Role of an acidic cluster/dileucine motif in cation-independent mannose 6-phosphate receptor traffic

The endocytic trafficking of the cation-independent mannose 6-phosphate receptor (CI-MPR) involves multiple sorting steps. A cluster of acidic amino acids followed by a dileucine motif in the cytoplasmic tail has been proposed to mediate receptor sorting from the trans Golgi network (TGN) to late endosomes. Mutations in this motif impair lysosomal enzyme sorting by preventing association of CI-MPR with coat proteins. The role of the acidic cluster/dileucine motif in the post-endocytic transport of the receptor was examined using the CI-MPR mutants, AC01 and D160E (Chen HJ, Yuan J, Lobel P. J Biol Chem 1997;272:7003-7012). Following internalization, wild type (WT) CI-MPR is transported through sorting endosomes into the endocytic recycling compartment (ERC), after which it traffics to the TGN and other organelles. However, the mutants localize mostly to the ERC and only a small portion reaches the TGN, suggesting that the sorting of the CI-MPR mutants from the ERC into the TGN is severely impaired. We observed no defect in receptor internalization or in the rate of tail mutant recycling to the cell surface compared to the WT. These results demonstrate that the acidic cluster/dileucine motif of CI-MPR is critical for receptor sorting at early stages of intracellular transport following endocytosis.     

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.     

Rabs and other small GTPases in ciliary transport

The non-motile primary cilium is a single, microtubule-based hair-like projection that emanates from most, if not all, non-dividing mammalian cells. Enriched in a variety of signalling receptors and accessories, the cilium mediates crucial sensory and regulatory functions during development and postnatal tissue homoeostasis. Maintenance of ciliary morphology and function requires continuous IFT (intraflagellar transport), and recent findings have shed light on some molecular details of how ciliogenesis is dependent on targeted exocytic membrane trafficking from the Golgi. The ARL [Arf (ADP ribosylation factor)-related] small GTPase Arf4 functions in TGN (trans-Golgi network) sorting of cilia-targeted rhodopsin into carrier vesicles, while Arl6 (Arf-like 6) and Arl13b regulate aspects of ciliary transport and IFT. Ciliogenesis and ciliary functions are also regulated by small Rabs. Rab8a, in conjunction with Rab11a, and via its interaction with a multitude of proteins associated with the ciliary basal body and axoneme/membrane, appears to be critical for ciliogenesis. Rab8's close homologue Rab10 may also play a ciliogenic role in some cells. Rab23, the depletion or inactivation of which affects cilia formation, may regulate specific ciliary protein targeting and turnover, particularly those involved in Shh (Sonic hedgehog) signalling. Recent findings have also implicated Ran, a small GTPase better known for nuclear import, in ciliary targeting of the KIF17 motor protein. We highlight and discuss recent findings on how Rabs and other small GTPases mediate ciliogenesis and ciliary traffic.     

The ubiquitin ligase RNF126 regulates the retrograde sorting of the cation-independent mannose 6-phosphate receptor

The ubiquitin proteasome system is central to the regulation of a number of intracellular sorting pathways in mammalian cells including quality control at the endoplasmic reticulum and the internalization and endosomal sorting of cell surface receptors. Here we describe that RNF126, an E3 ubiquitin ligase, is involved in the sorting of the cation-independent mannose 6-phosphate receptor (CI-MPR). In cells transiently depleted of RNF126, the CI-MPR is dispersed into Rab4 positive endosomes and the efficiency of retrograde sorting is delayed. Furthermore, the stable knockdown of RNF126 leads to the lysosomal degradation of CI-MPR and missorting of cathepsin D. RNF126 specifically regulates the sorting of the CI-MPR as other cargo that follow the retrograde sorting route including the cholera toxin, furin and TGN38 are unaffected in the absence of RNF126. Lastly we show that the RING finger domain of RNF126 is required to rescue the decrease in CI-MPR levels, suggesting that the ubiquitin ligase activity of RNF126 is required for CI-MPR sorting. Together, our data indicate that the ubiquitin ligase RNF126 has a role in the retrograde sorting of the CI-MPR     

A PACS-1, GGA3 and CK2 complex regulates CI-MPR trafficking

The cation-independent mannose-6-phosphate receptor (CI-MPR) follows a highly regulated sorting itinerary to deliver hydrolases from the trans-Golgi network (TGN) to lysosomes. Cycling of CI-MPR between the TGN and early endosomes is mediated by GGA3, which directs TGN export, and PACS-1, which directs endosome-to-TGN retrieval. Despite executing opposing sorting steps, GGA3 and PACS-1 bind to an overlapping CI-MPR trafficking motif and their sorting activity is controlled by the CK2 phosphorylation of their respective autoregulatory domains. However, how CK2 coordinates these opposing roles is unknown. We report a CK2-activated phosphorylation cascade controlling PACS-1- and GGA3-mediated CI-MPR sorting. PACS-1 links GGA3 to CK2, forming a multimeric complex required for CI-MPR sorting. PACS-1-bound CK2 stimulates GGA3 phosphorylation, releasing GGA3 from CI-MPR and early endosomes. Bound CK2 also phosphorylates PACS-1Ser(278), promoting binding of PACS-1 to CI-MPR to retrieve the receptor to the TGN. Our results identify a CK2-controlled cascade regulating hydrolase trafficking and sorting of itinerant proteins in the TGN/endosomal system.     

Transport of mannose-6-phosphate receptors from the trans-Golgi network to endosomes requires Rab31

Rab31, a protein that we originally cloned from a rat oligodendrocyte cDNA library, localizes in the trans-Golgi network (TGN) and endosomes. However, its function has not yet been established. Here we show the involvement of Rab31 in the transport of mannose 6-phosphate receptors (MPRs) from TGN to endosomes. We demonstrate the specific sorting of cation-dependent-MPR (CD-MPR), but not CD63 and vesicular stomatitis virus G (VSVG) protein, to Rab31-containing trans-Golgi network carriers. CD-MPR and Rab31 containing carriers originate from extending TGN tubules that also contain clathrin and GGA1 coats. Expression of constitutively active Rab31 reduced the content of CD-MPR in the TGN relative to that of endosomes, while expression of dominant negative Rab31 triggered reciprocal changes in CD-MPR distribution. Expression of dominant negative Rab31 also inhibited the formation of carriers containing CD-MPR in the TGN, without affecting the exit of VSVG from this compartment. Importantly, siRNA-mediated depletion of endogenous Rab31 caused the collapse of the Golgi apparatus. Our observations demonstrate that Rab31 is required for transport of MPRs from TGN to endosomes and for the Golgi/TGN organization.     

Overexpression of Rab22a hampers the transport between endosomes and the Golgi apparatus

The transport and sorting of soluble and membrane-associated macromolecules arriving at endosomal compartments require a complex set of Rab proteins. Rab22a has been localized to the endocytic compartment; however, very little is known about the function of Rab22a and inconsistent results have been reported in studies performed in different cell lines. To characterize the function of Rab22a in endocytic transport, the wild-type protein (Rab22a WT), a hydrolysis-deficient mutant (Rab22a Q64L), and a mutant with reduced affinity for GTP (Rab22a S19N) were expressed in CHO cells. None of the three Rab22a constructs affected the transport of rhodamine-dextran to lysosomes, the digestion of internalized proteins, or the lysosomal localization of cathepsin D. In contrast with the mild effect of Rab22a on the endosome-lysosome route, cells expressing Rab22a WT and Rab22a Q64L presented a strong delay in the retrograde transport of cholera toxin from endosomes to the Golgi apparatus. Moreover, these cells accumulated the cation independent mannose 6-phosphate receptor in endosomes. These observations indicate that Rab22a can affect the trafficking from endosomes to the Golgi apparatus probably by promoting fusion among endosomes and impairing the proper segregation of membrane domains required for targeting to the trans-Golgi network (TGN).     

Rab15 mediates an early endocytic event in Chinese hamster ovary cells.

Rab GTPases comprise a large family of monomeric proteins that regulate a diverse number of membrane trafficking events, including endocytosis. In this paper, we examine the subcellular distribution and function of the GTPase Rab15. Our biochemical and confocal immunofluorescence studies demonstrate that Rab15 associates with the transferrin receptor, a marker for the early endocytic pathway, but not with Rab7 or the cation-independent mannose 6-phosphate receptor, markers for late endosomal membranes. Furthermore, Rab15 colocalizes with Rab4 and -5 on early/sorting endosomes, as well as Rab11 on pericentriolar recycling endosomes. Consistent with its localization to early endosomal membranes, overexpression of the constitutively active mutant HArab15Q67L reduces receptor-mediated and fluid phase endocytosis. Therefore, our functional studies suggest that Rab15 may function as an inhibitory GTPase in early endocytic trafficking.     

Internalized Pseudomonas exotoxin A can exploit multiple pathways to reach the endoplasmic reticulum

Receptor-mediated internalization to the endoplasmic reticulum (ER) and subsequent retro-translocation to the cytosol are essential sequential processes required for the intoxication of mammalian cells by Pseudomonas exotoxin A (PEx). The toxin binds the alpha2-macroglobulin receptor/low-density lipoprotein receptor-related protein. Here, we show that in HeLa cells, PEx recruits a proportion of this receptor to detergent-resistant microdomains (DRMs). Uptake of receptor-bound PEx involves transport steps both directly from early endosomes to the trans-Golgi network (TGN) independently of Rab9 function and from late endosomes to the TGN in a Rab9-dependent manner. Furthermore, treatments that simultaneously perturb both Arf1-dependent and Rab6-dependent retrograde pathways show that PEx can use multiple routes to reach the ER. The Rab6-dependent route has only been described previously for cargo with lipid-sorting signals. These findings suggest that partial localization of PEx within DRM permits a choice of trafficking routes consistent with a model that DRM-associated toxins reach the ER on a lipid-dependent sorting pathway whilst non-DRM-associated PEx exploits the previously characterized KDEL receptor-mediated uptake pathway. Thus, unexpectedly, an ER-directed toxin with a proteinaceous receptor shows promiscuity in its intracellular trafficking pathways, exploiting routes controlled by both lipid- and protein-sorting signals.     

The DHR1 domain of DOCK180 binds to SNX5 and regulates cation-independent mannose 6-phosphate receptor transport

DOCK180 is the archetype of the DOCK180-family guanine nucleotide exchange factor for small GTPases Rac1 and Cdc42. DOCK180-family proteins share two conserved domains, called DOCK homology region (DHR)-1 and -2. Although the function of DHR2 is to activate Rac1, DHR1 is required for binding to phosphoinositides. To better understand the function of DHR1, we searched for its binding partners by direct nanoflow liquid chromatography/tandem mass spectrometry, and we identified sorting nexins (SNX) 1, 2, 5, and 6, which make up a multimeric protein complex mediating endosome-to-trans-Golgi-network (TGN) retrograde transport of the cation-independent mannose 6-phosphate receptor (CI-MPR). Among these SNX proteins, SNX5 was coimmunoprecipitated with DOCK180 most efficiently. In agreement with this observation, DOCK180 colocalized with SNX5 at endosomes. The RNA interference-mediated knockdowns of SNX5 and DOCK180, but not Rac1, resulted in the redistribution of CI-MPR from TGN to endosomes. Furthermore, expression of the DOCK180 DHR1 domain was sufficient to restore the perturbed CI-MPR distribution in DOCK180 knockdown cells. These data suggest that DOCK180 regulates CI-MPR trafficking via SNX5 and that this function is independent of its guanine nucleotide exchange factor activity toward Rac1.     

Rab10 is involved in basolateral transport in polarized Madin-Darby canine kidney cells

The sorting of newly synthesized membrane proteins to the cell surface is an important mechanism of cell polarity. To identify more of the molecular machinery involved, we investigated the function of the small GTPase Rab10 in polarized epithelial Madin-Darby canine kidney cells. We find that GFP-tagged Rab10 localizes primarily to the Golgi during early cell polarization. Expression of an activated Rab10 mutant inhibits biosynthetic transport from the Golgi and missorts basolateral cargo to the apical membrane. Depletion of Rab10 by RNA interference has only mild effects on biosynthetic transport and epithelial polarization, but simultaneous inhibition of Rab10 and Rab8a more strongly impairs basolateral sorting. These results indicate that Rab10 functions in trafficking from the Golgi at early stages of epithelial polarization, is involved in biosynthetic transport to the basolateral membrane and may co-operate with Rab8.     

Characterization of RIN3 as a guanine nucleotide exchange factor for the Rab5 subfamily GTPase Rab31

The small GTPase Rab5, which cycles between GDP-bound inactive and GTP-bound active forms, plays essential roles in membrane budding and trafficking in the early endocytic pathway. Rab5 is activated by various vacuolar protein sorting 9 (VPS9) domain-containing guanine nucleotide exchange factors. Rab21, Rab22, and Rab31 (members of the Rab5 subfamily) are also involved in the trafficking of early endosomes. Mechanisms controlling the activation Rab5 subfamily members remain unclear. RIN (Ras and Rab interactor) represents a family of multifunctional proteins that have a VPS9 domain in addition to Src homology 2 (SH2) and Ras association domains. We investigated whether RIN family members act as guanine nucleotide exchange factors (GEFs) for the Rab5 subfamily on biochemical and cell morphological levels. RIN3 stimulated the formation of GTP-bound Rab31 in cell-free and in cell GEF activity assays. RIN3 also formed enlarged vesicles and tubular structures, where it colocalized with Rab31 in HeLa cells. In contrast, RIN3 did not exhibit any apparent effects on Rab21. We also found that serine to alanine substitutions in the sequences between SH2 and RIN family homology domain of RIN3 specifically abolished its GEF action on Rab31 but not Rab5. We examined whether RIN3 affects localization of the cation-dependent mannose 6-phosphate receptor (CD-MPR), which is transported between trans-Golgi network and endocytic compartments. We found that RIN3 partially translocates CD-MPR from the trans-Golgi network to peripheral vesicles and that this is dependent on its Rab31-GEF activity. These results indicate that RIN3 specifically acts as a GEF for Rab31.     

Spatiotemporal Resolution of Rab9 and CI‐MPR Dynamics in the Endocytic Pathway

Rab9 is a small GTPase that localizes to the trans‐Golgi Network (TGN) and late endosomes. Its main function has long been connected to the recycling of mannose‐6‐phosphate receptors (MPRs). However, recent studies link Rab9 also to autophagy and lysosome biogenesis. In this paper, using confocal imaging, we characterize for the first time the live dynamics of the Rab9 constitutively active mutant, Rab9Q66L. We find that it localizes predominantly to late endosomes and that its expression in HeLa cells disperses TGN46 and cation‐independent (CI‐MPR) away from the Golgi yet, has no effect on the retrograde transport of CI‐MPR. We also show that CI‐MPR and Rab9 enter the endosomal pathway together at the transition stage between early, Rab5‐positive, and late, Rab7a‐positive, endosomes. CI‐MPR localizes transiently to separate domains on these endosomes, where vesicles carrying CI‐MPR attach and detach within seconds. Taken together, our results demonstrate that Rab9 mediates the delivery of CI‐MPR to the endosomal pathway, entering the maturing endosome at the early‐to‐late transition.      

Golgi-localizing, gamma-adaptin ear homology domain, ADP-ribosylation factor-binding (GGA) proteins interact with acidic dileucine sequences within the cytoplasmic domains of sorting receptors through their Vps27p/Hrs/STAM (VHS) domains

GGA (Golgi-localizing, gamma-adaptin ear homology domain, ARF-binding) proteins are potential effectors of ADP-ribosylation factors, are associated with the trans-Golgi network (TGN), and are involved in protein transport from this compartment. By yeast two-hybrid screening and subsequent two-hybrid and pull-down analyses, we have shown that GGA proteins, through their VHS (Vps27p/Hrs/STAM) domains, interact with acidic dileucine sequences found in the cytoplasmic domains of TGN-localized sorting receptors such as sortilin and mannose 6-phosphate receptor. A mutational analysis has revealed that a leucine pair and a cluster of acidic residues adjacent to the pair are mainly responsible for the interaction. A chimeric receptor with the sortilin cytoplasmic domain localizes to the TGN, whereas the chimeric receptor with a mutation at the leucine pair or the acidic cluster is mislocalized to punctate structures reminiscent of early endosomes. These results indicate that GGA proteins regulate the localization to or exit from the TGN of the sorting receptors.     

SNX1 defines an early endosomal recycling exit for sortilin and mannose 6-phosphate receptors

Mannose-6-phosphate receptors (MPRs) transport lysosomal hydrolases from the trans Golgi network (TGN) to endosomes. Recently, the multi-ligand receptor sortilin has also been implicated in this transport, but the transport carriers involved herein have not been identified. By quantitative immuno-electron microscopy, we localized endogenous sortilin of HepG2 cells predominantly to the TGN and endosomes. In the TGN, sortilin colocalized with MPRs in the same clathrin-coated vesicles. In endosomes, sortilin and MPRs concentrated in sorting nexin 1 (SNX1)-positive buds and vesicles. SNX1 depletion by small interfering RNA resulted in decreased pools of sortilin in the TGN and an increase in lysosomal degradation. These data indicate that sortilin and MPRs recycle to the TGN in SNX1-dependent carriers, which we named endosome-to-TGN transport carriers (ETCs). Notably, ETCs emerge from early endosomes (EE), lack recycling plasma membrane proteins and by three-dimensional electron tomography exhibit unique structural features. Hence, ETCs are distinct from hitherto described EE-derived membranes involved in recycling. Our data emphasize an important role of EEs in recycling to the TGN and indicate that different, specialized exit events occur on the same EE vacuole.     

The sortilin cytoplasmic tail conveys Golgi-endosome transport and binds the VHS domain of the GGA2 sorting protein

Sortilin belongs to a growing family of multiligand type-1 receptors with homology to the yeast receptor Vps10p. Based on structural features and sortilin's intracellular predominance, we have proposed it to be a sorting receptor for ligands in the synthetic pathway as well as on the cell membrane. To test this hypothesis we examine here the cellular trafficking of chimeric receptors containing constructs of the sortilin tail. We report that sorting signals conforming to YXX and dileucine motifs mediate rapid endocytosis of sortilin chimeras, which subsequently travel to the trans-Golgi network, showing little or no recycling. Furthermore, we found that cation-independent mannose 6-phosphate receptor (MPR300)-sortilin chimeras, expressed in mannose 6-phosphate receptor knockout cells, were almost as efficient as MPR300 itself for transport of newly synthesized beta-hexosaminidase and beta-glucuronidase to lysosomes, and established that the sortilin tail contains potent signals for Golgi-endosome sorting. Finally, we provide evidence suggesting that sortilin is the first example of a mammalian receptor targeted by the recently described GGA family of cytosolic sorting proteins, which condition the Vps10p-mediated sorting of yeast carboxypeptidase Y.     

Rab24 interacts with the Rab7/Rab interacting lysosomal protein complex to regulate endosomal degradation

Endocytosis is a multistep process engaged in extracellular molecules internalization. Several proteins including the Rab GTPases family coordinate the endocytic pathway. The small GTPase Rab7 is present in late endosome (LE) compartments being a marker of endosome maturation. The Rab interacting lysosomal protein (RILP) is a downstream effector of Rab7 that recruits the functional dynein/dynactin motor complex to late compartments. In the present study, we have found Rab24 as a component of the endosome‐lysosome degradative pathway. Rab24 is an atypical protein of the Rab GTPase family, which has been attributed a function in vesicle trafficking and autophagosome maturation. Using a model of transiently expressed proteins in K562 cells, we found that Rab24 co‐localizes in vesicular structures labeled with Rab7 and LAMP1. Moreover, using a dominant negative mutant of Rab24 or a siRNA‐Rab24 we showed that the distribution of Rab7 in vesicles depends on a functional Rab24 to allow DQ‐BSA protein degradation. Additionally, by immunoprecipitation and pull down assays, we have demonstrated that Rab24 interacts with Rab7 and RILP. Interestingly, overexpression of the Vps41 subunit from the homotypic fusion and protein‐sorting (HOPS) complex hampered the co‐localization of Rab24 with RILP or with the lysosomal GTPase Arl8b, suggesting that Vps41 would affect the Rab24/RILP association. In summary, our data strongly support the hypothesis that Rab24 forms a complex with Rab7 and RILP on the membranes of late compartments. Our work provides new insights into the molecular function of Rab24 in the last steps of the endosomal degradative pathway.      

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.     

Giantin interacts with both the small GTPase Rab6 and Rab1

The interaction of small GTPases of the Rab family and coiled coil proteins of the golgin family has been reported for example for the Rab1 GTPase and p115, GM130 and Giantin. We now show that Rab6A, a GTPase that controls retrograde trafficking within the Golgi back to the endoplasmic reticulum is also able to bind to Giantin in vivo and in vitro pointing to an interesting complex formation between Giantin and two different Rab GTPases. In Saccharomyces cerevisiae a genetic interaction between Ypt1 and Ypt6 has already been demonstrated, but in this paper we were able to describe that the mammalian Rab GTPases are able to interact on the same golgin protein, Giantin.