Proteins of the Wiskott-Aldrich syndrome protein (WASP) family function as nucleation-promoting factors for the ubiquitously expressed Arp2/3 complex, which drives the generation of branched actin ...filaments. Arp2/3-generated actin regulates diverse cellular processes, including the formation of lamellipodia and filopodia, endocytosis and/or phagocytosis at the plasma membrane, and the generation of cargo-laden vesicles from organelles including the Golgi, endoplasmic reticulum (ER) and the endo-lysosomal network. Recent studies have also identified roles for WASP family members in promoting actin dynamics at the centrosome, influencing nuclear shape and membrane remodeling events leading to the generation of autophagosomes. Interestingly, several WASP family members have also been observed in the nucleus where they directly influence gene expression by serving as molecular platforms for the assembly of epigenetic and transcriptional machinery. In this Cell Science at a Glance article and accompanying poster, we provide an update on the subcellular roles of WHAMM, JMY and WASH (also known as WASHC1), as well as their mechanisms of regulation and emerging functions within the cell.
Endocytic recycling of internalized transmembrane proteins is essential for many important physiological processes. Recent studies have revealed that retromer-related Sorting Nexin family ...(SNX)-Bin/Amphiphysin/Rvs (BAR) proteins can directly recognize cargoes like cation-independent mannose 6-phosphate receptor (CI-MPR) and Insulin-like growth factor 1 receptor (IGF1R); however, it remains poorly understood how SNX-BARs select specific cargo proteins and whether they recognize additional ligands. Here, we discovered that the binding between SNX-BARs and CI-MPR or IGF1R is mediated by the phox-homology (PX) domain of SNX5 or SNX6 and a bipartite motif, termed SNX-BAR-binding motif (SBM), in the cargoes. Using this motif, we identified over 70 putative SNX-BAR ligands, many of which play critical roles in apoptosis, cell adhesion, signal transduction, or metabolite homeostasis. Remarkably, SNX-BARs could cooperate with both SNX27 and retromer in the recycling of ligands encompassing the SBM, PDZ-binding motif, or both motifs. Overall, our studies establish that SNX-BARs function as a direct cargo-selecting module for a large set of transmembrane proteins transiting the endosome, in addition to their roles in phospholipid recognition and biogenesis of tubular structures.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The Arp2/3 complex regulates endocytosis, sorting, and trafficking, yet the Arp2/3-stimulating factors orchestrating these distinct events remain ill defined. WASH (Wiskott-Aldrich Syndrome Protein ...and SCAR Homolog) is an Arp2/3 activator with unknown function that was duplicated during primate evolution. We demonstrate that WASH associates with tubulin and localizes to early endosomal subdomains, which are enriched in Arp2/3, F-actin, and retromer components. Although WASH localized with activated receptors, it was not essential for endocytosis. However, WASH did regulate retromer-mediated retrograde CI-MPR trafficking, which required its association with endosomes, Arp2/3-directed F-actin regulation, and tubulin interaction. Moreover, WASH exists in a multiprotein complex containing FAM21, which links WASH to endosomes and is required for WASH-dependent retromer-mediated sorting. Significantly, without WASH, retromer tubulation was exaggerated, supporting a model wherein WASH links retromer-mediated cargo containing tubules to microtubules for Golgi-directed trafficking and generates F-actin-driven force for tubule scission.
Endosomal protein trafficking is an essential cellular process that is deregulated in several diseases and targeted by pathogens. Here, we describe a role for ubiquitination in this process. We find ...that the E3 RING ubiquitin ligase, MAGE-L2-TRIM27, localizes to endosomes through interactions with the retromer complex. Knockdown of MAGE-L2-TRIM27 or the Ube2O E2 ubiquitin-conjugating enzyme significantly impaired retromer-mediated transport. We further demonstrate that MAGE-L2-TRIM27 ubiquitin ligase activity is required for nucleation of endosomal F-actin by the WASH regulatory complex, a known regulator of retromer-mediated transport. Mechanistic studies showed that MAGE-L2-TRIM27 facilitates K63-linked ubiquitination of WASH K220. Significantly, disruption of WASH ubiquitination impaired endosomal F-actin nucleation and retromer-dependent transport. These findings provide a cellular and molecular function for MAGE-L2-TRIM27 in retrograde transport, including an unappreciated role of K63-linked ubiquitination and identification of an activating signal of the WASH regulatory complex.
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► MAGE-L2-TRIM27 E3 ubiquitin ligase localizes to retromer-positive endosomes ► Retrograde transport requires MAGE-L2-TRIM27, Ube2O, and K63-ubiquitination ► MAGE-L2-TRIM27, Ube2O, and K63-ubiquitination facilitate endosomal actin nucleation ► WASH is activated by K63-linked ubiquitination of WASH K220 by MAGE-L2-TRIM27
The E3 ubiquitin ligase MAGE-L2-TRIM27 associates with the retromer complex and promotes F-actin nucleation via ubiquitination of the WASH complex. This regulation is critical for retrograde endosomal trafficking.
The tubular endolysosomal network is a quality control system that ensures the proper delivery of internalized receptors to specific subcellular destinations in order to maintain cellular ...homeostasis. Although retromer was originally described in yeast as a regulator of endosome‐to‐Golgi receptor recycling, mammalian retromer has emerged as a central player in endosome‐to‐plasma membrane recycling of a variety of receptors. Over the past decade, information regarding the mechanism by which retromer facilitates receptor trafficking has emerged, as has the identification of numerous retromer‐associated molecules including the WASH complex, sorting nexins (SNXs) and TBC1d5. Moreover, the recent demonstration that several SNXs can directly interact with retromer cargo to facilitate endosome‐to‐Golgi retrieval has provided new insight into how these receptors are trafficked in cells. The mechanism by which SNX17 cargoes are recycled out of the endosomal system was demonstrated to involve a retromer‐like complex termed the retriever, which is recruited to WASH positive endosomes through an interaction with the COMMD/CCDC22/CCDC93 (CCC) complex. Lastly, the mechanisms by which bacterial and viral pathogens highjack this complex sorting machinery in order to escape the endolysosomal system or remain hidden within the cells are beginning to emerge. In this review, we will highlight recent studies that have begun to unravel the intricacies by which the retromer and associated molecules contribute to receptor trafficking and how deregulation at this sorting domain can contribute to disease or facilitate pathogen infection.
The proper delivery of internalized receptors to specific subcellular destinations is critical to normal cellular physiology. Several endosomal‐associated molecules including sorting nexins, the retromer and WASH complexes, as well as the recently identified retriever and COMMD/CCDC22/CCDC93 (CCC) complexes regulate receptor recycling through the tubular endolysosomal network. Herein, we highlight the intricacies by which the retromer and associated molecules contribute to receptor trafficking and how deregulation at this sorting domain can contribute to disease or facilitate pathogen infection.
T cell receptor (TCR)-mediated cytoskeletal reorganization is considered to be actin-related protein (Arp) 2/3 complex dependent. We therefore examined the requirement for Arp2/3- and ...formin-dependent F-actin nucleation during T cell activation. We demonstrated that without Arp2/3-mediated actin nucleation, stimulated T cells could not form an F-actin-rich lamellipod, but instead produced polarized filopodia-like structures. Moreover, the microtubule-organizing center (MTOC, or centrosome), which rapidly reorients to the immunological synapse through an unknown mechanism, polarized in the absence of Arp2/3. Conversely, the actin-nucleating formins, Diaphanous-1 (DIA1) and Formin-like-1 (FMNL1), did not affect TCR-stimulated F-actin-rich structures, but instead displayed unique patterns of centrosome colocalization and controlled TCR-mediated centrosome polarization. Depletion of FMNL1 or DIA1 in cytotoxic lymphocytes abrogated cell-mediated killing. Altogether, our results have identifed Arp2/3 complex-independent cytoskeletal reorganization events in T lymphocytes and indicate that formins are essential cytoskeletal regulators of centrosome polarity in T cells.
Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and ...the plasma membrane 1. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease 2, 3. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.
•VPS35(D620N) mutation leads to a defect in endosome-to-TGN transport•Mutant has a perturbed association with the actin-polymerizing WASH complex•Primary defect is a reduced affinity for binding WASH component FAM21•Provides new molecular insight into retromer deregulation in Parkinson disease
Using quantitative proteomics and functional analysis in patient-derived fibroblasts, McGough et al. define the primary molecular defect associated with the Parkinson disease-linked retromer VPS35(D620N) mutation and, by revealing effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in this disease.
Following endocytosis into the endosomal network, integral membrane proteins undergo sorting for lysosomal degradation or are retrieved and recycled back to the cell surface. Here we describe the ...discovery of an ancient and conserved multiprotein complex that orchestrates cargo retrieval and recycling and, importantly, is biochemically and functionally distinct from the established retromer pathway. We have called this complex 'retriever'; it is a heterotrimer composed of DSCR3, C16orf62 and VPS29, and bears striking similarity to retromer. We establish that retriever associates with the cargo adaptor sorting nexin 17 (SNX17) and couples to CCC (CCDC93, CCDC22, COMMD) and WASH complexes to prevent lysosomal degradation and promote cell surface recycling of α
β
integrin. Through quantitative proteomic analysis, we identify over 120 cell surface proteins, including numerous integrins, signalling receptors and solute transporters, that require SNX17-retriever to maintain their surface levels. Our identification of retriever establishes a major endosomal retrieval and recycling pathway.
Protein recycling through the endolysosomal system relies on molecular assemblies that interact with cargo proteins, membranes, and effector molecules. Among them, the COMMD/CCDC22/CCDC93 (CCC) ...complex plays a critical role in recycling events. While CCC is closely associated with retriever, a cargo recognition complex, its mechanism of action remains unexplained. Herein we show that CCC and retriever are closely linked through sharing a common subunit (VPS35L), yet the integrity of CCC, but not retriever, is required to maintain normal endosomal levels of phosphatidylinositol-3-phosphate (PI(3)P). CCC complex depletion leads to elevated PI(3)P levels, enhanced recruitment and activation of WASH (an actin nucleation promoting factor), excess endosomal F-actin and trapping of internalized receptors. Mechanistically, we find that CCC regulates the phosphorylation and endosomal recruitment of the PI(3)P phosphatase MTMR2. Taken together, we show that the regulation of PI(3)P levels by the CCC complex is critical to protein recycling in the endosomal compartment.
Wiskott-Aldrich syndrome protein (WASPs) control actin dynamics in cellular processes, including cell motility, receptor-mediated endocytosis, bacterial invasion, and vesicular trafficking. We ...demonstrated that WASH, a recently identified WASP family protein, colocalizes on endosomal subdomains with the cargo-selective complex (CSC) of the retromer, where it regulates retrograde sorting from endosomes in an actin-dependent manner. However, the mechanism of WASH recruitment to these retromer-enriched endosomal subdomains is unclear. Here we show that a component of the WASH regulatory complex (SHRC), FAM21, which contains 21 copies of a novel L-F-D/E(3-10)-L-F motif, directly interacts with the retromer CSC protein VPS35. Endosomal localization of FAM21 is VPS35 dependent and relies on multivalency of FAM21 repeat elements. Using a combination of pull-down assays and isothermal calorimetry, we demonstrate that individual repeats can bind CSC, and binding affinity varies among different FAM21 repeats. A high-affinity repeat can be converted into a low-affinity one by mutation of a hydrophobic residue within the motif. These in vitro data mirror the localization of FAM21 to retromer-coated vesicles in cells. We propose that multivalency enables FAM21 to sense the density of retromer on membranes, allowing coordination of SHRC recruitment, and consequent actin polymerization, with retromer sorting domain organization/maturation.
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