The oxysterol-binding protein homologue ORP1L interacts with Rab7 and alters functional properties of late endocytic compartments

ORP1L is a member of the human oxysterol-binding protein (OSBP) family. ORP1L localizes to late endosomes (LEs)/lysosomes, colocalizing with the GTPases Rab7 and Rab9 and lysosome-associated membrane protein-1. We demonstrate that ORP1L interacts physically with Rab7, preferentially with its GTP-bound form, and provide evidence that ORP1L stabilizes GTP-bound Rab7 on LEs/lysosomes. The Rab7-binding determinant is mapped to the ankyrin repeat (ANK) region of ORP1L. The pleckstrin homology domain (PHD) of ORP1L binds phosphoinositides with low affinity and specificity. ORP1L ANK- and ANK+PHD fragments induce perinuclear clustering of LE/lysosomes. This is dependent on an intact microtubule network and a functional dynein/dynactin motor complex. The dominant inhibitory Rab7 mutant T22N reverses the LE clustering, suggesting that the effect is dependent on active Rab7. Transport of fluorescent dextran to LEs is inhibited by overexpression of ORP1L. Overexpression of ORP1L, and in particular the N-terminal fragments of ORP1L, inhibits vacuolation of LE caused by Helicobacter pylori toxin VacA, a process also involving Rab7. The present study demonstrates that ORP1L binds to Rab7, modifies its functional cycle, and can interfere with LE/lysosome organization and endocytic membrane trafficking. This is the first report of a direct connection between the OSBP-related protein family and the Rab GTPases.     

Rab11BP/Rabphilin-11, a downstream target of rab11 small G protein implicated in vesicle recycling.

Rab11 small G protein has been implicated in vesicle recycling, but its upstream regulators or downstream targets have not yet been identified. We isolated here a downstream target of Rab11, named rabphilin-11, from bovine brain. Moreover, we isolated from a rat brain cDNA library its cDNA, which encoded a protein with a M(r) of 100,946 and 908 amino acids (aa). Rabphilin-11 bound GTP-Rab11 more preferentially than GDP-Rab11 at the N-terminal region and was specific for Rab11 and inactive for other Rab and Rho small G proteins. Both GTP-Rab11 and rabphilin-11 were colocalized at perinuclear regions, presumably the Golgi complex and recycling endosomes, in Madin-Darby canine kidney cells. In HeLa cells cultured on fibronectin, both the proteins were localized not only at perinuclear regions but also along microtubules, which were oriented toward membrane lamellipodia. Treatment of HeLa cells with nocodazole caused disruption of microtubules and dispersion of GTP-Rab11 and rabphilin-11. Overexpression of the C-terminal fragment of rabphilin-11 (aa 607-730), lacking the GTP-Rab11 binding domain, in HeLa cells reduced accumulation of transferrin at perinuclear regions and cell migration. Rabphilin-11 turned out to be a rat counterpart of recently reported bovine Rab11BP. These results indicate that rabphilin-11 is a downstream target of Rab11 which is involved in vesicle recycling.     

ORP10, a cholesterol binding protein associated with microtubules, regulates apolipoprotein B-100 secretion

ORP10/OSBPL10 is a member of the oxysterol-binding protein family, and genetic variation in OSBPL10 is associated with dyslipidemias and peripheral artery disease. In this study we investigated the ligand binding properties of ORP10 in vitro as well as its localization and function in human HuH7 hepatocytes. The pleckstrin homology (PH) domain of ORP10 selectively interacts with phosphatidylinositol-4-phosphate, while the C-terminal ligand binding domain binds cholesterol and several acidic phospholipids. Full-length ORP10 decorates microtubules (MT), while the ORP10 N-terminal fragment (aa 1-318) localizes at Golgi membranes. Removal of the C-terminal aa 712-764 of ORP10 containing a predicted coiled-coil segment abolishes the MT association, but allows partial Golgi targeting. A PH domain-GFP fusion protein is distributed mainly in the cytosol and the plasma membrane, indicating that the Golgi affinity of ORP10 involves other determinants in addition to the PH domain. HuH7 cells expressing ORP10-specific shRNA display increased accumulation of apolipoprotein B-100 (apoB-100), but not of albumin, in culture medium, and contain reduced levels of intracellular apoB-100. Pulse-chase analysis of cellular [(35)S]apoB-100 demonstrates enhanced apoB-100 secretion by cells expressing ORP10-specific shRNA. The apoB-100 secretion phenotype is replicated in HepG2 cells transduced with the ORP10 shRNA lentiviruses. As a conclusion, the present study dissects the determinants of ORP10 association with MT and the Golgi complex and provides evidence for a specific role of this protein in β-lipoprotein secretion by human hepatocytes.     

Subfamily III of mammalian oxysterol-binding protein (OSBP) homologues: the expression and intracellular localization of ORP3, ORP6, and ORP7

The human OSBP related protein (ORP) family consists of 12 members, which can be divided into six subfamilies based on the genomic organization and amino acid homology. Here we performed basic characterization of subfamily III, which consists of three members: ORP3, ORP6, and ORP7. According to cDNA hybridization, the three genes are expressed in a tissue-specific manner. While ORP3 mRNA is most abundant in kidney, lymph nodes, and thymus, ORP6 shows highest expression in brain and skeletal muscle, and ORP7 in the gastrointestinal tract. Using monospecific peptide antibodies, we confirmed the presence of the three proteins in human and mouse tissues. ORP6 gene expression was induced upon differentiation of F9 embryonic carcinoma cells into parietal endoderm, while ORP3 and ORP7 mRNA levels were unchanged. In the F9 cells, endogenous ORP6 associated predominantly with the nuclear envelope. When expressed from the cDNA in cultured cells, the three proteins were distributed between the cytosol and endoplasmic reticulum (ER) membranes, with a minor portion found at the plasma membrane. Experiments with truncated constructs showed that the N-terminal portion of the proteins, containing a pleckstrin homology (PH) domain, has markedly strong plasma membrane targeting specificity, while the C-terminal half remains largely cytosolic. The expression data demonstrates that ORP3, -6, and -7 are not merely redundant gene products but show marked quantitative differences in tissue expression, suggesting tissue-specific aspects in their function. The dual targeting of the proteins indicates a putative role in communication between the ER and the plasma membrane.This study was supported by the Clinical Research Fund of Helsinki University Central Hospital (J.T.), the Academy of Finland (grant 51883 to M.L.; grants 49987, 50641, and 54301 to V.M.O.), the Sigrid Juselius Foundation, and the Finnish Cultural Foundation     

Rab1b interacts with GBF1 and modulates both ARF1 dynamics and COPI association

Assembly of the cytosolic coat protein I (COPI) complex at the ER-Golgi interface is directed by the ADP ribosylation factor1 (Arf1) and its guanine nucleotide exchange factor (GBF1). Rab1b GTPase modulates COPI recruitment, but the molecular mechanism underlying this action remains unclear. Our data reveal that in vivo expression of the GTP-restricted Rab1b mutant (Rab1Q67L) increased the association of GBF1 and COPI to peripheral structures localized at the ER exit sites (ERES) interface. Active Rab1b also stabilized Arf1 on Golgi membranes. Furthermore, we characterized GBF1 as a new Rab1b effector, and showed that its N-terminal domain was involved in this interaction. Rab1b small interfering RNA oligonucleotide assays suggested that Rab1b was required for GBF1 membrane association. To further understand how Rab1b functions in ER-to-Golgi transport, we analyzed GFP-Rab1b dynamics in HeLa cells. Time-lapse microscopy indicated that the majority of the Rab1b-labeled punctuated structures are relatively short-lived with limited-range movements. FRAP of Golgi GFP-Rab1bwt showed rapid recovery (t(1/2) 120 s) with minimal dependence on microtubules. Our data support a model where Rab1b-GTP induces GBF1 recruitment at the ERES interface and at the Golgi complex where it is required for COPII/COPI exchange or COPI vesicle formation, respectively.     

Thank ORP9 for FFAT: With endosomal ORP10, it’s fission accomplished!

Heterogeneity in endosomal membrane phospholipid content is emerging as a regulator of endocytic trafficking pathways. Kawasaki et al. (2021. J. Cell. Biol. https://doi.org/10.1083/jcb.202103141) demonstrate exchange of endosomal PI4P for PS by ORP10 at ER–endosome contact sites, with the consequent recruitment of endosomal fission factors.

Most cellular lipids are synthesized in the ER, often undergoing rapid redistribution to other cellular membranes, thereby maintaining low concentrations at the ER. Consequently, lipids exiting the ER may need to be transported against their concentration gradient. Lipid flow along a gradient to the ER can drive countertransport of ER-derived lipid to membranes with a higher lipid concentration. This nonvesicular lipid exchange occurs at membrane contact sites (MCS), where different organelles are closely apposed, providing a platform for lipid transport proteins including oxysterol-binding protein (OSBP)-related proteins (ORPs). Lipid specificity, which varies between ORPs, is defined by the OSBP-related domain (ORD). The ORD of ORP10 shares phosphatidylinositol-4-phosphate (PI4P) and phosphatidylserine (PS) binding residues with ORP5/8 and can bind and extract PS from liposomes (1), suggesting a potential role in PI4P-PS counter transport, analogous to that of ORP5/8 at ER–plasma membrane MCS (2). ORPs are targeted to specific organelles by interaction between their PH domain and membrane phospholipids. Most ORPs also possess a FFAT motif (two phenylalanines in an acidic tract), which simultaneously targets the ORP to ER-localized VAMP-associated proteins (VAPs) at MCS between the ER and other organelles. ORP10, however, lacks a FFAT motif, yet was found to stabilize ER–Golgi MCSs (Fig. 1 A) for PI4P transport to the ER (2). Kawasaki et al. have now uncovered a novel function for ORP10 in PI4P–PS lipid exchange at the ER–endosome interface (Fig. 1 B), with downstream effects on endosomal fission and retrograde transport (3).Open in a separate windowFigure 1.Regulation of retrograde and secretory traffic by ORP10-mediated lipid exchange. (A) ORP10 interacts with VAP-bound ORP9 at ER–endosome and ER–Golgi MCSs, with downstream effects on retrograde transport of mannose 6-phosphate receptor (M6PR). Boxed region (detailed in B) depicts ORP10 at the ER–endosome interface. (B) ORP10 functions in lipid exchange between the ER and endosomes, transporting endosomal PI4P to the ER in exchange for ER-derived PS. Production of PI4P in endosomes by PI4KIIα-dependent phosphorylation of phosphatidylinositol (PI), coupled with its consumption in the ER by ER-localized Sac1, generates a PI4P concentration gradient from the endosome to the ER. Low membrane PS concentrations in the ER are maintained by PS inhibition of PS synthesis from phosphatidylcholine (PC) by Pss1 or from PE by Pss2, with PS synthesis at ER–endosome contact sites promoting rapid PS export from the ER in yeast (not yet known if a similar mechanism operates in mammalian cells). ORP10 mediates PI4P transport along its gradient to the ER, driving countertransport of PS by ORP10 against its concentration gradient to the endosome. PS enrichment at the endosome leads to recruitment of the ATPase EHD1 to facilitate endosome fission for retrograde transport. (C) Depletion of ORP10 prevents lipid exchange at ER–endosome contact sites, resulting in a loss of retrograde transport of M6PR. Additionally, ER–Golgi MCSs are diminished, and secretion of ApoB-100 is increased.The PH domain of ORP10 selectively binds PI4P and is required for ORP10 recruitment to the TGN (2) and endosomes (3), both home to PI4KIIα, a PI4P-producing kinase. Rapid PI4P degradation at the ER by the phosphatase Sac1 generates a PI4P gradient at the ER–endosome or TGN interface, with PI4P flow to the ER driving countertransport of PS to the endosome (as also predicted for the Golgi). Activity of endosomal PI4P phosphatase Sac2 (4) may hamper formation of an endosome–ER PI4P gradient, but since ORP10 did not colocalise well with Sac2 (3), they likely function at different endosome populations.PS synthesis at MCSs may also contribute to ORP10-mediated lipid exchange. Targeting PS synthase to ER:mitochondria contacts in yeast was found topromote PS transport out of the ER to mitochondria (5). Similarly, ER to endosome PS transport was increased when PS synthase was targeted to ER:endosome MCS. Localized PS gradients from PS synthesis in the ER at MCSs, coupled with rapid decarboxylation of PS to phosphatidylethanolamine (PE) in mitochondria/endosomes by yeast PS decarboxylases Psd1/Psd2, could contribute to lipid exchange. In mammalian cells, though, since no endosomal decarboxylase has been identified, ORP10-mediated lipid exchange is likely to be primarily driven by the PI4P gradient. Whether this process is facilitated by localized activation of PS synthase at MCS has not yet been demonstrated. Since PS synthase activity is negatively regulated by PS, exit from the ER is a key factor in its biogenesis. Recruitment of specific ORPs to endosomes/TGN by PI4P for ER tethering and consequent lipid exchange provides an elegant regulatory pathway for PI4P–PS homeostasis in cellular membranes.ORP10 shares functional similarities with ORP11: both proteins comprise an N-terminal PH domain and a C-terminal ORD, with a linker region in between harboring a coiled-coiled domain. Unlike other ORPs, ORP10 and ORP11 possess neither a FFAT motif nor a membrane spanning domain to enable ER interaction, but heterotypic interaction with ORP9, which does contain a FFAT motif, has been demonstrated for both proteins. Kawasaki et al. identified an ORP9-ORP10 interaction at ER–endosome MCSs that is dependent on the ORP10 linker region. ORP9 was also implicated in ORP10-mediated lipid exchange at the TGN, where it may play a redundant role with OSBP in maintaining ER contact. Similarly, ORP11 is also recruited to the TGN and, to a lesser extent, the endosome, by ORP9, with the interaction depending on the linker region of both proteins (6).The finely tuned regulation of PI4P/PS is emerging as an important determinant of endocytic traffic. Previous studies have shown that endosomal PI4P accumulation inhibits retrograde transport from endosomes to the TGN (7), while endosomal PS regulates endosome to Golgi retrograde traffic. As depicted in Fig. 1 B, Kawasaki et al. have built on this to show that through interaction with VAP-bound ORP9, ORP10 mediates lipid countertransport at ER–endosome MCSs, removing PI4P from, and supplying PS to, the endosome, with consequent recruitment of the membrane scission protein EHD1 to control endosomal fission and retrograde transport (3). Spatial and temporal regulation of endosome fission by ER–endosome MCSs involves recruitment of the ER membrane protein TMCC1 to the budding endosome by the actin regulator Coronin 1C, stabilizing the MCS (7), but the mechanism by which MCS might effect scission has remained elusive. The findings of Kawaski et al. present an explanation: by providing a platform for lipid exchange, MCS promote the recruitment of EHD1, which belongs to a conserved class of ATPases that can oligomerise in ring-like structures around tubules to mediate fission (8). VAP interaction with OSBP at ER–endosome MCSs is also required for retrograde transport (7), but potential redundancy between ORP9/OSBP in ORP10-mediated lipid exchange, or if ORP10 functions at Coronin 1C/TMCC1-regulated MCS is not yet established.Interestingly, ORP10 function at the TGN has been implicated in regulating ApoB-100 secretion (Fig. 1 C), with hypersecretion reported in ORP10-depleted cells (9). FFAP1, which promotes PI4P consumption by Sac1 at ER:TGN contacts, also negatively regulates ApoB-100 exit from the TGN in a PI4KIIIβ-dependent manner, suggesting direct regulation of ApoB-100 secretion by PI4P at the TGN (9). Could PI4P coordinate nutrient sensing with cargo sorting and secretion at the TGN? PI4P has been described as lipid biosensor of cytosolic pH, with protonation of its head group regulating protein interactions (10). The influence of cytosolic pH on ORP10-PI4P interaction may provide an additional layer of regulation of lipoprotein secretion in response to changes in cellular energy/pH.How ORP10 function is coordinated at Golgi and endosomal membranes and the significance of potential redundancy with ORP11 remains unclear. The regulation of Sac2 activity and how it relates to ER-endosome lipid exchange is also intriguing. While questions still remain, an important role for ORP10 is emerging in maintaining homoeostasis between endosome maturation, retrograde traffic and secretory transport.     

Oxysterol-binding proteins: Sterol and phosphoinositide sensors coordinating transport,signaling and metabolism

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a family of sterol and phosphoinositide binding proteins conserved in eukaryotes. The mechanisms of ORP function have remained incompletely understood. However, several ORPs are present at membrane contact sites and control the activity of enzymatic effectors or assembly of protein complexes, with impacts on signaling, vesicle transport, and lipid metabolism. An increasing number of protein interaction partners of ORPs have been identified, providing clues of their involvement in multiple aspects of cell regulation.The functions assigned for mammalian ORPs include coordination of sterol and sphingolipid metabolism and mitogenic signaling (OSBP), control of ER-late endosome (LE) contacts and LE motility (ORP1L), neutral lipid metabolism (ORP2), cell adhesion (ORP3), cholesterol eggress from LE (ORP5), macrophage lipid homeostasis, migration and high-density lipoprotein metabolism (ORP8), apolipoprotein B-100 secretion (ORP10), and adipogenesis (ORP11). The anti-proliferative ORPphilin compounds target OSBP and ORP4, revealing a function of ORPs in cell proliferation and survival. The Saccharomyces cerevisiae OSBP homologue (Osh) proteins execute multifaceted functions in sterol and sphingolipid homeostasis, post-Golgi vesicle transport, as well as phosphatidylinositol-4-phosphate and target of rapamycin complex 1 (TORC1) signaling. These observations identify ORPs as coordinators of lipid signals with an unforeseen variety of cellular processes.     

Activation of endosomal dynein motors by stepwise assembly of Rab7-RILP-p150Glued, ORP1L, and the receptor betalll spectrin

The small GTPase Rab7 controls late endocytic transport by the minus end-directed motor protein complex dynein-dynactin, but how it does this is unclear. Rab7-interacting lysosomal protein (RILP) and oxysterol-binding protein-related protein 1L (ORP1L) are two effectors of Rab7. We show that GTP-bound Rab7 simultaneously binds RILP and ORP1L to form a RILP-Rab7-ORP1L complex. RILP interacts directly with the C-terminal 25-kD region of the dynactin projecting arm p150(Glued), which is required for dynein motor recruitment to late endocytic compartments (LEs). Still, p150(Glued) recruitment by Rab7-RILP does not suffice to induce dynein-driven minus-end transport of LEs. ORP1L, as well as betaIII spectrin, which is the general receptor for dynactin on vesicles, are essential for dynein motor activity. Our results illustrate that the assembly of microtubule motors on endosomes involves a cascade of linked events. First, Rab7 recruits two effectors, RILP and ORP1L, to form a tripartite complex. Next, RILP directly binds to the p150(Glued) dynactin subunit to recruit the dynein motor. Finally, the specific dynein motor receptor Rab7-RILP is transferred by ORP1L to betaIII spectrin. Dynein will initiate translocation of late endosomes to microtubule minus ends only after interacting with betaIII spectrin, which requires the activities of Rab7-RILP and ORP1L.     

Small GTPase Rab40c Associates with Lipid Droplets and Modulates the Biogenesis of Lipid Droplets

The subcellular location and cell biological function of small GTPase Rab40c in mammalian cells have not been investigated in detail. In this study, we demonstrated that the exogenously expressed GFP-Rab40c associates with lipid droplets marked by neutral lipid specific dye Oil red or Nile red, but not with the Golgi or endosomal markers. Further examination demonstrated that Rab40c is also associated with ERGIC-53 containing structures, especially under the serum starvation condition. Rab40c is increasingly recruited to the surface of lipid droplets during lipid droplets formation and maturation in HepG2 cells. Rab40c knockdown moderately decreases the size of lipid droplets, suggesting that Rab40c is involved in the biogenesis of lipid droplets. Stimulation for adipocyte differentiation increases the expression of Rab40c in 3T3-L1 cells. Rab40c interacts with TIP47, and is appositionally associated with TIP47-labeled lipid droplets. In addition, over-expression of Rab40c causes the clustering of lipid droplets independent of its GTPase activity, but completely dependent of the intact SOCS box domain of Rab40c. In addition, Rab40c displayed self-interaction as well as interaction with TIP47 and the SOCS box is essential for its ability to induce clustering of lipid droplets. Our results suggest that Rab40c is a novel Rab protein associated with lipid droplets, and is likely involved in modulating the biogenesis of lipid droplets.     

Identification and characterization of a family of Rab11-interacting proteins

Rab11a is a small GTP-binding protein enriched in the pericentriolar plasma membrane recycling systems. We hypothesized that Rab11a-binding proteins exist as downstream effectors of its action. Here we define a family of four Rab11-interacting proteins: Rab11-Family Interacting Protein 1 (Rab11-FIP1), Rab11-Family Interacting Protein 2 (Rab11-FIP2), Rab11-Family Interacting Protein 3 (Rab11-FIP3), and pp75/Rip11. All four interacting proteins associated with wild type Rab11a and dominant active Rab11a (Rab11aS20V) as well as Rab11b and Rab25. Rab11-FIP2 also interacted with dominant negative Rab11a (Rab11aS25N) and the tail of myosin Vb. The binding of Rab11-FIP1, Rab11-FIP2, and Rab11-FIP3 to Rab11a was dependent upon a conserved carboxyl-terminal amphipathic alpha-helix. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 colocalized with Rab11a in plasma membrane recycling systems in both non-polarized HeLa cells and polarized Madin-Darby canine kidney cells. GFP-Rab11-FIP3 also colocalized with Rab11a in HeLa cells. Rab11-FIP1, Rab11-FIP2, and pp75/Rip11 also coenriched with Rab11a and H(+)K(+)-ATPase on parietal cell tubulovesicles, and Rab11-FIP1 and Rab11-FIP2 translocated with Rab11a and the H(+)K(+)-ATPase upon stimulating parietal cells with histamine. The results suggest that the function of Rab11a in plasma membrane recycling systems is dependent upon a compendium of protein effectors.     

The roles of the human lipid-binding proteins ORP9S and ORP10S in vesicular transport.

Inactivation of the yeast oxysterol binding protein related protein (ORP) family member Kes1p allows yeast cells to survive in the absence of Sec14p, a phospholipid transfer protein required for cell viability because of the role it plays in transporting vesicles from the Golgi. We expressed human ORP9S and ORP10S in yeast lacking Sec14p and Kes1p function, and found that ORP9S completely complemented Kes1p function, whereas ORP10S possessed only a weak ability to replace Kes1p function. Purified ORP9S protein bound several phosphoinositides, whereas ORP10 bound specifically to phosphatidylinositol 3-phosphate. The combined evidence demonstrates that only a subset of human ORP proteins can function as negative regulators of Golgi-derived vesicular transport.     

Induction of autophagy causes dramatic changes in the subcellular distribution of GFP-Rab24

Rab GTPases comprises a large family of proteins, with more than 50 gene products localized in distinct subcellular compartments. Rab24 is a member of this family whose function is not presently known. In order to elucidate the role of this protein we have generated a GFP-tagged Rab24 and studied the distribution of this chimera by fluorescence microscopy. GFP-Rab24 showed a perinuclear reticular localization that often encircled the nucleus. This reticular pattern partially overlapped with ER markers, cis-Golgi, and the ER-Golgi intermediate compartment. Surprisingly, when GFP-Rab24-transfected cells were starved to induce autophagy the distribution of the protein changed dramatically. GFP-Rab24 localized in large dots, cup-shaped structures and ring-shaped vesicles. Some of these vesicles were labeled with monodansylcadaverine , a specific autophagosome marker. In the presence of vinblastine, an agent that induces the formation of very large autophagic vesicles, GFP-Rab24 accumulated in the large vacuoles that were also labeled by monodansylcadaverine. Furthermore, Rab24 colocalized with LC3, a mammalian homolog of the yeast protein Apg8/Aut7, an essential gene for autophagy. This is the first report indicating that Rab24 localizes on autophagosomes, suggesting that this Rab protein is involved in the autophagic pathway.     

VAMP-associated protein-A regulates partitioning of oxysterol-binding protein-related protein-9 between the endoplasmic reticulum and Golgi apparatus

We recently showed that oxysterol-binding protein (OSBP), one of twelve related PH domain containing proteins with lipid and sterol binding activity, interacts with VAMP-associated protein (VAP)-A on the endoplasmic reticulum (ER). In addition to OSBP, seven OSBP-related proteins (ORPs) bind VAP-A via a conserved E-F/Y-F/Y-DA 'FFAT' motif. We focused on this interaction for ORP9, which is expressed as a full-length (ORP9L) or truncated version missing the PH domain (ORP9S). Mutation analysis showed that the interaction required the ORP9 FFAT motif and the N-terminal conserved domain of VAP. Endogenous ORP9L displayed Golgi localization, which was partially mediated by the PH domain based on limited localization of OPR9-PH-GFP with the Golgi apparatus. When inducibly overexpressed, ORP9S and ORP9L colocalized with VAP-A and caused vacuolation of the ER as well as retention of the ER-Golgi intermediate compartment marker ERGIC-53/p58 in the ER. ORP9L mutated in the VAP-A binding domain (ORP9L-FY-->AA) did not localize to the ER but appeared with giantin and Sec31 on large vesicular structures, suggesting the presence of a hybrid Golgi-COPII compartment. Normal Golgi localization was also observed for ORP9L-FY-->AA. Results show that VAP binding and PH domains target ORP9 to the ER and a Golgi-COPII compartment, respectively, and that ORP9L overexpression in these compartments severely perturbed their organization.     

Rab11 promotes docking and fusion of multivesicular bodies in a calcium-dependent manner

Multivesicular bodies (MVBs) are membranous structures within 60-100 nm diameter vesicles accumulate. MVBs are generated after invagination and pinching off of the endosomal membrane in the lumen of the vacuole. In certain cell types, fusion of MVBs with the plasma membrane results in the release of the internal vesicles called exosomes. In this report we have examined how an increase in cytosolic calcium affects the development of MVBs and exosome release in K562 cells overexpressing GFP-Rab11 wt or its mutants. In cells overexpressing the Rab11Q70 L mutant or Rab11 wt, an increase in the cytosolic calcium concentration induced by monensin caused a marked enlargement of the MVBs. This effect was abrogated by the membrane permeant calcium chelator BAPTA-AM. We also examined the behavior of MVBs in living cells by time lapse confocal microscopy. Many MVBs, decorated by wt or Q70L mutant GFP-Rab11, were docked and ready to fuse in the presence of a calcium chelator. This observation suggests that Rab11 is acting in the tethering/docking of MVBs to promote homotypic fusion, but that the final fusion reaction requires the presence of calcium. Additionally, a rise in intracellular calcium concentration enhanced exosome secretion in Rab11 wt overexpressing cells and reversed the inhibition of the mutants. The results suggest that both Rab11 and calcium are involved in the homotypic fusion of MVBs.     

Enforced Expression of Oxygen-Regulated Protein,ORP150, Induces Vacuolar Degeneration in Mouse Myocardium

Although the 150 kDa oxygen-regulated protein (ORP150) is known as a protein induced by low oxygen tension or ischemical insult, its possible role has not been fully investigated in vivo. To investigate the intracellular function of this protein, we generated the ORP150 over-expressing transgenic mice (ORP-Tg mice) under -actin promoter, and established three independent lines of the transgene expressed mice. All lines invariably showed growth retardation. Over-expression of ORP150 was confirmed by western blotting in heart, brain, spleen, skeletal muscle, pancreas, lung, thymus, and kidney. To ascertain the relationship between the over-expression of the ORP150 and the growth retardation in the transgenic mice, we examined pathological changes in the transgenics. In the ORP-Tg mice, vacuolar degeneration appeared in the heart. The degeneration in the myocytes became conspicuous with advancing age. Immunostaining demonstrated ORP150 in the vacuoles of degenerating myocytes. Electron microscopical findings revealed striking development of intracellular membrane system, for example, rough endoplasmic reticula (rER), vacuoles and Golgi bodies, swelling of sarcoplasmic reticulum, and lysis of myofibrils and mitochondria. These findings indicate that ORP150 may locate in the rER and other outer compartment of ER, and that constitutive over-expression of ORP150 in the heart induces vacuolar degeneration in myocytes, resulting in growth retardation of the transgenics.     

Cholesterol-binding molecules MLN64 and ORP1L mark distinct late endosomes with transporters ABCA3 and NPC1

Cholesterol is an essential lipid in eukaryotic cells and is present in membranes of all intracellular compartments. A major source for cellular cholesterol is internalized lipoprotein particles that are transported toward acidic late endosomes (LE) and lysosomes. Here the lipoprotein particles are hydrolyzed, and free cholesterol is redistributed to other organelles. The LE can contain over half of the cellular cholesterol and, as a major sorting station, can contain many cholesterol-binding proteins from the ABCA, STARD, and ORP families. Here, we show that metastatic lymph node 64 (MLN64, STARD3) and oxysterol-binding protein-related protein 1L (ORP1L) define two subpopulations of LE. MLN64 is present on a LE containing the cholesterol transporter ABCA3, whereas ORP1L localizes to another population of LE containing Niemann Pick type C1 (NPC1), a cholesterol exporter. Endocytosed cargo passes through MLN64/ABCA3-positive compartments before it reaches ORP1L/NPC1-positive LE. The MLN64/ABCA3 compartments cycle between LE and plasma membrane and frequently contact “later” ORP1L/NPC1-containing LE. We propose two stages of cholesterol handling in late endosomal compartments: first, cholesterol enters MLN64/ABCA3-positive compartments from where it can be recycled to the plasma membrane, and later, cholesterol enters ORP1L/NPC1 endosomes that mediate cholesterol export to the endoplasmic reticulum.     

Rab1 defines a novel pathway connecting the pre-Golgi intermediate compartment with the cell periphery

The function of the pre-Golgi intermediate compartment (IC) and its relationship with the endoplasmic reticulum (ER) and Golgi remain only partially understood. Here, we report striking segregation of IC domains in polarized PC12 cells that develop neurite-like processes. Differentiation involves expansion of the IC and movement of Rab1-containing tubules to the growth cones of the neurites, whereas p58- and COPI-positive IC elements, like rough ER and Golgi, remain in the cell body. Exclusion of Rab1 effectors p115 and GM130 from the neurites further indicated that the centrifugal, Rab1-mediated pathway has functions that are not directly related to ER-to-Golgi trafficking. Disassembly of COPI coats did not affect this pathway but resulted in missorting of p58 to the neurites. Live cell imaging showed that green fluorescent protein (GFP)-Rab1A-containing IC elements move bidirectionally both within the neurites and cell bodies, interconnecting different ER exit sites and the cis-Golgi region. Moreover, in nonpolarized cells GFP-Rab1A-positive tubules moved centrifugally towards the cell cortex. Hydroxymethylglutaryl-CoA reductase, the key enzyme of cholesterol biosynthesis, colocalized with slowly sedimenting, Rab1-enriched membranes when the IC subdomains were separated by velocity sedimentation. These results reveal a novel pathway directly connecting the IC with the cell periphery and suggest that this Rab1-mediated pathway is linked to the dynamics of smooth ER.     

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.     

Oxysterol-binding protein-related protein (ORP) 9 is a PDK-2 substrate and regulates Akt phosphorylation

The oxysterol-binding protein and oxysterol-binding protein-related protein family has been implicated in lipid transport and metabolism, vesicle trafficking and cell signaling. While investigating the phosphorylation of Akt/protein kinase B in stimulated bone marrow-derived mast cells, we observed that a monoclonal antibody directed against phospho-S473 Akt cross-reacted with oxysterol-binding protein-related protein 9 (ORP9). Further analysis revealed that mast cells exclusively express ORP9S, an N-terminal truncated version of full-length ORP9L. A PDK-2 consensus phosphorylation site in ORP9L and OPR9S at S287 (VPEFS(287)Y) was confirmed by site-directed mutagenesis. In contrast to Akt, increased phosphorylation of ORP9S S287 in stimulated mast cells was independent of phosphatidylinositol 3-kinase but sensitive to inhibition of conventional PKC isotypes. PKC-beta dependence was confirmed by lack of ORP9S phosphorylation at S287 in PKC-beta-deficient, but not PKC-alpha-deficient, mast cells. Moreover, co-immunoprecipitation of PKC-beta and ORP9S, and in vitro phosphorylation of ORP9S in this complex, argued for direct phosphorylation of ORP9S by PKC-beta, introducing ORP9S as a novel PKC-beta substrate. Akt was also detected in a PKC-beta/ORP9S immune complex and phosphorylation of Akt on S473 was delayed in PKC-deficient mast cells. In HEK293 cells, RNAi experiments showed that depletion of ORP9L increased Akt S473 phosphorylation 3-fold without affecting T308 phosphorylation in the activation loop. Furthermore, mammalian target of rapamycin was implicated in ORP9L phosphorylation in HEK293 cells. These studies identify ORP9 as a PDK-2 substrate and negative regulator of Akt phosphorylation at the PDK-2 site.     

OSBP-related protein 8 (ORP8) interacts with Homo sapiens sperm associated antigen 5 (SPAG5) and mediates oxysterol interference of HepG2 cell cycle

We earlier identified OSBP-related protein 8 (ORP8) as an endoplasmic reticulum/nuclear envelope oxysterol-binding protein implicated in cellular lipid homeostasis, migration, and organization of the microtubule cytoskeleton. Here, a yeast two-hybrid screen identified Homo sapiens sperm associated antigen 5 (SPAG5)/Astrin as interaction partner of ORP8. The putative interaction was further confirmed by pull-down and co-immunoprecipitation assays. ORP8 did not colocalize with kinetochore-associated SPAG5 in mitotic HepG2 or HuH7 cells, but overexpressed ORP8 was capable of recruiting SPAG5 onto endoplasmic reticulum membranes in interphase cells. In our experiments, 25-hydroxycholesterol (25OHC) retarded the HepG2 cell cycle, causing accumulation in G2/M phase; ORP8 overexpression resulted in the same phenotype. Importantly, ORP8 knock-down dramatically inhibited the oxysterol effect on HepG2 cell cycle, suggesting a mediating role of ORP8. Furthermore, knock-down of SPAG5 significantly reduced the effects of both ORP8 overexpression and 25OHC on the cell cycle, placing SPAG5 downstream of the two cell-cycle interfering factors. Taken together, the present results suggest that ORP8 may via SPAG5 mediate oxysterol interference of the HepG2 cell cycle.