The role of motor proteins in endosomal sorting

Microtubule motor proteins play key roles in the spatial organization of intracellular organelles as well as the transfer of material between them. This is well illustrated both by the vectorial transfer of biosynthetic cargo from the endoplasmic reticulum to the Golgi apparatus as well as the sorting of secretory and endocytic cargo in the endosomal system. Roles have been described for dynein and kinesin motors in each of these steps. Cytoplasmic dynein is a highly complex motor comprising multiple subunits that provide functional specialization. The family of human kinesins includes over 40 members. This complexity provides immense functional diversity, yet little is known of the specific requirements and functions of individual motors during discrete membrane trafficking steps. In the present paper, we describe some of the latest findings in this area that seek to define the mechanisms of recruitment and control of activity of microtubule motors in spatial organization and cargo trafficking through the endosomal network.     

Phosphoinositides and the regulation of tubular-based endosomal sorting

From the pioneering work of Mabel and Lowell Hokin in the 1950s, the biology of this specific isomer of hexahydroxycyclohexane and its phosphorylated derivatives, in the form of inositol phosphates and phosphoinositides, has expanded to fill virtually every corner of cell biology, whole-organism physiology and development. In the present paper, I give a personal view of the role played by phosphoinositides in regulating the function of the endosomal network, and, in so doing, highlight some of the basic properties through which phosphoinositides regulate cell function.     

Membrane transport: ubiquitylation in endosomal sorting.

In yeast, membrane proteins from the biosynthetic and endocytic pathways must be ubiquitylated for sorting to inward-budding vesicles in late endosomes, which give rise to multivesicular bodies. A conserved protein complex containing the yeast Vps23p or its mammalian counterpart Tsg101 may act as the ubiquitin receptor.     

Determinants of the endosomal localization of sorting nexin 1

The sorting nexin (SNX) family of proteins is characterized by sequence-related phox homology (PX) domains. A minority of PX domains bind with high affinity to phosphatidylinositol 3-phosphate [PI(3)P], whereas the majority of PX domains exhibit low affinity that is insufficient to target them to vesicles. SNX1 is located on endosomes, but its low affinity PX domain fails to localize in vivo. The NMR structure of the PX domain of SNX1 reveals an overall fold that is similar to high-affinity PX domains. However, the phosphatidylinositol (PI) binding pocket of the SNX1 PX domain is incomplete; regions of the pocket that are well defined in high-affinity PX domains are highly mobile in SNX1. Some of this mobility is lost upon binding PI(3)P. The C-terminal domain of SNX1 is a long helical dimer that localizes to vesicles but not to the early endosome antigen-1-containing vesicles where endogenous SNX1 resides. Thus, the obligate dimerization of SNX1 that is driven by the C-terminal domain creates a high-affinity PI binding species that properly targets the holo protein to endosomes.     

Sequence-dependent sorting of recycling proteins by actin-stabilized endosomal microdomains

The functional consequences of signaling receptor endocytosis are determined by the endosomal sorting of receptors between degradation and recycling pathways. How receptors recycle efficiently, in a sequence-dependent manner that is distinct from bulk membrane recycling, is not known. Here, in live cells, we visualize the sorting of a prototypical sequence-dependent recycling receptor, the beta-2 adrenergic receptor, from bulk recycling proteins and the degrading delta-opioid receptor. Our results reveal a remarkable diversity in recycling routes at the level of individual endosomes, and indicate that sequence-dependent recycling is an active process mediated by distinct endosomal subdomains distinct from those mediating bulk recycling. We identify a specialized subset of tubular microdomains on endosomes, stabilized by a highly localized but dynamic actin machinery, that mediate this sorting, and provide evidence that these actin-stabilized domains provide the physical basis for a two-step kinetic and affinity-based model for protein sorting into the sequence-dependent recycling pathway.     

Regulation of Notch signalling by endocytosis and endosomal sorting

Cell-cell signalling is an essential process in the formation of multicellular organisms. Notch is the receptor of an evolutionarily conserved signalling pathway regulating numerous developmental decisions. Indeed, its misregulation is linked to multiple developmental and physiological disorders. Notch and its ligands are distributed widely throughout development, yet Notch activity is highly controlled and restricted in time and space. Recent advances have highlighted that endocytosis followed by endosomal sorting of both the Notch receptor and its ligands is an essential mechanism by which Notch-mediated signalling is developmentally controlled.     

Double-sided ubiquitin binding of Hrs-UIM in endosomal protein sorting

Hrs has an essential role in sorting of monoubiquitinated receptors to multivesicular bodies for lysosomal degradation, through recognition of ubiquitinated receptors by its ubiquitin-interacting motif (UIM). Here, we present the structure of a complex of Hrs-UIM and ubiquitin at 1.7-A resolution. Hrs-UIM forms a single alpha-helix, which binds two ubiquitin molecules, one on either side. These two ubiquitin molecules are related by pseudo two-fold screw symmetry along the helical axis of the UIM, corresponding to a shift by two residues on the UIM helix. Both ubiquitin molecules interact with the UIM in the same manner, using the Ile44 surface, with equal binding affinities. Mutational experiments show that both binding sites of Hrs-UIM are required for efficient degradative protein sorting. Hrs-UIM belongs to a new subclass of double-sided UIMs, in contrast to its yeast homolog Vps27p, which has two tandem single-sided UIMs.     

The tetraspanin CD63 regulates ESCRT-independent and -dependent endosomal sorting during melanogenesis

Cargo sorting to intraluminal vesicles (ILVs) of multivesicular endosomes is required for lysosome-related organelle (LRO) biogenesis. PMEL-a component of melanocyte LROs (melanosomes)-is sorted to ILVs in an ESCRT-independent manner, where it is proteolytically processed and assembled into functional amyloid fibrils during melanosome maturation. Here we show that the tetraspanin CD63 directly participates in ESCRT-independent sorting of the PMEL luminal domain, but not of traditional ESCRT-dependent cargoes, to ILVs. Inactivating CD63 in cell culture or in mice impairs amyloidogenesis and downstream melanosome morphogenesis. Whereas CD63 is required for normal PMEL luminal domain sorting, the disposal of the remaining PMEL transmembrane fragment requires functional ESCRTs but not CD63. In the absence of CD63, the PMEL luminal domain follows this fragment and is targeted for ESCRT-dependent degradation. Our data thus reveal a tight interplay regulated by CD63 between two distinct endosomal ILV sorting processes for a single cargo during LRO biogenesis.     

New component of ESCRT-I regulates endosomal sorting complex assembly

The endosomal sorting complex required for transport (ESCRT) complexes play a critical role in receptor down-regulation and retroviral budding. Although the crystal structures of two ESCRT complexes have been determined, the molecular mechanisms underlying the assembly and regulation of the ESCRT machinery are still poorly understood. We identify a new component of the ESCRT-I complex, multivesicular body sorting factor of 12 kD (Mvb12), and demonstrate that Mvb12 binds to the coiled-coil domain of the ESCRT-I subunit vacuolar protein sorting 23 (Vps23). We show that ESCRT-I adopts an oligomeric state in the cytosol, the formation of which requires the coiled-coil domain of Vps23, as well as Mvb12. Loss of Mvb12 results in the disassembly of the ESCRT-I oligomer and the formation of a stable complex of ESCRT-I and -II in the cytosol. We propose that Mvb12 stabilizes ESCRT-I in an oligomeric, inactive state in the cytosol to ensure that the ordered recruitment and assembly of ESCRT-I and -II is spatially and temporally restricted to the surface of the endosome after activation of the MVB sorting reaction.     

SCF acts in endosomal sorting of the GH receptor

The ubiquitin ligase SCF^TrCP is required for internalisation of the growth hormone receptor (GHR) and acts via a direct interaction with the ubiquitin-dependent endocytosis motif. Details of how the ligase communicates its information to the clathrin-mediated internalisation machinery are unknown. For the EGF receptor, c-Cbl acts both at the cell surface and in endosomes. We hypothesised that SCF^TrCP is required for GHR degradation at both sites. This was tested by truncating GHR after a di-leucine-based internalisation motif (GHR349). This receptor enters the cells via the adapter complex AP2. We show that TrCP acts in an early stage of cargo selection: both TrCP silencing and mutation of the ubiquitin-dependent endocytosis motif force the GHR to recycle between endosomes and the plasma membrane, together with the transferrin receptor. Depletion of Tsg101 (ESCRT-I) has the same effect, while silencing of Hrs (ESCRT-0) prevents GH recycling. GH passes through late endosomal vesicles, marked by Lamp1. Coexpressing GHR and EGFR demonstrates that both receptors use the same route to the lysosomes. We show for the first time that SCF^TrCP is involved in cargo-specific sorting at endosomes and that Tsg101 rather than Hrs might direct the cargo into the ESCRT machinery.     

Differential beta-arrestin trafficking and endosomal sorting of somatostatin receptor subtypes

The physiological responses of somatostatin are mediated by five different G protein-coupled receptors. Although agonist-induced endocytosis of the various somatostatin receptor subtypes (sst(1)-sst(5)) has been studied in detail, little is known about their postendocytic trafficking. Here we show that somatostatin receptors profoundly differ in patterns of beta-arrestin mobilization and endosomal sorting. The beta-arrestin-dependent trafficking of the sst(2A) somatostatin receptor resembled that of a class B receptor in that upon receptor activation, beta-arrestin and the receptor formed stable complexes and internalized together into the same endocytic vesicles. This pattern was dependent on GRK2 (G protein-coupled receptor kinase 2)-mediated phosphorylation of a cluster of phosphate acceptor sites within the cytoplasmic tail of the sst(2A) receptor. Unlike other class B receptors, however, the sst(2A) receptor was rapidly resensitized and recycled to the plasma membrane. The beta-arrestin mobilization of the sst(3) and the sst(5) somatostatin receptors resembled that of a class A receptor in that upon receptor activation, beta-arrestin and the receptor formed relatively unstable complexes that dissociated at or near the plasma membrane. Consequently, beta-arrestin was excluded from sst(3)-containing vesicles. Unlike other class A receptors, a large proportion of sst(3) receptors was subject to ubiquitin-dependent lysosomal degradation and did not rapidly recycle to the plasma membrane. The sst(4) somatostatin receptor is unique in that it did not exhibit agonist-dependent receptor phosphorylation and beta-arrestin recruitment. Together, these findings may provide important clues about the regulation of receptor responsiveness during long-term administration of somatostatin analogs.     

Molecular basis for SNX-BAR-mediated assembly of distinct endosomal sorting tubules

Sorting nexins (SNXs) are regulators of endosomal sorting. For the SNX‐BAR subgroup, a Bin/Amphiphysin/Rvs (BAR) domain is vital for formation/stabilization of tubular subdomains that mediate cargo recycling. Here, by analysing the in vitro membrane remodelling properties of all 12 human SNX‐BARs, we report that some, but not all, can elicit the formation of tubules with diameters that resemble sorting tubules observed in cells. We reveal that SNX‐BARs display a restricted pattern of BAR domain‐mediated dimerization, and by resolving a 2.8 Å structure of a SNX1‐BAR domain homodimer, establish that dimerization is achieved in part through neutralization of charged residues in the hydrophobic BAR‐dimerization interface. Membrane remodelling also requires functional amphipathic helices, predicted to be present in all SNX‐BARs, and the formation of high order SNX‐BAR oligomers through selective ‘tip–loop’ interactions. Overall, the restricted and selective nature of these interactions provide a molecular explanation for how distinct SNX‐BAR‐decorated tubules are nucleated from the same endosomal vacuole, as observed in living cells. Our data provide insight into the molecular mechanism that generates and organizes the tubular endosomal network.     

Intracellular receptor/ligand sorting based on endosomal retention components

Endocytosed molecules are sorted in endosomes to different cellular destinations (e.g., to lysosomes or to the plasma membrane). Diverse endosomal sorting results have been reported for different ligands and receptors in a variety of cell types, but the general principles governing these sorting outcomes are not well understood. For example, we observed a wide range of sorting outcomes with the epidermal growth factor (EGF)/receptor system in fibroblasts using several members of the EGF family and site-directed ligand and receptor mutants. In this article we describe a mechanistic mathematical model of endosomal sorting based on the hypothesis that receptors may be selectively retained by the endosomal sorting apparatus and that this process may be modulated by receptor occupancy. Our results show that this single mechanism can account for the wide variety of observed sorting outcomes. By providing a conceptual framework for understanding endosomal sorting, this model not only helps interpret our experimental results for the EGF/receptor system, but also provides some insight into the principles governing sorting. For example, the model predicts that the influence of selective endosomal retention of receptor/ligand complexes is seen in deviations of ligand sorting outcomes from pure fluid phase sorting behavior. Furthermore, the model suggests that selective endosomal retention of complexes within endosomes gives rise to three sorting regimes characterized by distinguishable qualitative trends in the dependence of ligand sorting fractions on intracellular ligand concentrations. (c) 1996 John Wiley & Sons, Inc.     

The clathrin heavy chain isoform CHC22 functions in a novel endosomal sorting step

Clathrin heavy chain 22 (CHC22) is an isoform of the well-characterized CHC17 clathrin heavy chain, a coat component of vesicles that mediate endocytosis and organelle biogenesis. CHC22 has a distinct role from CHC17 in trafficking glucose transporter 4 (GLUT4) in skeletal muscle and fat, though its transfection into HEK293 cells suggests functional redundancy. Here, we show that CHC22 is eightfold less abundant than CHC17 in muscle, other cell types have variably lower amounts of CHC22, and endogenous CHC22 and CHC17 function independently in nonmuscle and muscle cells. CHC22 was required for retrograde trafficking of certain cargo molecules from endosomes to the trans-Golgi network (TGN), defining a novel endosomal-sorting step distinguishable from that mediated by CHC17 and retromer. In muscle cells, depletion of syntaxin 10 as well as CHC22 affected GLUT4 targeting, establishing retrograde endosome–TGN transport as critical for GLUT4 trafficking. Like CHC22, syntaxin 10 is not expressed in mice but is present in humans and other vertebrates, implicating two species-restricted endosomal traffic proteins in GLUT4 transport.     

The role of the endosomal sorting complexes required for transport (ESCRT) in tumorigenesis

The endosomal sorting complexes required for transport (ESCRT) are needed for three distinct cellular functions in higher eukaryotes: (i) Multivesicular body formation for the degradation of transmembrane proteins in lysosomes, (ii) midbody abscission during cytokinesis and (iii) retroviral budding. Not surprisingly, loss of ESCRT function has severe consequences, which include the failure to down-regulate growth factor receptors leading to deregulated mitogenic signaling. While it is clear that the function of the ESCRT machinery is important for embryonic development, its role in cancer is more controversial. Various experimental approaches in different model organisms arrive at partially divergent conclusions regarding the contribution of ESCRTs to tumorigenesis. Therefore the aim of this review is to provide an overview on different model systems used to study the role of the ESCRT machinery in cancer development, to highlight common grounds and present certain controversies in the field.     

The role of ceroid lipofuscinosis neuronal protein 5 (CLN5) in endosomal sorting

Mutations in the gene encoding CLN5 are the cause of Finnish variant late infantile Neuronal Ceroid Lipofuscinosis (NCL), and the gene encoding CLN5 is 1 of 10 genes (encoding CLN1 to CLN9 and cathepsin D) whose germ line mutations result in a group of recessive disorders of childhood. Although CLN5 localizes to the lysosomal compartment, its function remains unknown. We have uncovered an interaction between CLN5 and sortilin, the lysosomal sorting receptor. However, CLN5, unlike prosaposin, does not require sortilin to localize to the lysosomal compartment. We demonstrate that in CLN5-depleted HeLa cells, the lysosomal sorting receptors sortilin and cation-independent mannose 6-phosphate receptor (CI-MPR) are degraded in lysosomes due to a defect in recruitment of the retromer (an endosome-to-Golgi compartment trafficking component). In addition, we show that the retromer recruitment machinery is also affected by CLN5 depletion, as we found less loaded Rab7, which is required to recruit retromer. Taken together, our results support a role for CLN5 in controlling the itinerary of the lysosomal sorting receptors by regulating retromer recruitment at the endosome.     

Endothelin-converting enzyme-1 regulates endosomal sorting of calcitonin receptor-like receptor and beta-arrestins

Although cell surface metalloendopeptidases degrade neuropeptides in the extracellular fluid to terminate signaling, the function of peptidases in endosomes is unclear. We report that isoforms of endothelin-converting enzyme-1 (ECE-1a–d) are present in early endosomes, where they degrade neuropeptides and regulate post-endocytic sorting of receptors. Calcitonin gene-related peptide (CGRP) co-internalizes with calcitonin receptor-like receptor (CLR), receptor activity-modifying protein 1 (RAMP1), β-arrestin2, and ECE-1 to early endosomes, where ECE-1 degrades CGRP. CGRP degradation promotes CLR/RAMP1 recycling and β-arrestin2 redistribution to the cytosol. ECE-1 inhibition or knockdown traps CLR/RAMP1 and β-arrestin2 in endosomes and inhibits CLR/RAMP1 recycling and resensitization, whereas ECE-1 overexpression has the opposite effect. ECE-1 does not regulate either the resensitization of receptors for peptides that are not ECE-1 substrates (e.g., angiotensin II), or the recycling of the bradykinin B₂ receptor, which transiently interacts with β-arrestins. We propose a mechanism by which endosomal ECE-1 degrades neuropeptides in endosomes to disrupt the peptide/receptor/β-arrestin complex, freeing internalized receptors from β-arrestins and promoting recycling and resensitization.