Lipid droplet biogenesis.

Lipid droplets (LDs) store neutral lipids in their core as an energy source when nutrients are scarce. The center of an LD is hydrophobic, and hence it is surrounded by a phospholipid monolayer, unlike other organelles that have an aqueous interior and are bounded by a phospholipid bilayer. LDs arise from the ER, where neutral lipid synthesis enzymes are localized. A combination of biophysical analysis and modeling, in vitro reconstitution and cell biological analyses has provided a great deal of information over the past few years on the process of LD biogenesis from the ER. In addition to lipid composition, four protein families (seipin proteins, perilipins, FIT proteins and ER shaping proteins) are crucial for LD biogenesis. Recent studies have shown that LDs preferentially arise, along with peroxisomes, at special ER sites marked by the reticulon-like Pex30/MCTP2 protein. New functions for perilipins and FIT family proteins have been uncovered, and the cryo-electron microscopy structure of seipin coupled with high resolution imaging in cells has provided a more comprehensive picture of its function in LD biogenesis. Seipin, along with other proteins such as Rab18 and its effector NRZ, have been shown to carry out their functions at least in part through regulation of ER-LD contact sites, whose establishment and maintenance have emerged as an essential component of LD biogenesis and maturation.

Functional and Physical Interaction between the Arf Activator GBF1 and Hepatitis C Virus NS3 Protein.

GBF1 has emerged as a host factor required for the genome replication of RNA viruses of different families. During the hepatitis C virus (HCV) life cycle, GBF1 performs a critical function at the onset of genome replication but is dispensable when the replication is established. To better understand how GBF1 regulates HCV infection, we have looked for interactions between GBF1 and HCV proteins. NS3 was found to interact with GBF1 in yeast two-hybrid, coimmunoprecipitation, and proximity ligation assays and to interfere with GBF1 function and alter GBF1 intracellular localization in cells expressing NS3. The interaction was mapped to the Sec7 domain of GBF1 and the protease domain of NS3. A reverse yeast two-hybrid screen to identify mutations altering NS3-GBF1 interaction yielded an NS3 mutant (N77D, Con1 strain) that is nonreplicative despite conserved protease activity and does not interact with GBF1. The mutated residue is exposed at the surface of NS3, suggesting it is part of the domain of NS3 that interacts with GBF1. The corresponding mutation in strain JFH-1 (S77D) produces a similar phenotype. Our results provide evidence for an interaction between NS3 and GBF1 and suggest that an alteration of this interaction is detrimental to HCV genome replication.IMPORTANCE Single-stranded, positive-sense RNA viruses rely to a significant extent on host factors to achieve the replication of their genome. GBF1 is such a cellular protein that is required for the replication of several RNA viruses, but its mechanism of action during viral infections is not yet defined. In this study, we investigated potential interactions that GBF1 might engage in with proteins of HCV, a GBF1-dependent virus. We found that GBF1 interacts with NS3, a nonstructural protein involved in HCV genome replication, and our results suggest that this interaction is important for GBF1 function during HCV replication. Interestingly, GBF1 interaction with HCV appears different from its interaction with enteroviruses, another group of GBF1-dependent RNA viruses, in keeping with the fact that HCV and enteroviruses use different functions of GBF1.

GBF1 and Arf1 interact with Miro and regulate mitochondrial positioning within cells.

The spatial organization of cells depends on coordination between cytoskeletal systems and intracellular organelles. The Arf1 small G protein and its activator GBF1 are important regulators of Golgi organization, maintaining its morphology and function. Here we show that GBF1 and its substrate Arf1 regulate the spatial organization of mitochondria in a microtubule-dependent manner. Miro is a mitochondrial membrane protein that interacts through adaptors with microtubule motor proteins such as cytoplasmic dynein, the major microtubule minus end directed motor. We demonstrate a physical interaction between GBF1 and Miro, and also between the active GTP-bound form of Arf1 and Miro. Inhibition of GBF1, inhibition of Arf1 activation, or overexpression of Miro, caused a collapse of the mitochondrial network towards the centrosome. The change in mitochondrial morphology upon GBF1 inhibition was due to a two-fold increase in the time engaged in retrograde movement compared to control conditions. Electron tomography revealed that GBF1 inhibition also resulted in larger mitochondria with more complex morphology. Miro silencing or drug inhibition of cytoplasmic dynein activity blocked the GBF1-dependent repositioning of mitochondria. Our results show that blocking GBF1 function promotes dynein- and Miro-dependent retrograde mitochondrial transport along microtubules towards the microtubule-organizing center, where they form an interconnected network.

Membrane Trafficking: A Little Flexibility Helps Vesicles Get into Shape.

Formation of a transport vesicle in membrane trafficking pathways requires deformation of the membrane to form a highly curved structure. A recent study reveals a crucial function for the conical lipid lysophosphatidylinositol in reducing the bending rigidity of the membrane during COPII vesicle budding in the early secretory pathway.

Inheritance of the Golgi Apparatus and Cytokinesis Are Controlled by Degradation of GBF1.

Although much is known about how chromosome segregation is coupled to cell division, how intracellular organelles partition during mitotic division is poorly understood. We report that the phosphorylation-dependent degradation of the ARFGEF GBF1 regulates organelle trafficking during cell division. We show that, in mitosis, GBF1 is phosphorylated on Ser292 and Ser297 by casein kinase-2 allowing recognition by the F-box protein ßTrCP. GBF1 interaction with ßTrCP recruits GBF1 to the SCFßTrCP ubiquitin ligase complex, triggering its degradation. Phosphorylation and degradation of GBF1 occur along microtubules at the intercellular bridge of telophase cells and are required for Golgi membrane positioning and postmitotic Golgi reformation. Indeed, expression of a non-degradable GBF1 mutant inhibits the transport of the Golgi cluster adjacent to the midbody toward the Golgi twin positioned next to the centrosome and results in defective Golgi reassembly and cytokinesis failure. These findings define a mechanism that controls postmitotic Golgi reassembly and inheritance.

Activators and Effectors of the Small G Protein Arf1 in Regulation of Golgi Dynamics During the Cell Division Cycle.

When eukaryotic cells divide, they must faithfully segregate not only the genetic material but also their membrane-bound organelles into each daughter cell. To assure correct partitioning of cellular contents, cells use regulatory mechanisms to verify that each stage of cell division has been correctly accomplished before proceeding to the next step. A great deal is known about mechanisms that regulate chromosome segregation during cell division, but we know much less about the mechanisms by which cellular organelles are partitioned, and how these processes are coordinated. The Golgi apparatus, the central sorting and modification station of the secretory pathway, disassembles during mitosis, a process that depends on Arf1 and its regulators and effectors. Prior to total disassembly, the Golgi ribbon in mammalian cells, composed of alternating cisternal stacks and tubular networks, undergoes fission of the tubular networks to produce individual stacks. Failure to carry out this unlinking leads to cell division arrest at late G2 prior to entering mitosis, an arrest that can be relieved by inhibition of Arf1 activation. The level of active Arf1-GTP drops during mitosis, due to inactivation of the major Arf1 guanine nucleotide exchange factor at the Golgi, GBF1. Expression of constitutively active Arf1 prevents Golgi disassembly, and leads to defects in chromosome segregation and cytokinesis. In this review, we describe recent advances in understanding the functions of Arf1 regulators and effectors in the crosstalk between Golgi structure and cell cycle regulation.

A giant amphipathic helix from a perilipin that is adapted for coating lipid droplets.

How proteins are targeted to lipid droplets (LDs) and distinguish the LD surface from the surfaces of other organelles is poorly understood, but many contain predicted amphipathic helices (AHs) that are involved in targeting. We have focused on human perilipin 4 (Plin4), which contains an AH that is exceptional in terms of length and repetitiveness. Using model cellular systems, we show that AH length, hydrophobicity, and charge are important for AH targeting to LDs and that these properties can compensate for one another, albeit at a loss of targeting specificity. Using synthetic lipids, we show that purified Plin4 AH binds poorly to lipid bilayers but strongly interacts with pure triglycerides, acting as a coat and forming small oil droplets. Because Plin4 overexpression alleviates LD instability under conditions where their coverage by phospholipids is limiting, we propose that the Plin4 AH replaces the LD lipid monolayer, for example during LD growth.

Identification of GBF1 as a cellular factor required for hepatitis E virus RNA replication.

The hepatitis E virus (HEV) genome is a single-stranded, positive-sense RNA that encodes three proteins including the ORF1 replicase. Mechanisms of HEV replication in host cells are unclear, and only a few cellular factors involved in this step have been identified so far. Here, we used brefeldin A (BFA) that blocks the activity of the cellular Arf guanine nucleotide exchange factors GBF1, BIG1, and BIG2, which play a major role in reshuffling of cellular membranes. We showed that BFA inhibits HEV replication in a dose-dependent manner. The use of siRNA and Golgicide A identified GBF1 as a host factor critically involved in HEV replication. Experiments using cells expressing a mutation in the catalytic domain of GBF1 and overexpression of wild type GBF1 or a BFA-resistant GBF1 mutant rescuing HEV replication in BFA-treated cells, confirmed that GBF1 is the only BFA-sensitive factor required for HEV replication. We demonstrated that GBF1 is likely required for the activity of HEV replication complexes. However, GBF1 does not colocalise with the ORF1 protein, and its subcellular distribution is unmodified upon infection or overexpression of viral proteins, indicating that GBF1 is likely not recruited to replication sites. Together, our results suggest that HEV replication involves GBF1-regulated mechanisms.

GBF1 and Arf1 function in vesicular trafficking, lipid homoeostasis and organelle dynamics.

The ADP-ribosylation factor (Arf) small G proteins act as molecular switches to coordinate multiple downstream pathways that regulate membrane dynamics. Their activation is spatially and temporally controlled by the guanine nucleotide exchange factors (GEFs). Members of the evolutionarily conserved GBF/Gea family of Arf GEFs are well known for their roles in formation of coat protein complex I (COPI) vesicles, essential for maintaining the structure and function of the Golgi apparatus. However, studies over the past 10 years have found new functions for these GEFs, along with their substrate Arf1, in lipid droplet metabolism, clathrin-independent endocytosis, signalling at the plasma membrane, mitochondrial dynamics and transport along microtubules. Here, we describe these different functions, focussing in particular on the emerging theme of GFB1 and Arf1 regulation of organelle movement on microtubules.

Interdigitation between Triglycerides and Lipids Modulates Surface Properties of Lipid Droplets.

Intracellular lipid droplets (LDs) are the main cellular site of metabolic energy storage. Their structure is unique inside the cell, with a core of esterified fatty acids and sterols, mainly triglycerides and sterol esters, surrounded by a single monolayer of phospholipids. Numerous peripheral proteins, including several that were previously associated with intracellular compartments surrounded by a lipid bilayer, have been recently shown to target the surface of LDs, but how they are able to selectively target this organelle remains largely unknown. Here, we use atomistic and coarse-grained molecular dynamics simulations to investigate the molecular properties of the LD surface and to characterize how it differs from that of a lipid bilayer. Our data suggest that although several surface properties are remarkably similar between the two structures, key differences originate from the interdigitation between surface phospholipids and core neutral lipids that occurs in LDs. This property is extremely sensitive to membrane undulations, unlike in lipid bilayers, and it strongly affects both lipid-packing defects and the lateral pressure profile. We observed a marked change in overall surface properties for surface tensions >10 mN/m, indicative of a bimodal behavior. Our simulations provide a comprehensive molecular characterization of the unique surface properties of LDs and suggest how the molecular properties of the surface lipid monolayer can be modulated by the underlying neutral lipids.

Lipids and Their Trafficking: An Integral Part of Cellular Organization.

An evolutionarily conserved feature of cellular organelles is the distinct phospholipid composition of their bounding membranes, which is essential to their identity and function. Within eukaryotic cells, two major lipid territories can be discerned, one centered on the endoplasmic reticulum and characterized by membranes with lipid packing defects, the other comprising plasma-membrane-derived organelles and characterized by membrane charge. We discuss how this cellular lipid organization is maintained, how lipid flux is regulated, and how perturbations in cellular lipid homeostasis can lead to disease.

ORP5/ORP8 localize to endoplasmic reticulum-mitochondria contacts and are involved in mitochondrial function.

The oxysterol-binding protein (OSBP)-related proteins ORP5 and ORP8 have been shown recently to transport phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) at ER-PM contact sites. PS is also transferred from the ER to mitochondria where it acts as precursor for mitochondrial PE synthesis. Here, we show that, in addition to ER-PM contact sites, ORP5 and ORP8 are also localized to ER-mitochondria contacts and interact with the outer mitochondrial membrane protein PTPIP51. A functional lipid transfer (ORD) domain was required for this localization. Interestingly, ORP5 and ORP8 depletion leads to defects in mitochondria morphology and respiratory function.

Identification of class II ADP-ribosylation factors as cellular factors required for hepatitis C virus replication.

GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G-proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf-guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA-mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.

INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.

In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles.

Fatty acid metabolism meets organelle dynamics.

Upon nutrient deprivation, cells metabolize fatty acids (FAs) in mitochondria to supply energy, but how FAs, stored as triacylglycerols in lipid droplets, reach mitochondria has been mysterious. Rambold et al. (2015) now show that FA mobilization depends on triacylglycerol lipolysis, whereas autophagy feeds the lipid droplet pool for continued fueling of mitochondria.

Arfs at a glance.

The Arf small G proteins regulate protein and lipid trafficking in eukaryotic cells through a regulated cycle of GTP binding and hydrolysis. In their GTP-bound form, Arf proteins recruit a specific set of protein effectors to the membrane surface. These effectors function in vesicle formation and tethering, non-vesicular lipid transport and cytoskeletal regulation. Beyond fundamental membrane trafficking roles, Arf proteins also regulate mitosis, plasma membrane signaling, cilary trafficking and lipid droplet function. Tight spatial and temporal regulation of the relatively small number of Arf proteins is achieved by their guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs), which catalyze GTP binding and hydrolysis, respectively. A unifying function of Arf proteins, performed in conjunction with their regulators and effectors, is sensing, modulating and transporting the lipids that make up cellular membranes. In this Cell Science at a Glance article and the accompanying poster, we discuss the unique features of Arf small G proteins, their functions in vesicular and lipid trafficking in cells, and how these functions are modulated by their regulators, the GEFs and GAPs. We also discuss how these Arf functions are subverted by human pathogens and disease states.

The SNARE Sec22b has a non-fusogenic function in plasma membrane expansion.

Development of the nervous system requires extensive axonal and dendritic growth during which neurons massively increase their surface area. Here we report that the endoplasmic reticulum (ER)-resident SNARE Sec22b has a conserved non-fusogenic function in plasma membrane expansion. Sec22b is closely apposed to the plasma membrane SNARE syntaxin1. Sec22b forms a trans-SNARE complex with syntaxin1 that does not include SNAP23/25/29, and does not mediate fusion. Insertion of a long rigid linker between the SNARE and transmembrane domains of Sec22b extends the distance between the ER and plasma membrane, and impairs neurite growth but not the secretion of VSV-G. In yeast, Sec22 interacts with lipid transfer proteins, and inhibition of Sec22 leads to defects in lipid metabolism at contact sites between the ER and plasma membrane. These results suggest that close apposition of the ER and plasma membrane mediated by Sec22 and plasma membrane syntaxins generates a non-fusogenic SNARE bridge contributing to plasma membrane expansion, probably through non-vesicular lipid transfer.

GEF-effector interactions.

Members of the Arf family of small GTP-binding proteins, or GTPases, are activated by guanine nucleotide exchange factors (GEFs) that catalyze GDP release from their substrate Arf, allowing GTP to bind. In the secretory pathway, Arf1 is first activated by GBF1 at the cis-Golgi, then by BIG1 and BIG2 at the trans-Golgi and trans-Golgi network (TGN). Upon activation, Arf1-GTP interacts with effectors such as coat complexes, and is able to recruit different coat complexes to different membrane sites in cells. The COPI coat is primarily recruited to cis-Golgi membranes, whereas other coats, such as AP-1/clathrin, and GGA/clathrin, are recruited to the trans-Golgi and the TGN. Although Arf1-GTP is required for stable association of these various coats to membranes, and is sufficient in vitro, other molecules, such as vesicle cargo and coat receptors on the membrane, contribute to specificity of coat recruitment in cells. Another mechanism to achieve specificity is interaction of effectors such as coats with the GEF itself, which would increase the concentration of a given coat in proximity to the site where Arf is activated, thus favoring its recruitment. This interaction between a GEF and an effector could also provide a mechanism for spatial organization of vesicle budding sites, similar to that described for Cdc42-mediated establishment of polarity sites such as the emerging bud in yeast. Another factor affecting the amount of freely diffusible Arf1-GTP in membranes is the GEF(s) themselves acting as effectors. Sec7p, the yeast homolog of mammalian BIG1 and BIG2, and Arno/cytohesin 2, a PM-localized Arf1 GEF, both bind to Arf1-GTP. This binding to the products of the exchange reaction establishes a positive feedback loop for activation.