A functional role for the GCC185 golgin in mannose 6-phosphate receptor recycling

Mannose 6-phosphate receptors (MPRs) deliver newly synthesized lysosomal enzymes to endosomes and then recycle to the Golgi. MPR recycling requires Rab9 GTPase; Rab9 recruits the cytosolic adaptor TIP47 and enhances its ability to bind to MPR cytoplasmic domains during transport vesicle formation. Rab9-bearing vesicles then fuse with the trans-Golgi network (TGN) in living cells, but nothing is known about how these vesicles identify and dock with their target. We show here that GCC185, a member of the Golgin family of putative tethering proteins, is a Rab9 effector that is required for MPR recycling from endosomes to the TGN in living cells, and in vitro. GCC185 does not rely on Rab9 for its TGN localization; depletion of GCC185 slightly alters the Golgi ribbon but does not interfere with Golgi function. Loss of GCC185 triggers enhanced degradation of mannose 6-phosphate receptors and enhanced secretion of hexosaminidase. These data assign a specific pathway to an interesting, TGN-localized protein and suggest that GCC185 may participate in the docking of late endosome-derived, Rab9-bearing transport vesicles at the TGN.     

Quantitative analysis of TIP47-receptor cytoplasmic domain interactions: implications for endosome-to-trans Golgi network trafficking

TIP47 (tail-interacting protein of 47 kDa) binds to the cytoplasmic domains of the cation-independent and cation-dependent mannose 6-phosphate receptors and is required for their transport from late endosomes to the trans Golgi network in vitro and in vivo. We report here a quantitative analysis of the interaction of recombinant TIP47 with mannose 6-phosphate receptor cytoplasmic domains. Recombinant TIP47 binds more tightly to the cation-independent mannose 6-phosphate receptor (K(D) = 1 microm) than to the cation-dependent mannose 6-phosphate receptor (K(D) = 3 microm). In addition, TIP47 fails to interact with the cytoplasmic domains of the hormone-processing enzymes, furin, phosphorylated furin, and metallocarboxypeptidase D, as well as the cytoplasmic domain of TGN38, proteins that are also transported from endosomes to the trans Golgi network. Although these proteins failed to bind TIP47, furin and TGN38 were readily recognized by the clathrin adaptor, AP-2. These data suggest that TIP47 recognizes a very select set of cargo molecules. Moreover, our data suggest unexpectedly that furin, TGN38, and carboxypeptidase D may use a distinct vesicular carrier and perhaps a distinct route for transport between endosomes and the trans Golgi network.     

A Novel Rab9 Effector Required for Endosome-to-TGN Transport

Rab9 GTPase is required for the transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network in living cells, and in an in vitro system that reconstitutes this process. We have used the yeast two-hybrid system to identify proteins that interact preferentially with the active form of Rab9. We report here the discovery of a 40-kD protein (p40) that binds Rab9–GTP with roughly fourfold preference to Rab9–GDP. p40 does not interact with Rab7 or K-Ras; it also fails to bind Rab9 when it is bound to GDI. The protein is found in cytosol, yet a significant fraction (~30%) is associated with cellular membranes. Upon sucrose density gradient flotation, membrane- associated p40 cofractionates with endosomes containing mannose 6-phosphate receptors and the Rab9 GTPase. p40 is a very potent transport factor in that the pure, recombinant protein can stimulate, significantly, an in vitro transport assay that measures transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network. The functional importance of p40 is confirmed by the finding that anti-p40 antibodies inhibit in vitro transport. Finally, p40 shows synergy with Rab9 in terms of its ability to stimulate mannose 6-phosphate receptor transport. These data are consistent with a model in which p40 and Rab9 act together to drive the process of transport vesicle docking.     

Visualization of Rab9-mediated vesicle transport from endosomes to the trans-Golgi in living cells

Mannose 6-phosphate receptors (MPRs) are transported from endosomes to the trans-Golgi via a transport process that requires the Rab9 GTPase and the cargo adaptor TIP47. We have generated green fluorescent protein variants of Rab9 and determined their localization in cultured cells. Rab9 is localized primarily in late endosomes and is readily distinguished from the trans-Golgi marker galactosyltransferase. Coexpression of fluorescent Rab9 and Rab7 revealed that these two late endosome Rabs occupy distinct domains within late endosome membranes. Cation-independent mannose 6-phosphate receptors are enriched in the Rab9 domain relative to the Rab7 domain. TIP47 is likely to be present in this domain because it colocalizes with the receptors in fixed cells, and a TIP47 mutant disrupted endosome morphology and sequestered MPRs intracellularly. Rab9 is present on endosomes that display bidirectional microtubule-dependent motility. Rab9-positive transport vesicles fuse with the trans-Golgi network as followed by video microscopy of live cells. These data provide the first indication that Rab9-mediated endosome to trans-Golgi transport can use a vesicle (rather than a tubular) intermediate. Our data suggest that Rab9 remains vesicle associated until docking with the Golgi complex and is rapidly removed concomitant with or just after membrane fusion.     

Endosome to Golgi Transport of Ricin Is Independent of Clathrin and of the Rab9- and Rab11-GTPases

The plant toxin ricin is transported to the Golgi and the endoplasmic reticulum before translocation to the cytosol where it inhibits protein synthesis. The toxin can therefore be used to investigate pathways leading to the Golgi apparatus. Except for the Rab9-mediated transport of mannose 6-phosphate receptors from endosomes to the trans-Golgi network (TGN), transport routes between endosomes and the Golgi apparatus are still poorly characterized. To investigate endosome to Golgi transport, we have used here a modified ricin molecule containing a tyrosine sulfation site and quantified incorporation of radioactive sulfate, a TGN modification. A tetracycline-inducible mutant Rab9S21N HeLa cell line was constructed and characterized to study whether Rab9 was involved in transport of ricin to the TGN and, if not, to further investigate the route used by ricin. Induced expression of Rab9S21N inhibited Golgi transport of mannose 6-phosphate receptors but did not affect the sulfation of ricin, suggesting that ricin is transported to the TGN via a Rab9-independent pathway. Moreover, because Rab11 is present in the endosomal recycling compartment and the TGN, studies of transient transfections with mutant Rab11 were performed. The results indicated that routing of ricin from endosomes to the TGN occurs by a Rab11-independent pathway. Finally, because clathrin has been implicated in early endosome to TGN transport, ricin transport was investigated in cells with inducible expression of antisense to clathrin heavy chain. Importantly, endosome to TGN transport (sulfation of endocytosed ricin) was unchanged when clathrin function was abolished. In conclusion, ricin is transported from endosomes to the Golgi apparatus by a Rab9-, Rab11-, and clathrin-independent pathway.     

Mapmodulin, Cytoplasmic Dynein, and Microtubules Enhance the Transport of Mannose 6-Phosphate Receptors from Endosomes to the Trans-Golgi Network

Late endosomes and the Golgi complex maintain their cellular localizations by virtue of interactions with the microtubule-based cytoskeleton. We study the transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network in vitro. We show here that this process is facilitated by microtubules and the microtubule-based motor cytoplasmic dynein; transport is inhibited by excess recombinant dynamitin or purified microtubule-associated proteins. Mapmodulin, a protein that interacts with the microtubule-associated proteins MAP2, MAP4, and tau, stimulates the microtubule- and dynein-dependent localization of Golgi complexes in semi-intact Chinese hamster ovary cells. The present study shows that mapmodulin also stimulates the initial rate with which mannose 6-phosphate receptors are transported from late endosomes to the trans-Golgi network in vitro. These findings represent the first indication that mapmodulin can stimulate a vesicle transport process, and they support a model in which the microtubule-based cytoskeleton enhances the efficiency of vesicle transport between membrane-bound compartments in mammalian cells.     

Rab 7: an important regulator of late endocytic membrane traffic

Rab5 and rab7 proteins belong to a superfamily of small molecular weight GTPases known to be associated with early and late endosomes, respectively. The rab5 protein plays an important regulatory role in early endocytosis, yet the function of rab7 protein was previously uncharacterized. This question was addressed by comparing the kinetics of vesicular stomatitis virus (VSV) G protein internalization in baby hamster kidney cells overexpressing wild-type or dominant negative mutant forms of the rab7 protein (rab7N125I and rab7T22N). Overexpression of wild-type rab7 protein allowed normal transport to late endosomes (mannose 6-phosphate receptor positive), while the rab7N125I mutant caused the VSV G protein to accumulate specifically in early (transferrin receptor positive) endosomes. Horseradish peroxidase and paramyxovirus SV5 hemagglutinin-neuraminidase (HN) were used in quantitative biochemical assays to further demonstrate that rab7 function was not required for early internalization events, but was crucial in downstream degradative events. The characteristic cleavage of SV5 HN in the late endosome distinguishes internalization from transport to later stages of the endocytic pathway. Mutant rab7N125I or rab7T22N proteins had no effect on the internalization of either horseradish peroxidase or SV5 HN protein. In contrast, the mutant proteins markedly inhibited the subsequent cleavage of the SV5 HN protein. Taken together, these data support a key role for rab7, downstream of rab5, in regulating membrane transport leading from early to late endosomes. We compare our findings to those obtained for the yeast homologues Ypt51p, Ypt52p, Ypt53p, and Ypt7p.     

RUTBC2 protein, a Rab9A effector and GTPase-activating protein for Rab36

Rab GTPases regulate vesicle budding, motility, docking, and fusion. In cells, their cycling between active, GTP-bound states and inactive, GDP-bound states is regulated by the action of opposing enzymes called guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). The substrates for most RabGAPs are unknown, and the potential for cross-talk between different membrane trafficking pathways remains uncharted territory. Rab9A and its effectors regulate recycling of mannose 6-phosphate receptors from late endosomes to the trans Golgi network. We show here that RUTBC2 is a TBC domain-containing protein that binds to Rab9A specifically both in vitro and in cultured cells but is not a GAP for Rab9A. Biochemical screening of Rab protein substrates for RUTBC2 revealed highest GAP activity toward Rab34 and Rab36. In cells, membrane-associated RUTBC2 co-localizes with Rab36, and expression of wild type RUTBC2, but not the catalytically inactive, RUTBC2 R829A mutant, decreases the amount of membrane-associated Rab36 protein. These data show that RUTBC2 can act as a Rab36 GAP in cells and suggest that RUTBC2 links Rab9A function to Rab36 function in the endosomal system.     

RUTBC1 protein, a Rab9A effector that activates GTP hydrolysis by Rab32 and Rab33B proteins

Rab GTPases regulate all steps of membrane trafficking. Their interconversion between active, GTP-bound states and inactive, GDP-bound states is regulated by guanine nucleotide exchange factors and GTPase-activating proteins. The substrates for most Rab GTPase-activating proteins (GAPs) are unknown. Rab9A and its effectors regulate transport of mannose 6-phosphate receptors from late endosomes to the trans-Golgi network. We show here that RUTBC1 is a Tre2/Bub2/Cdc16 domain-containing protein that binds to Rab9A-GTP both in vitro and in cultured cells, but is not a GTPase-activating protein for Rab9A. Biochemical screening of RUTBC1 Rab protein substrates revealed highest in vitro GTP hydrolysis-activating activity with Rab32 and Rab33B. Catalysis required Arg-803 of RUTBC1, and RUTBC1 could activate a catalytically inhibited Rab33B mutant (Q92A), in support of a dual finger mechanism for RUTBC1 action. Rab9A binding did not influence GAP activity of bead-bound RUTBC1 protein. In cells and cell extracts, RUTBC1 influenced the ability of Rab32 to bind its effector protein, Varp, consistent with a physiological role for RUTBC1 in regulating Rab32. In contrast, binding of Rab33B to its effector protein, Atg16L1, was not influenced by RUTBC1 in cells or extracts. The identification of a protein that binds Rab9A and inactivates Rab32 supports a model in which Rab9A and Rab32 act in adjacent pathways at the boundary between late endosomes and the biogenesis of lysosome-related organelles.     

Characterization of the in vitro retrograde transport of MPR46

The mannose 6-phosphate receptor MPR46 mediates sorting of lysosomal enzymes and recycles between the trans -Golgi network and endosomes. We characterized the retrograde transport of MPR46 from endosomes to the TGN by an in vitro transport assay using mouse fibroblast cell lines. Sulfation of a modified MPR46 upon entering the TGN is measured. The in vitro retrograde transport is time-, temperature-, ATP- and cytosol-dependent. Transport requires the SNARE proteins Vti1a and Syntaxin 16 and the Rab family member Rab6. The transport is sensitive to GTPγS, brefeldin A and independent of TIP47. These data indicate that MPR46 follows an early endosome-to-TGN route. Transport is inhibited by MPR46 tail peptide comprising the acidic cluster-di-leucine sorting motif to which adaptor proteins AP-1 and AP-3 bind. Transport depends on cytosolic AP-3, but not on cytosolic AP-1. Residual membrane-associated AP-1 may have masked a requirement for cytosolic AP-1. The competence of membranes from AP-1-deficient cells for endosome-to-TGN transport in vitro was severely compromised.     

Selective recycling of the mannose 6-phosphate/IGF-II receptor to the trans Golgi network in vitro

Mannose 6-phosphate receptors carry soluble lysosomal enzymes from the trans Golgi network (TGN) to prelysosomes, and then return to the TGN for another round of lysosomal enzyme sorting. We describe here a complementation scheme that detects the vesicular transport of the 300 kd mannose 6-phosphate/IGF-II receptor from prelysosomes to the TGN in cell extracts. In vitro transport displays the same selectivity observed in living cells in that the transferrin receptor traverses to the TGN at a much lower rate than mannose 6-phosphate receptors. Furthermore, recycling of mannose 6-phosphate/IGF-II receptors to the TGN requires GTP hydrolysis and can be distinguished biochemically from the constitutive transport of proteins between Golgi cisternae by its resistance to the weak base, primaquine.     

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).     

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.     

Rab9 GTPase regulates late endosome size and requires effector interaction for its stability

Rab9 GTPase resides in a late endosome microdomain together with mannose 6-phosphate receptors (MPRs) and the tail-interacting protein of 47 kDa (TIP47). To explore the importance of Rab9 for microdomain establishment, we depleted the protein from cultured cells. Rab9 depletion decreased late endosome size and reduced the numbers of multilamellar and dense-tubule-containing late endosomes/lysosomes, but not multivesicular endosomes. The remaining late endosomes and lysosomes were more tightly clustered near the nucleus, implicating Rab9 in endosome localization. Cells displayed increased surface MPRs and lysosome-associated membrane protein 1. In addition, cells showed increased MPR synthesis in conjunction with MPR missorting to the lysosome. Surprisingly, Rab9 stability on late endosomes required interaction with TIP47. Rabs are thought of as independent, prenylated entities that reside either on membranes or in cytosol, bound to GDP dissociation inhibitor. These data show that Rab9 stability is strongly influenced by a specific effector interaction. Moreover, Rab9 and the proteins with which it interacts seem critical for the maintenance of specific late endocytic compartments and endosome/lysosome localization.     

The double-play of PP17/TIP47

A missing link in the understanding of the mechanisms of transport of the mannose 6-phosphate receptors has recently been discovered, following the identification of the protein TIP47. In association with Rab9-GTP, this protein is responsible for the return of the receptors from the late endosomes back to the trans-Golgi network. Curiously, the same protein called PP17b, was described as a placental protein twenty years ago, and more recently, as a blood marker for human uterine cervical cancer. The sequence of PP17b/TIP47 displays not only a strong homology with those of adipophilin and the perilipins, two proteins known to be involved in the intracellular traffic of lipid droplets but also PP17b/TIP47 is associated with the later. How this ubiquitous protein could participate in processes as different as the mannose 6-phosphate receptors traffic and the formation and/or traffic of lipid droplets? A tentative hypothesis is put forward.     

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.      

Ric1-Rgp1 Complex Is a Guanine Nucleotide Exchange Factor for the Late Golgi Rab6A GTPase and an Effector of the Medial Golgi Rab33B GTPase

Rab GTPases are master regulators of membrane trafficking events and template the directionality of protein transport through the secretory and endocytic pathways. Certain Rabs recruit the guanine nucleotide exchange factor (GEF) that activates a subsequent acting Rab protein in a given pathway; this process has been termed a Rab cascade. We show here that the medial Golgi-localized Rab33B GTPase has the potential to link functionally to the late Golgi, Rab6 GTPase, by its capacity for association with Ric1 and Rgp1 proteins. In yeast, Ric1p and Rgp1p form a complex that catalyzes guanine nucleotide exchange by Ypt6p, the Rab6 homolog. Human Ric1 and Rgp1 both bind Rab6A with preference for the GDP-bound conformation, characteristic of a GEF. Nevertheless, both Ric1 and Rgp1 proteins are needed to catalyze nucleotide exchange on Rab6A protein. Ric1 and Rgp1 form a complex, but unlike their yeast counterparts, most of the subunits are not associated, and most of the proteins are cytosolic. Loss of Ric1 or Rgp1 leads to destabilization of Rab6, concomitant with a block in Rab6-dependent retrograde transport of mannose 6-phosphate receptors to the Golgi. The C terminus of Ric1 protein contains a distinct binding site for Rab33B-GTP, supporting the existence of a Rab cascade between the medial and trans Golgi. This study thus identifies a GEF for Rab6A in human cells.     

Endosome to Golgi transport of ricin is regulated by cholesterol

We have here studied the role of cholesterol in transport of ricin from endosomes to the Golgi apparatus. Ricin is endocytosed even when cells are depleted for cholesterol by using methyl-beta-cyclodextrin (m beta CD). However, as here shown, the intracellular transport of ricin from endosomes to the Golgi apparatus, measured by quantifying sulfation of a modified ricin molecule, is strongly inhibited when the cholesterol content of the cell is reduced. On the other hand, increasing the level of cholesterol by treating cells with mbetaCD saturated with cholesterol (m beta CD/chol) reduced the intracellular transport of ricin to the Golgi apparatus even more strongly. The intracellular transport routes affected include both Rab9-independent and Rab9-dependent pathways to the Golgi apparatus, since both sulfation of ricin after induced expression of mutant Rab9 (mRab9) to inhibit late endosome to Golgi transport and sulfation of a modified mannose 6-phosphate receptor (M6PR) were inhibited after removal or addition of cholesterol. Furthermore, the structure of the Golgi apparatus was affected by increased levels of cholesterol, as visualized by pronounced vesiculation and formation of smaller stacks. Thus, our results indicate that transport of ricin from endosomes to the Golgi apparatus is influenced by the cholesterol content of the cell.     

Lysosome biogenesis requires Rab9 function and receptor recycling from endosomes to the trans-Golgi network

Newly synthesized lysosomal enzymes bind to mannose 6-phosphate receptors (MPRs) in the TGN, and are carried to prelysosomes, where they are released. MPRs then return to the TGN for another round of transport. Rab9 is a ras-like GTPase which facilitates MPR recycling to the TGN in vitro. We show here that a dominant negative form of rab9, rab9 S21N, strongly inhibited MPR recycling in living cells. The block was specific in that the rates of biosynthetic protein transport, fluid phase endocytosis and receptor-mediated endocytosis were unchanged. Expression of rab9 S21N was accompanied by a decrease in the efficiency of lysosomal enzyme sorting. Cells compensated for the presence of the mutant protein by inducing the synthesis of both soluble and membrane- associated lysosomal enzymes, and by internalizing lysosomal enzymes that were secreted by default. These data show that MPRs are limiting in the secretory pathway of cells expressing rab9 S21N and document the importance of MPR recycling and the rab9 GTPase for efficient lysosomal enzyme delivery.     

Live Salmonella modulate expression of Rab proteins to persist in a specialized compartment and escape transport to lysosomes

We investigated the intracellular route of Salmonella in macrophages to determine a plausible mechanism for their survival in phagocytes. Western blot analysis of isolated phagosomes using specific antibodies revealed that by 5 min after internalization dead Salmonella-containing phagosomes acquire transferrin receptors (a marker for early endosomes), whereas by 30 min the dead bacteria are found in vesicles carrying the late endosomal markers cation-dependent mannose 6-phosphate receptors, Rab7 and Rab9. In contrast, live Salmonella-containing phagosomes (LSP) retain a significant amount of Rab5 and transferrin receptor until 30 min, selectively deplete Rab7 and Rab9, and never acquire mannose 6-phosphate receptors even 90 min after internalization. Retention of Rab5 and Rab18 and selective depletion of Rab7 and Rab9 presumably enable the LSP to avoid transport to lysosomes through late endosomes. The presence of immature cathepsin D (48 kDa) and selective depletion of the vacuolar ATPase in LSP presumably contributes to the less acidic pH of LSP. In contrast, proteolytically processed cathepsin D (M(r) 17,000) was detected by 30 min on the dead Salmonella-containing phagosomes. Morphological analysis also revealed that after uptake by macrophages, the dead Salmonella are transported to lysosomes, whereas the live bacteria persist in compartments that avoid fusion with lysosomes, indicating that live Salmonella bypass the normal endocytic route targeted to lysosomes and mature in a specialized compartment.