammals, endosomes are also known to recycle vacuolar cargo receptors back to the trans Golgi network and sort membrane proteins for degradation in the vacuole/lysosome. Some of these sorting mechanisms are mediated by the retromer and endosomal sorting complex required for transport (ESCRT) complexes. Plants contain orthologs of all major retromer and ESCRT complex subunits, but they have also evolved variations in endosomal functions connected to plant-specific features such as the diversity of vacuolar transport pathways. This review focuses on recent studies in plants dealing with the regulation of endosomal recycling functions, architecture and formation of multivesicular bodies, ligand-mediated endocytosis and receptor signaling from endosomes as well as novel endosomal markers and the function of endosomes in the transport and processing of soluble vacuolar proteins.     

Recent advances in retromer biology

The endosomal network is an organized array of intracellular, membranous compartments that function as sorting sites for endosomal and biosynthetic cargo. The fate of endocytic cargo is reliant upon interactions with a number of molecularly distinct sorting complexes, which tightly control the relationship between sorting of their respective cargo and the physical process of membrane re-scuplturing required for the formation of transport carries. One such complex, retromer, mediates retrograde transport from endosomes to the trans-Golgi network (TGN). Disregulation of retromer has been implicated in a host of disease states including late-onset Alzheimer's. Rather than give a broad overview of retromer biology, here we aim to outline the recent advances in understanding this complex, focussing on the involvement of both clathrin and the cytoskeleton in retromer function.     

Analyses of sorting nexins reveal distinct retromer-subcomplex functions in development and protein sorting in Arabidopsis thaliana

Sorting nexins (SNXs) are conserved eukaryotic proteins that associate with three types of vacuolar protein sorting (VPS) proteins to form the retromer complex. How SNXs act in this complex and whether they might work independently of the retromer remains elusive. Here, we show by genetic and cell imaging approaches that the Arabidopsis thaliana SNX1 protein recruits SNX2 at the endosomal membrane, a process required for SNX1-SNX2 dimer activity. We report that, in contrast with the mammalian retromer, SNXs are dispensable for membrane binding and function of the retromer complex. We also show that VPS retromer components can work with or independently of SNXs in the trafficking of seed storage proteins, which reveals distinct functions for subcomplexes of the plant retromer. Finally, we provide compelling evidence that the combined loss of function of SNXs and VPS29 leads to embryo or seedling lethality, underlining the essential role of these proteins in development.     

VPS29 is not an active metallo-phosphatase but is a rigid scaffold required for retromer interaction with accessory proteins

VPS29 is a key component of the cargo-binding core complex of retromer, a protein assembly with diverse roles in transport of receptors within the endosomal system. VPS29 has a fold related to metal-binding phosphatases and mediates interactions between retromer and other regulatory proteins. In this study we examine the functional interactions of mammalian VPS29, using X-ray crystallography and NMR spectroscopy. We find that although VPS29 can coordinate metal ions Mn(2+) and Zn(2+) in both the putative active site and at other locations, the affinity for metals is low, and lack of activity in phosphatase assays using a putative peptide substrate support the conclusion that VPS29 is not a functional metalloenzyme. There is evidence that structural elements of VPS29 critical for binding the retromer subunit VPS35 may undergo both metal-dependent and independent conformational changes regulating complex formation, however studies using ITC and NMR residual dipolar coupling (RDC) measurements show that this is not the case. Finally, NMR chemical shift mapping indicates that VPS29 is able to associate with SNX1 via a conserved hydrophobic surface, but with a low affinity that suggests additional interactions will be required to stabilise the complex in vivo. Our conclusion is that VPS29 is a metal ion-independent, rigid scaffolding domain, which is essential but not sufficient for incorporation of retromer into functional endosomal transport assemblies.     

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.     

Regulation of endosomal clathrin and retromer‐mediated endosome to Golgi retrograde transport by the J‐domain protein RME‐8

After endocytosis, most cargo enters the pleiomorphic early endosomes in which sorting occurs. As endosomes mature, transmembrane cargo can be sequestered into inwardly budding vesicles for degradation, or can exit the endosome in membrane tubules for recycling to the plasma membrane, the recycling endosome, or the Golgi apparatus. Endosome to Golgi transport requires the retromer complex. Without retromer, recycling cargo such as the MIG‐14/Wntless protein aberrantly enters the degradative pathway and is depleted from the Golgi. Endosome‐associated clathrin also affects the recycling of retrograde cargo and has been shown to function in the formation of endosomal subdomains. Here, we find that the Caemorhabditis elegans endosomal J‐domain protein RME‐8 associates with the retromer component SNX‐1. Loss of SNX‐1, RME‐8, or the clathrin chaperone Hsc70/HSP‐1 leads to over‐accumulation of endosomal clathrin, reduced clathrin dynamics, and missorting of MIG‐14 to the lysosome. Our results indicate a mechanism, whereby retromer can regulate endosomal clathrin dynamics through RME‐8 and Hsc70, promoting the sorting of recycling cargo into the retrograde pathway.     

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.     

Rab5-family guanine nucleotide exchange factors bind retromer and promote its recruitment to endosomes

The retromer complex facilitates the sorting of integral membrane proteins from the endosome to the late Golgi. In mammalian cells, the efficient recruitment of retromer to endosomes requires the lipid phosphatidylinositol 3-phosphate (PI3P) as well as Rab5 and Rab7 GTPases. However, in yeast, the role of Rabs in recruiting retromer to endosomes is less clear. We identified novel physical interactions between retromer and the Saccharomyces cerevisiae VPS9-domain Rab5-family guanine nucleotide exchange factors (GEFs) Muk1 and Vps9. Furthermore, we identified a new yeast VPS9 domain-containing protein, VARP-like 1 (Vrl1), which is related to the human VARP protein. All three VPS9 domain–containing proteins show localization to endosomes, and the presence of any one of them is necessary for the endosomal recruitment of retromer. We find that expression of an active VPS9-domain protein is required for correct localization of the phosphatidylinositol 3-kinase Vps34 and the production of endosomal PI3P. These results suggest that VPS9 GEFs promote retromer recruitment by establishing PI3P-enriched domains at the endosomal membrane. The interaction of retromer with distinct VPS9 GEFs could thus link GEF-dependent regulatory inputs to the temporal or spatial coordination of retromer assembly or function.     

Mechanisms Governing the Endosomal Membrane Recruitment of the Core Retromer in Arabidopsis

The retromer complex localizes to endosomal membranes and is involved in protein trafficking. In mammals, it is composed of a dimer of sorting nexins and of the core retromer consisting of vacuolar protein sorting (VPS)26, VPS29, and VPS35. Although homologs of these proteins have been identified in plants, how the plant retromer functions remains elusive. To better understand the role of VPS components in the assembly and function of the core retromer, we characterize here Arabidopsis vps26-null mutants. We show that impaired VPS26 function has a dramatic effect on VPS35 levels and causes severe phenotypic defects similar to those observed in vps29-null mutants. This implies that functions of plant VPS26, VPS29, and VPS35 are tightly linked. Then, by combining live-cell imaging with immunochemical and genetic approaches, we report that VPS35 alone is able to bind to endosomal membranes and plays an essential role in VPS26 and VPS29 membrane recruitment. We also show that the Arabidopsis Rab7 homolog RABG3f participates in the recruitment of the core retromer to the endosomal membrane by interacting with VPS35. Altogether our data provide original information on the molecular interactions that mediate assembly of the core retromer in plants.     

Trying to make sense of retromer

Retromer is a cytosolic protein complex which binds to post-Golgi organelles involved in the trafficking of proteins to the lytic compartment of the cell. In non-plant organisms, retromer mediates the recycling of acid hydrolase receptors from early endosomal (EE) compartments. In plants, retromer components are required for the targeting of vacuolar storage proteins, and for the recycling of endocytosed PIN proteins. However, there are contradictory reports as to the localization of the sorting nexins and the core subunit of retromer. There is also uncertainty as to the identity of the organelles from which vacuolar sorting receptors (VSRs) and endocytosed plasma membrane (PM) proteins are recycled. In this review we try to resolve some of these conflicting observations.     

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.     

Identification of the functional domains of yeast sorting nexins Vps5p and Vps17p

Sorting nexins (Snxs) are a recently discovered family of conserved hydrophilic cytoplasmic proteins that have been found associated with membranes of the endocytic system and that are implicated in the trafficking of many endosomal membrane proteins, including the epidermal growth factor receptor and transferrin receptor. Snx proteins are partly defined by the presence of a p40 phox homology domain that has recently been shown to bind phosphatidylinositol 3-phosphate. Most Snx proteins also contain a predicted coiled-coils domain in the carboxyl-terminal half of the protein and have been shown to form dimers with other members of the Snx family. The yeast sorting nexins Vps5p and Vps17p form a dimer and are also components of the retromer complex that mediates endosome-to-Golgi transport of the carboxypeptidase Y receptor Vps10p. To functionally define the different domains of the yeast sorting nexins Vps5p and Vps17p, we have generated various truncations to examine the role that the different domains of Vps5p/Vps17p play in their respective functions. Herein, we show that the C-terminal halves of Vps5p and Vps17p, which contain the coiled-coils domains, are necessary and sufficient for their interaction. We have also mapped the retromer assembly domain to the N-terminal half of Vps5p and found that binding of Vps5p by Vps17p synergizes the interaction between Vps5p and other retromer components. Additionally, we have examined which domain(s) of Vps5p is necessary for membrane association.     

SNX–BAR proteins in phosphoinositide-mediated,tubular-based endosomal sorting

The endocytic network is morphologically characterized by a wide variety of membrane bound compartments that are able to undergo dynamic re-modeling through tubular and vesicular structures. The precise molecular mechanisms governing such re-modeling, and the events that co-ordinated this with the major role of endosomes, cargo sorting, remain unclear. That said, recent work on a protein family of sorting nexins (SNX) - especially a subfamily of SNX that contain a BAR domain (SNX-BARs) – has begun to shed some much needed light on these issues and in particular the process of tubular-based endosomal sorting. SNX-BARs are evolutionary conserved in endosomal protein complexes such as retromer, where they co-ordinate membrane deformation with cargo selection. Furthermore a central theme emerges of SNX-BARs linking the forming membrane carrier to cytoskeletal elements for transport through motor proteins such as dynein. By studying these SNX-BARs, we are gaining an increasingly detailed appreciation of the mechanistic basis of endosomal sorting and how this highly dynamic process functions in health and disease.     

Fission of SNX-BAR–coated endosomal retrograde transport carriers is promoted by the dynamin-related protein Vps1

Retromer is an endosomal sorting device that orchestrates capture and packaging of cargo into transport carriers coated with sorting nexin BAR domain proteins (SNX-BARs). We report that fission of retromer SNX-BAR–coated tubules from yeast endosomes is promoted by Vps1, a dynamin-related protein that localizes to endosomes decorated by retromer SNX-BARs and Mvp1, a SNX-BAR that is homologous to human SNX8. Mvp1 exhibits potent membrane remodeling activity in vitro, and it promotes association of Vps1 with the endosome in vivo. Retrograde transport carriers bud from the endosome coated by retromer and Mvp1, and cargo export is deficient in mvp1- and vps1-null cells, but with distinct endpoints; cargo export is delayed in mvp1-null cells, but cargo export completely fails in vps1-null cells. The results indicate that Mvp1 promotes Vps1-mediated fission of retromer- and Mvp1-coated tubules that bud from the endosome, revealing a functional link between the endosomal sorting and fission machineries to produce retrograde transport carriers.     

Assembly and Solution Structure of the Core Retromer Protein Complex

Retromer is a peripheral membrane protein complex that has pleiotropic roles in endosomal membrane trafficking. The core of retromer possesses three subunits, VPS35, VPS29 and VPS26, that play different roles in binding to cargo, regulatory proteins and complex stabilization. We have performed an investigation of the thermodynamics of core retromer assembly using isothermal titration calorimetry (ITC) demonstrating that VPS35 acts as the central subunit to which VPS29 and VPS26 bind independently. Furthermore, we confirm that the conserved PRLYL motif of the large VPS35 subunit is critical for direct VPS26 interaction. Heat capacity measurements of VPS29 and VPS26 binding to VPS35 indicate extensive binding interfaces and suggest conformational alterations in VPS29 or VPS35 upon complex formation. Solution studies of the retromer core using small‐angle X‐ray scattering allow us to propose a model whereby VPS35 forms an extended platform with VPS29 and VPS26 bound at distal ends, with the potential for forming dimeric assemblies.     

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.     

Control of RAB7 activity and localization through the retromer‐TBC1D5 complex enables RAB7‐dependent mitophagy

Retromer is an endosomal multi‐protein complex that organizes the endocytic recycling of a vast range of integral membrane proteins. Here, we establish an additional retromer function in controlling the activity and localization of the late endosomal small GTPase RAB7. Surprisingly, we found that RAB7 not only decorates late endosomes or lysosomes, but is also present on the endoplasmic reticulum, trans‐Golgi network, and mitochondrial membranes, a localization that is maintained by retromer and the retromer‐associated RAB7‐specific GAP TBC1D5. In the absence of either TBC1D5 or retromer, RAB7 activity state and localization are no longer controlled and hyperactivated RAB7 expands over the entire lysosomal domain. This lysosomal accumulation of hyperactivated RAB7 results in a striking loss of RAB7 mobility and overall depletion of the inactive RAB7 pool on endomembranes. Functionally, we establish that this control of RAB7 activity is not required for the recycling of retromer‐dependent cargoes, but instead enables the correct sorting of the autophagy related transmembrane protein ATG9a and autophagosome formation around damaged mitochondria during Parkin‐mediated mitophagy.