The cholesterol trafficking protein NPC1 is required for Drosophila spermatogenesis

Niemann–Pick C (NPC) disease is a lethal neurodegenerative disorder affecting cellular sterol trafficking. Besides neurodegeneration, NPC patients also exhibit other pleiotropic conditions, indicating that NPC protein is required for other physiological processes. Previous studies indicated that a sterol shortage that in turn leads to a shortage of steroid hormones (for example, ecdysone in Drosophila) is likely to be the cause of NPC disease pathology. We have shown that mutations in Drosophila npc1, one of the two NPC disease-related genes, leads to larval lethal and male infertility. Here, we reported that npc1 mutants are defective in spermatogenesis and in particular in the membrane-remodeling individualization process. Interestingly, we found that ecdysone, the steroid hormone responsible for the larval lethal phenotype in npc1 mutants, is not required for individualization. However, supplying 7-dehydrocholesterol can partially rescue the male infertility of npc1 mutants, suggesting that a sterol shortage is responsible for the spermatogenesis defects. In addition, the individualization defects of npc1 mutants were enhanced at high temperature, suggesting that the sterol shortage may lead to temperature-sensitive defects in the membrane-remodeling process. Together, our study reveals a sterol-dependent, ecdysone-independent mechanism of NPC1 function in Drosophila spermatogenesis.     

Membrane phospholipid asymmetry counters the adverse effects of sterol overloading in the Golgi membrane of Drosophila

Cholesterol and phospholipids serve as structural and functional components of cellular membranes in all eukaryotes. Heterogeneity in cholesterol and phospholipid content both within and between different organelles is an important characteristic of eukaryotic membranes. How this heterogeneity is achieved and orchestrated to maintain proper cellular physiology remains poorly understood. We previously found that overexpression of the Drosophila oxysterol-binding protein (OSBP) leads to sterol accumulation in the Golgi apparatus. Here, we show that Osbp overexpression in a set of neuroendocrine neurons compromises the function of the Golgi apparatus. It impairs trafficking of the neuropeptide bursicon and results in post-eclosion behavior defects characterized by unexpanded wings. We performed a genetic screen to identify modifiers that suppress the unexpanded wing phenotype. A putative phospholipid flippase-encoding gene, CG33298, was validated, suggesting that a membrane-asymmetry-directed mechanism balances cholesterol chaos within the Golgi membranes. Since the functional connection between cholesterol metabolism and the activity of phospholipid flippase has been implicated in studies in yeast and worms, our findings here support an evolutionarily conserved causal link between cholesterol homeostasis and phospholipid asymmetry that maintains normal cellular physiology.     

Overlapping functions of the yeast oxysterol-binding protein homologues

The Saccharomyces cerevisiae genome encodes seven homologues of the mammalian oxysterol-binding protein (OSBP), a protein implicated in lipid trafficking and sterol homeostasis. To determine the functions of the yeast OSBP gene family (OSH1-OSH7), we used a combination of genetics, genomics, and sterol lipid analysis to characterize OSH deletion mutants. All 127 combinations and permutations of OSH deletion alleles were constructed. Individual OSH genes were not essential for yeast viability, but the elimination of the entire gene family was lethal. Thus, the family members shared an essential function. In addition, the in vivo depletion of all Osh proteins disrupted sterol homeostasis. Like mutants that affect ergosterol production, the viable combinations of OSH deletion alleles exhibited specific sterol-related defects. Although none of the single OSH deletion mutants was defective for growth, gene expression profiles revealed that each mutant had a characteristic molecular phenotype. Therefore, each gene performed distinct nonessential functions and contributed to a common essential function. Our findings indicated that OSH genes performed a multitude of nonessential roles defined by specific subsets of the genes and that most shared at least one essential role potentially linked to changes in sterol lipid levels.     

Drosophila NPC1b promotes an early step in sterol absorption from the midgut epithelium

The NPC1 family of proteins plays crucial roles in the intestinal absorption and intracellular trafficking of sterols. The Drosophila genome encodes two NPC1 homologs, one of which, NPC1a, is required for intracellular sterol trafficking in many tissues. Here we show that the other Drosophila NPC1 family member, NPC1b, is expressed in the midgut epithelium and that NPC1b is essential for growth during the early larval stages of development. NPC1b mutants are severely defective in sterol absorption, and the midgut epithelium of NPC1b mutants is deficient in sterols and sterol trafficking intermediates. By contrast, NPC1a mutants absorb sterols more efficiently than wild-type animals, and, unexpectedly, NPC1b;NPC1a double mutants absorb sterols as efficiently as wild-type animals. Together, these findings suggest that NPC1b plays an early role in sterol absorption, although sterol absorption continues at high efficiency through an NPC1a- and NPC1b-independent mechanism under conditions of impaired intracellular sterol trafficking.     

Nonvesicular sterol movement from plasma membrane to ER requires oxysterol-binding protein-related proteins and phosphoinositides

Sterols are moved between cellular membranes by nonvesicular pathways whose functions are poorly understood. In yeast, one such pathway transfers sterols from the plasma membrane (PM) to the endoplasmic reticulum (ER). We show that this transport requires oxysterol-binding protein (OSBP)-related proteins (ORPs), which are a large family of conserved lipid-binding proteins. We demonstrate that a representative member of this family, Osh4p/Kes1p, specifically facilitates the nonvesicular transfer of cholesterol and ergosterol between membranes in vitro. In addition, Osh4p transfers sterols more rapidly between membranes containing phosphoinositides (PIPs), suggesting that PIPs regulate sterol transport by ORPs. We confirmed this by showing that PM to ER sterol transport slows dramatically in mutants with conditional defects in PIP biosynthesis. Our findings argue that ORPs move sterols among cellular compartments and that sterol transport and intracellular distribution are regulated by PIPs.     

Yeast oxysterol-binding proteins: sterol transporters or regulators of cell polarization?

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) are a conserved family of soluble cytoplasmic proteins that can bind sterols, translocate between membrane compartments, and affect sterol trafficking. These properties make ORPs attractive candidates for lipid transfer proteins (LTPs) that directly mediate nonvesicular sterol transfer to the plasma membrane. To test whether yeast ORPs (the Osh proteins) are sterol LTPs, we studied endoplasmic reticulum (ER)-to-plasma membrane (PM) sterol transport in OSH deletion mutants lacking one, several, or all Osh proteins. In conditional OSH mutants, ER-PM ergosterol transport slowed ~20-fold compared with cells expressing a full complement of Osh proteins. Although this initial finding suggested that Osh proteins act as sterol LTPs, the situation is far more complex. Osh proteins have established roles in Rho small GTPase signaling. Osh proteins reinforce cell polarization and they specifically affect the localization of proteins involved in polarized cell growth such as septins, and the GTPases Cdc42p, Rho1p, and Sec4p. In addition, Osh proteins are required for a specific pathway of polarized secretion to sites of membrane growth, suggesting that this is how Osh proteins affect Cdc42p- and Rho1p-dependent polarization. Our findings suggest that Osh proteins integrate sterol trafficking and sterol-dependent cell signaling with the control of cell polarization.     

Oxysterol-binding protein and vesicle-associated membrane protein-associated protein are required for sterol-dependent activation of the ceramide transport protein

Sphingomyelin (SM) and cholesterol are coregulated metabolically and associate physically in membrane microdomains involved in cargo sorting and signaling. One mechanism for regulation of this metabolic interface involves oxysterol binding protein (OSBP) via high-affinity binding to oxysterol regulators of cholesterol homeostasis and activation of SM synthesis at the Golgi apparatus. Here, we show that OSBP regulation of SM synthesis involves the endoplasmic reticulum (ER)-to-Golgi ceramide transport protein (CERT). RNA interference (RNAi) experiments in Chinese hamster ovary (CHO)-K1 cells revealed that OSBP and vesicle-associated membrane protein-associated protein (VAP) were required for stimulation of CERT-dependent ceramide transport and SM synthesis by 25-hydroxycholesterol and cholesterol depletion in response to cyclodextrin. Additional RNAi experiments in human embryonic kidney 293 cells supported OSBP involvement in oxysterol-activated SM synthesis and also revealed a role for OSBP in basal SM synthesis. Activation of ER-to-Golgi ceramide transport in CHO-K1 cells required interaction of OSBP with the ER and Golgi apparatus, OSBP-dependent Golgi translocation of CERT, and enhanced CERT-VAP interaction. Regulation of CERT by OSBP, sterols, and VAP reveals a novel mechanism for integrating sterol regulatory signals with ceramide transport and SM synthesis in the Golgi apparatus.     

Vesicle-associated membrane protein-associated protein-A (VAP-A) interacts with the oxysterol-binding protein to modify export from the endoplasmic reticulum

Oxysterol-binding protein (OSBP) is 1 of 12 related proteins implicated in the regulation of vesicle transport and sterol homeostasis. A yeast two-hybrid screen using full-length OSBP as bait was undertaken to identify partner proteins that would provide clues to the function of OSBP. This resulted in the cloning of vesicle-associated membrane protein-associated protein-A (VAP-A), a syntaxin-like protein implicated in endoplasmic reticulum (ER)/Golgi vesicle transport, and phospholipid regulation in mammalian cells and yeast, respectively. By using a combination of yeast two-hybrid, glutathione S-transferase pull-down and immunoprecipitation experiments, the VAP-A-binding region in OSBP was localized to amino acids 351-442. This region did not include the pleckstrin homology (PH) domain but overlapped with the N terminus of the oxysterol binding and OSBP homology domains. C- and N-terminal truncations or deletions of VAP prevented interaction with OSBP but did not affect VAP multimerization. Although the OSBP PH domain was not necessary for VAP-A binding in vitro, interaction with VAP-A was enhanced in cells by mutation of the conserved PH domain tryptophan (OSBP W174A) or deletion of the C-terminal half of the PH domain (OSBP Delta 132-182). OSBP W174A retained oxysterol binding activity, association with phospholipid vesicles via the PH domain, and localized with VAP in unusual ER-associated structures. At 40 degrees C, misfolded ts045-vesicular stomatitis virus G protein fused to green fluorescent protein was co-localized with VAP-A/OSBP W174A structures on the ER but was exported to the Golgi when folded normally at 32 degrees C. A fluorescent ceramide analogue also accumulated in these ER inclusions, and export to the Golgi was partially inhibited as indicated by decreased Golgi staining and a 30% reduction in sphingomyelin synthesis. These studies show that OSBP binding to the ER and Golgi apparatus is regulated by its PH domain and VAP interactions, and the complex is involved at a stage of protein and ceramide transport from the ER.     

Oxysterol Binding Protein–related Protein 9 (ORP9) Is a Cholesterol Transfer Protein That Regulates Golgi Structure and Function

Oxysterol-binding protein (OSBP) and OSBP-related proteins (ORPs) constitute a large gene family that differentially localize to organellar membranes, reflecting a functional role in sterol signaling and/or transport. OSBP partitions between the endoplasmic reticulum (ER) and Golgi apparatus where it imparts sterol-dependent regulation of ceramide transport and sphingomyelin synthesis. ORP9L also is localized to the ER–Golgi, but its role in secretion and lipid transport is unknown. Here we demonstrate that ORP9L partitioning between the trans-Golgi/trans-Golgi network (TGN), and the ER is mediated by a phosphatidylinositol 4-phosphate (PI-4P)-specific PH domain and VAMP-associated protein (VAP), respectively. In vitro, both OSBP and ORP9L mediated PI-4P–dependent cholesterol transport between liposomes, suggesting their primary in vivo function is sterol transfer between the Golgi and ER. Depletion of ORP9L by RNAi caused Golgi fragmentation, inhibition of vesicular somatitus virus glycoprotein transport from the ER and accumulation of cholesterol in endosomes/lysosomes. Complete cessation of protein transport and cell growth inhibition was achieved by inducible overexpression of ORP9S, a dominant negative variant lacking the PH domain. We conclude that ORP9 maintains the integrity of the early secretory pathway by mediating transport of sterols between the ER and trans-Golgi/TGN.     

LUMP is a putative double-stranded RNA binding protein required for male fertility in Drosophila melanogaster

In animals, male fertility requires the successful development of motile sperm. During Drosophila melanogaster spermatogenesis, 64 interconnected spermatids descended from a single germline stem cell are resolved into motile sperm in a process termed individualization. Here we identify a putative double-stranded RNA binding protein LUMP that is required for male fertility. lump(1) mutants are male-sterile and lack motile sperm due to defects in sperm individualization. We show that one dsRNA binding domains (dsRBD) is essential for LUMP function in male fertility. These findings reveal LUMP is a novel factor required for late stages of male germline differentiation.     

Regulation of Oxysterol-binding Protein Golgi Localization through Protein Kinase D–mediated Phosphorylation

Protein kinase D (PKD) plays a critical role at the trans-Golgi network by regulating the fission of transport carriers destined for the plasma membrane. Two known Golgi-localized PKD substrates, PI4-kinase IIIβ and the ceramide transfer protein CERT, mediate PKD signaling to influence vesicle trafficking to the plasma membrane and sphingomyelin synthesis, respectively. PKD is recruited and activated at the Golgi through interaction with diacylglycerol, a pool of which is generated as a by-product of sphingomyelin synthesis from ceramide. Here we identify a novel substrate of PKD at the Golgi, the oxysterol-binding protein OSBP. Using a substrate-directed phospho-specific antibody that recognizes the optimal PKD consensus motif, we show that PKD phosphorylates OSBP at Ser240 in vitro and in cells. We further show that OSBP phosphorylation occurs at the Golgi. Phosphorylation of OSBP by PKD does not modulate dimerization, sterol binding, or affinity for PI(4)P. Instead, phosphorylation attenuates OSBP Golgi localization in response to 25-hydroxycholesterol and cholesterol depletion, impairs CERT Golgi localization, and promotes Golgi fragmentation.     

The hsp60B gene of Drosophila melanogaster is essential for the spermatid individualization process

The 60-kDa heat shock protein family (Hsp60) is found in prokaryotes, mitochondria, and chloroplasts. The Hsp60 proteins promote proper protein folding by preventing aggregation. In Drosophila melanogaster, the hsp60 gene is essential for a variety of developmental processes, beginning at early embryogenesis. In this study we show that an additional member of the Drosophila hsp60 gene family, hsp60B, is essential in male fertility. In males homozygous for a mutation of the hsp60B gene, developmental processes appeared normal throughout most of spermatogenesis, including spermatocyte growth, meiosis, and spermatid elongation. At these stages, mitochondria also displayed a differentiation process similar to wild-types. However, we found that the mutation disrupted a late stage of spermatogenesis, the spermatid individualization process. In this process, the individualization complex is assembled at spermatid nuclear heads, traverses along spermatid tails, and generates membranes for each of the spermatids in a cyst. Our analysis further shows that the individualization complex in sterile males displayed abnormal morphology as it was traveling along the spermatid tails. The Drosophila Hsp60 proteins are believed to be exclusively localized in the mitochondria. Our observation that the hsp60B mutation displayed no apparent defect in mitochondrial differentiation during spermatogenesis suggests that the Hsp60B protein may operate in a nonmitochondrial location.     

ORP2, a homolog of oxysterol binding protein, regulates cellular cholesterol metabolism

Oxysterol binding protein (OSBP) related proteins (ORPs) constitute a family that has at least 12 members in humans. In the present study we characterize one of the novel OSBP homologs, ORP2, which we show to be expressed ubiquitously in mammalian tissues. The ORP2 cDNA encodes a deduced 55 kDa protein that lacks a pleckstrin homology (PH) domain, a feature found in the other family members. Sucrose gradient centrifugation analysis of Chinese hamster ovary (CHO) cell post-nuclear supernatant demonstrated that ORP2 is distributed in soluble and membrane-bound fractions. Immunofluorescence microscopy of the endogenous and overexpressed ORP2 in CHO cells suggested that the membrane-bound fraction of the protein localizes to the Golgi apparatus. Stably transfected CHO cells that overexpress ORP2 showed an increase in [14C]cholesterol efflux to serum, apolipoprotein A-I (apoA-I), and phosphatidyl choline vesicles. The proportion of cellular [14C]cholesterol that is esterified and the ACAT activity measured as [14C]oleyl-CoA conversion into cholesteryl [14C]oleate by the cellular membranes, were markedly decreased in the ORP2 expressing cells. Transient high level overexpression of ORP2 interfered with the clearance of a secretory pathway protein marker from the Golgi complex. The results implicate ORP2 as a novel regulator of cellular sterol homeostasis and intracellular membrane trafficking.     

Vps74 gives phosphatase directions

Synaptic demise and accumulation of dysfunctional proteins are thought of as common features in neurodegeneration. However, the mechanisms by which synaptic proteins turn over remain elusive. In this paper, we study Drosophila melanogaster lacking active TBC1D24/Skywalker (Sky), a protein that in humans causes severe neurodegeneration, epilepsy, and DOOR (deafness, onychdystrophy, osteodystrophy, and mental retardation) syndrome, and identify endosome-to-lysosome trafficking as a mechanism for degradation of synaptic vesicle-associated proteins. In fly sky mutants, synaptic vesicles traveled excessively to endosomes. Using chimeric fluorescent timers, we show that synaptic vesicle-associated proteins were younger on average, suggesting that older proteins are more efficiently degraded. Using a genetic screen, we find that reducing endosomal-to-lysosomal trafficking, controlled by the homotypic fusion and vacuole protein sorting (HOPS) complex, rescued the neurotransmission and neurodegeneration defects in sky mutants. Consistently, synaptic vesicle proteins were older in HOPS complex mutants, and these mutants also showed reduced neurotransmission. Our findings define a mechanism in which synaptic transmission is facilitated by efficient protein turnover at lysosomes and identify a potential strategy to suppress defects arising from TBC1D24 mutations in humans.     

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.     

The role of Gilgamesh protein kinase in Drosophila melanogaster spermatogenesis

The cellular function of the gilgamesh mutation (89B9-12) of casein kinase gene in Drosophila spermatogenesis was studied. It was demonstrated that the sterility resulting from this mutation is connected with the abnormalities in spermatid individualization. A phylogenetic study of the protein sequences of casein kinases 1 from various organisms was conducted. The Gilgamesh protein was shown to be phylogenetically closer to the cytoplasmic casein kinase family, represented by the YCK3, YCK2, and YCK1 proteins of Saccharomyces cerevisiae and animal gamma-casein kinases. It is known that these yeast casein kinases are involved in vesicular trafficking, which, in turn, is related in its genetic control to the cell membrane remodeling during spermatid individualization. Thus, the data of phylogenetic analysis fit well the results obtained by studying the mutation phenotype.     

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.     

Characterization of the Sterol and Phosphatidylinositol 4-Phosphate Binding Properties of Golgi-Associated OSBP-Related Protein 9 (ORP9)

Oxysterol binding protein (OSBP) and OSBP-related proteins (ORPS) have a conserved lipid-binding fold that accommodates cholesterol, oxysterols and/or phospholipids. The diversity of OSBP/ORPs and their potential ligands has complicated the analysis of transfer and signalling properties of this mammalian gene family. In this study we explored the use of the fluorescent sterol cholestatrienol (CTL) to measure sterol binding by ORP9 and competition by other putative ligands. Relative to cholesterol, CTL and dehydroergosterol (DHE) were poor ligands for OSBP. In contrast, both long (ORP9L) and short (ORP9S) variants of ORP9 rapidly extracted CTL, and to a lesser extent DHE, from liposomes. ORP9L and ORP9S also extracted [³²P]phosphatidylinositol 4-phosphate (PI-4P) from liposomes, which was inhibited by mutating two conserved histidine residues (HH_488,489AA) at the entrance to the binding pocket but not by a mutation in the lid region that inhibited cholesterol binding. Results of direct binding and competition assays showed that phosphatidylserine was poorly extracted from liposomes by ORP9 compared to CTL and PI-4P. ORP9L and PI-4P did not co-localize in the trans-Golgi/TGN of HeLa cells, and siRNA silencing of ORP9L expression did not affect PI-4P distribution in the Golgi apparatus. However, transient overexpression of ORP9L or ORP9S in CHO cells, but not the corresponding PI-4P binding mutants, prevented immunostaining of Golgi-associated PI-4P. The apparent sequestration of Golgi PI-4P by ORP9S was identified as a possible mechanism for its growth inhibitory effects. These studies identify ORP9 as a dual sterol/PI-4P binding protein that could regulate PI-4P in the Golgi apparatus.