Endosomal receptor trafficking: Retromer and beyond

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.      

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.     

The structure and function of the retromer protein complex

The transport of transmembrane proteins and associated ligands through the endosomal system is governed by a number of different protein assemblies. One such assembly is retromer, a peripheral membrane protein complex that has important roles in endosomal sorting of a variety of cargo molecules. Retromer was first shown to control the endosome-to-Golgi retrieval of lysosomal hydrolase receptors, and over the past few years, it has been found to play a similar role in the transport of many other proteins in all eukaryotes from simple amoeba to plants and mammals. Recent structural studies of the core retromer complex have revealed both unexpected similarities and intriguing differences between retromer and other regulators of membrane trafficking and are beginning to open the door to a mechanistic understanding of retromer-mediated endosomal transport.     

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.      

Towards a molecular understanding of endosomal trafficking by Retromer and Retriever

Endosomes are dynamic intracellular compartments that control the sorting of a constant stream of different transmembrane cargos either for ESCRT‐mediated degradation or for egress and recycling to compartments such as the Golgi and the plasma membrane. The recycling of cargos occurs within tubulovesicular membrane domains and is facilitated by peripheral membrane protein machineries that control both membrane remodelling and selection of specific transmembrane cargos. One of the primary sorting machineries is the Retromer complex, which controls the recycling of a large array of different cargo molecules in cooperation with various sorting nexin (SNX) adaptor proteins. Recently a Retromer‐like complex was also identified that controls plasma membrane recycling of cargos including integrins and lipoprotein receptors. Termed “Retriever,” this complex uses a different SNX family member SNX17 for cargo recognition, and cooperates with the COMMD/CCDC93/CCDC22 (CCC) complex to form a larger assembly called “Commander” to mediate endosomal trafficking. In this review we focus on recent advances that have begun to provide a molecular understanding of these two distantly related transport machineries.     

Expression of Sorting Nexin 18 (SNX18) Is Dynamically Regulated in Developing Spinal Motor Neurons

The sorting nexin (SNX) family proteins, which contain a Phox homology (PX) domain, play crucial roles in regulating the intracellular membrane trafficking of the endocytic pathway. The proper coordination of this pathway is important for axonal elongation; however, little is known about the expression and intracellular dynamics of the SNX members during the formation of the nervous system. Here the authors found that SNX18, which belongs to the Src-homology-3-PX-Bin/Amphiphysin/Rvs domain-containing SNX subfamily, was specifically expressed in differentiating motor neurons in the chick and mouse embryonic spinal cord. The expression of SNX18 in embryonic spinal motor neurons was transient and was downregulated as the neurons matured. The authors further demonstrated that the localization of EGFP-SNX18 in growth cones was dynamically regulated and accumulated especially at areas in contact with permissive substrates. These findings collectively suggest that SNX18 may play an active role in axonal elongation.     

The emerging roles of retromer and sorting nexins in the life cycle of viruses

Retromer and sorting nexins (SNXs) transport cargoes from endosomes to the trans-Golgi network or plasma membrane. Recent studies have unveiled the emerging roles for retromer and SNXs in the life cycle of viruses, including members of Coronaviridae, Flaviviridae and Retroviridae. Key components of retromer/SNXs, such as Vps35, Vps26, SNX5 and SNX27, can affect multiple steps of the viral life cycle, including facilitating the entry of viruses into cells, participating in viral replication, and promoting the assembly of virions. Here we present a comprehensive updated review on the interplay between retromer/SNXs and virus, which will shed mechanistic insights into controlling virus infection.     

Phosphoinositide-regulated retrograde transport of ricin: crosstalk between hVps34 and sorting nexins

The plant toxin ricin is transported from the plasma membrane via early endosomes and the Golgi apparatus to the endoplasmic reticulum. From this compartment, it enters the cytosol and inhibits protein synthesis. Lipid phosphorylation is an important regulator of vesicular transport, and in the present study we have investigated the role of the phosphatidylinositol (PI) 3-kinase hVps34 in retrograde transport of ricin. Our data demonstrate that transport of ricin from endosomes to the Golgi apparatus in human embryonic kidney cells (HEK 293) is dependent on PI(3)P. By using PI 3-kinase inhibitors, by sequestering the hVps34 product PI(3)P and by expressing mutants of hVps34 or small interfering RNA targeted against its messenger RNA, we show that hVps34 and its product PI(3)P are involved in transport of ricin from endosome to Golgi apparatus. Furthermore, we identify two effector proteins in the hVps34-dependent pathway, namely sorting nexin (SNX) 2 and SNX4. Knockdown of SNX2 or SNX4 inhibits ricin transport to the Golgi apparatus to the same extent as when hVps34 is perturbed. Furthermore, inhibition or knockdown of hVps34 redistributes these proteins. Interestingly, knocking down both SNX2 and SNX4 results in a better inhibition than knocking down only one of them, suggesting that they may act on separate pathways.     

Multiple Roles of VARP in Endosomal Trafficking: Rabs,Retromer Components and R‐SNARE VAMP7 Meet on VARP

VARP (VPS9‐ankyrin‐repeat protein, also known as ANKRD27) was originally identified as an N‐terminal VPS9 (vacuolar protein sorting 9)‐domain‐containing protein that possesses guanine nucleotide exchange factor (GEF) activity toward small GTPase Rab21 and contains two ankyrin repeat (ANKR) domains in its central region. A number of VARP‐interacting molecules have been identified during the past five years, and considerable attention is now being directed to the multiple roles of VARP in endosomal trafficking. More specifically, VARP is now known to interact with three different types of key membrane trafficking regulators, i.e. small GTPase Rabs (Rab32, Rab38 and Rab40C), the retromer complex (a sorting nexin dimer, VPS26, VPS29 and VPS35) and R‐SNARE VAMP7. By binding to several of these molecules, VARP regulates endosomal trafficking, which underlies a variety of cellular events, including melanogenic enzyme trafficking to melanosomes, dendrite outgrowth of melanocytes, neurite outgrowth and retromer‐mediated endosome‐to‐plasma membrane sorting of transmembrane proteins.      

Receptor-mediated Endocytosis 8 Utilizes an N-terminal Phosphoinositide-binding Motif to Regulate Endosomal Clathrin Dynamics

Receptor-mediated endocytosis 8 (RME-8) is a DnaJ domain containing protein implicated in translocation of Hsc70 to early endosomes for clathrin removal during retrograde transport. Previously, we have demonstrated that RME-8 associates with early endosomes in a phosphatidylinositol 3-phosphate (PI(3)P)-dependent fashion. In this study, we have now identified amino acid determinants required for PI(3)P binding within a region predicted to adopt a pleckstrin homology-like fold in the N terminus of RME-8. The ability of RME-8 to associate with PI(3)P and early endosomes is largely abolished when residues Lys¹⁷, Trp²⁰, Tyr²⁴, or Arg²⁶ are mutated resulting in diffuse cytoplasmic localization of RME-8 while maintaining the ability to interact with Hsc70. We also provide evidence that RME-8 PI(3)P binding regulates early endosomal clathrin dynamics and alters the steady state localization of the cation-independent mannose 6-phosphate receptor. Interestingly, RME-8 endosomal association is also regulated by the PI(3)P-binding protein SNX1, a member of the retromer complex. Wild type SNX1 restores endosomal localization of RME-8 W20A, whereas a SNX1 variant deficient in PI(3)P binding disrupts endosomal localization of wild type RME-8. These results further highlight the critical role for PI(3)P in the RME-8-mediated organizational control of various endosomal activities, including retrograde transport.     

Structure and Membrane Binding Properties of the Endosomal Tetratricopeptide Repeat (TPR) Domain-containing Sorting Nexins SNX20 and SNX21

Sorting nexins (SNX) orchestrate membrane trafficking and signaling events required for the proper distribution of proteins within the endosomal network. Their phox homology (PX) domain acts as a phosphoinositide (PI) recognition module that targets them to specific endocytic membrane domains. The modularity of SNX proteins confers a wide variety of functions from signaling to membrane deformation and cargo binding, and many SNXs are crucial modulators of endosome dynamics and are involved in a myriad of physiological and pathological processes such as neurodegenerative diseases, cancer, and inflammation. Here, we have studied the poorly characterized SNX20 and its paralogue SNX21, which contain an N-terminal PX domain and a C-terminal PX-associated B (PXB) domain of unknown function. The two proteins share similar PI-binding properties and are recruited to early endosomal compartments by their PX domain. The crystal structure of the SNX21 PXB domain reveals a tetratricopeptide repeat (TPR)-fold, a module that typically binds short peptide motifs, with three TPR α-helical repeats. However, the C-terminal capping helix adopts a highly unusual and potentially self-inhibitory topology. SAXS solution structures of SNX20 and SNX21 show that these proteins adopt a compact globular architecture, and membrane interaction analyses indicate the presence of overlapping PI-binding sites that may regulate their intracellular localization. This study provides the first structural analysis of this poorly characterized subfamily of SNX proteins, highlighting a likely role as endosome-associated scaffolds.     

Transient surface delivery of invariant chain-MHC II complexes via endosomes: a quantitative study

Newly synthesized major histocompatibility complex class II needs to be directed to late endocytic compartments to combine with peptide antigens. Efficient transport requires complexes of major histocompatibility complex class II and invariant chain (αβIi). Since such complexes have been detected on the plasma membrane in human cells, this compartment was proposed as the primary destination for αβIi exiting the trans-Golgi network. Here, I have used density gradient electrophoresis and selective biotinylation to investigate the trafficking route of αβIi quantitatively. Density gradient electrophoresis analysis showed that αβIi was transported from the trans-Golgi network to endosomes at ∼ 1.7% min⁻¹. Surface delivery of αβIi was delayed relative to endosome transport by ∼ 10 min and showed slower kinetics (∼ 0.4% min⁻¹), suggesting that αβIi reached the plasma membrane only after arrival in endosomes. A biotinylation assay revealed that 20–40% of endosomal αβIi was delivered to the plasma membrane at steady state, suggesting that surface αβIi was entirely derived from endosomes. Surface αβIi was rapidly re-internalized and either returned to the cell surface or accessed degradative compartments. Peptide loading commenced ∼ 30 min after delivery to endosomes. Thus αβIi directly traffics from trans-Golgi network to endosomes and enters an endosome–plasma membrane 'carousel' until transport to peptide-loading compartments ensues .     

Sorting nexin 27 interactome in T‐lymphocytes identifies zona occludens‐2 dynamic redistribution at the immune synapse

T Lymphocyte recognition of antigens leads to the formation of a highly organized structure termed immune synapse (IS) by analogy with the neuronals synapse. Sorting nexin 27 (SNX27) controls the endosomal traffic of PSD95, Dlg1, ZO‐1 (PDZ) domain‐interacting proteins, and its alteration is associated with impaired synaptic function and neurological diseases. In T‐lymphocytes, SNX27‐positive vesicles polarize to the IS, the identity of SNX27 interactors in these conditions nonetheless remains unknown. Here we used proteomics to analyze the SNX27 interactome purified from IS‐forming T cells, and confirmed the conserved nature of the SNX27/WASH/retromer association in hematopoietic cells. Furthermore, our comparative interactome analysis of SNX27 wild‐type and a mutant‐deficient for PDZ cargo recognition identified the epithelial cell‐cell junction protein zona occludens‐2 (ZO‐2) as an IS component. Biochemistry and microscopy approaches in T cells confirmed SNX27/ZO‐2 PDZ‐dependent interaction, and demonstrated its role controlling the dynamic localization of ZO‐2 at the IS. This study broadens our knowledge of SNX27 function in T lymphocytes, and suggests that pathways that delimit polarized structures in nervous and epithelial systems also participate in IS regulation.      

病毒劫持ESCRT系统促进自身复制的研究进展

内吞体分选转运复合体（endosomal sorting complex required for transport，ESCRT）系统驱动细胞的不同生命进程，包括内体分选、细胞器生物发生、囊泡运输、维持质膜完整性、细胞质分裂期间的膜裂变、有丝分裂后的核膜重组、自噬过程中吞噬孔的封闭以及包膜病毒出芽等。越来越多的证据表明，ESCRT系统能够被不同家族病毒劫持用于自身增殖。在病毒生命周期的不同阶段，病毒可以通过各种方式干扰或利用ESCRT系统介导的生理过程，最大限度地提高感染宿主的机会。此外，许多逆转录病毒和RNA病毒蛋白具有“晚期结构域”基序，可招募宿主ESCRT亚基蛋白帮助病毒内吞、运输、复制、出芽以及外排。因此，病毒“晚期结构域”基序和ESCRT亚基蛋白可能是病毒感染治疗中具有广泛应用前景的药物靶点。本文重点综述了ESCRT系统的组成及功能，ESCRT亚基和病毒“晚期结构域”基序对病毒复制的影响以及ESCRT介导的抗病毒作用，以期为抗病毒药物的开发和利用提供参考。     

The ESCRT‐III protein CeVPS‐32 is enriched in domains distinct from CeVPS‐27 and CeVPS‐23 at the endosomal membrane of epithelial cells

Background information. Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. Results. In the present study, we characterize the ESCRT‐III protein CeVPS‐32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS‐27, CeVPS‐23 and CeVPS‐4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps‐32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps‐32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS‐32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS‐32 and CeVPS‐27/Hrs (hepatocyte‐growth‐factor‐regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS‐27‐positive subdomains are also enriched for the ESCRT‐I protein CeVPS‐23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS‐27 subdomains is not affected by the depletion of CeVPS‐23, CeVPS‐32 or the ATPase CeVPS‐4. Conclusion. Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.     

Dysregulation of Ack1 inhibits down-regulation of the EGF receptor

The protein tyrosine kinase Ack1 has been linked to cancer when over-expressed. Ack1 has also been suggested to function in clathrin-mediated endocytosis and in down-regulation of the epidermal growth factor (EGF) receptor (EGFR). We have studied the intracellular localization of over-expressed Ack1 and found that Ack1 co-localizes with the EGFR upon EGF-induced endocytosis in cells with moderate over-expression of Ack. This co-localization is mainly observed in early endosomes. Furthermore, we found that over-expression of Ack1 retained the EGFR at the limiting membrane of early endosomes, inhibiting sorting to inner vesicles of multivesicular bodies. Down-regulation of Ack1 in HeLa cells resulted in reduced rate of (125)I-EGF internalization, whereas internalization of (125)I-transferrin was not affected. In cells where Ack1 had been knocked down by siRNA, recycling of internalized (125)I-EGF was increased, while degradation of (125)I-EGF was inhibited. Together, these data suggest that Ack1 is involved in an early step of EGFR desensitization.     

Synthesis and characterization of a high spin d Fe(II) complex with the tridentate ligand dipyrido(2,3-a:3,2-j)phenazine (dpop)

A new bis-(N-tridentate) Fe(II) complex [Fe(dpop^′)₂](PF₆)₂ (dpop^′=dipyrido(2,3-a:3^′,2^′-j)phenazine) was prepared and studied. The magnetic moment of the solid was determined as μ=5.2-4.9 BM and in CH₃CN solution as μ=4.9 BM and indicate the high spin Fe(II) state. The electronic absorption spectrum displays a broad weak absorption MLCT transition at 602 nm (ε=3.8×10³ M⁻¹ cm⁻¹), consistent with CT absorptions of other Fe(II) HS complexes. The cyclic voltammogram of the complex shows an irreversible Fe^2+/3+ oxidation at +1.55 V and two dpop^′0/−1 centered reductions at −0.20 and −0.59 V versus Ag/AgCl.