Acute metabolic changes in plasma membrane (PM) lipids, such as those mediating signalling reactions, are rapidly compensated by homeostatic responses whose molecular basis is poorly understood. Here we show that the extended synaptotagmins (E-Syts), endoplasmic reticulum (ER) proteins that function as PtdIns(4,5)P2- and Ca(2+)-regulated tethers to the PM, participate in these responses. E-Syts transfer glycerolipids between bilayers in vitro, and this transfer requires Ca(2+) and their lipid-harbouring SMP domain. Genome-edited cells lacking E-Syts do not exhibit abnormalities in the major glycerolipids at rest, but exhibit enhanced and sustained accumulation of PM diacylglycerol following PtdIns(4,5)P2 hydrolysis by PLC activation, which can be rescued by expression of E-Syt1, but not by mutant E-Syt1 lacking the SMP domain. The formation of E-Syt-dependent ER-PM tethers in response to stimuli that cleave PtdIns(4,5)P2 and elevate Ca(2+) may help reverse accumulation of diacylglycerol in the PM by transferring it to the ER for metabolic recycling.

Sealing of phagosomes is accompanied by the disappearance of phosphatidylinositol (4,5)-bisphosphate (PtdIns(4,5)P(2)) from their cytoplasmic leaflet. Elimination of PtdIns(4,5)P(2), which is required for actin remodeling during phagosome formation, has been attributed to hydrolysis by phospholipase C and phosphorylation by phosphatidylinositol 3-kinase. We found that two inositol 5-phosphatases, OCRL and Inpp5B, become associated with nascent phagosomes. Both phosphatases, which are Rab5 effectors, associate with the adaptor protein APPL1, which is recruited to the phagosomes by active Rab5. Knockdown of APPL1 or inhibition of Rab5 impairs association of OCRL and Inpp5B with phagosomes and prolongs the presence of PtdIns(4,5)P(2) and actin on their membranes. Even though APPL1 can serve as an anchor for Akt, its depletion accentuated the activation of the kinase, likely by increasing the amount of PtdIns(4,5)P(2) available to generate phosphatidylinositol (3,4,5)-trisphosphate. Thus, inositol 5-phosphatases are important contributors to the phosphoinositide remodeling and signaling that are pivotal for phagocytosis.

Complexin prevents SNAREs from releasing neurotransmitters until an action potential arrives at the synapse. To understand the mechanism for this inhibition, we determined the structure of complexin bound to a mimetic of a prefusion SNAREpin lacking the portion of the v-SNARE that zippers last to trigger fusion. The 'central helix' of complexin is anchored to one SNARE complex, while its 'accessory helix' extends away at ~45° and bridges to a second complex, occupying the vacant v-SNARE binding site to inhibit fusion. We expected the accessory helix to compete with the v-SNARE for t-SNARE binding but found instead that the interaction occurs intermolecularly. Thus, complexin organizes the SNAREs into a zigzag topology that, when interposed between the vesicle and plasma membranes, is incompatible with fusion.

The retroviral Gag protein is the central player in the process of virion assembly at the plasma membrane, and is sufficient to induce the formation and release of virus-like particles. Recent evidence suggests that Gag may co-opt the host cell's endocytic machinery to facilitate retroviral assembly and release.

The GTPase dynamin, a key player in endocytic membrane fission, interacts with numerous proteins that regulate actin dynamics and generate/sense membrane curvature. To determine the functional relationship between these proteins and dynamin, we have analyzed endocytic intermediates that accumulate in cells that lack dynamin (derived from dynamin 1 and 2 double conditional knockout mice). In these cells, actin-nucleating proteins, actin, and BAR domain proteins accumulate at the base of arrested endocytic clathrin-coated pits, where they support the growth of dynamic long tubular necks. These results, which we show reflect the sequence of events in wild-type cells, demonstrate a concerted action of these proteins prior to, and independent of, dynamin and emphasize similarities between clathrin-mediated endocytosis in yeast and higher eukaryotes. Our data also demonstrate that the relationship between dynamin and actin is intimately connected to dynamin's endocytic role and that dynamin terminates a powerful actin- and BAR protein-dependent tubulating activity.

The multimeric membrane-tethering complexes TRAPPI and TRAPPII share seven subunits, of which four (Bet3p, Bet5p, Trs23p, and Trs31p) are minimally needed to activate the Rab GTPase Ypt1p in an event preceding membrane fusion. Here, we present the structure of a heteropentameric TRAPPI assembly complexed with Ypt1p. We propose that TRAPPI facilitates nucleotide exchange primarily by stabilizing the nucleotide-binding pocket of Ypt1p in an open, solvent-accessible form. Bet3p, Bet5p, and Trs23p interact directly with Ypt1p to stabilize this form, while the C terminus of Bet3p invades the pocket to participate in its remodeling. The Trs31p subunit does not interact directly with the GTPase but allosterically regulates the TRAPPI interface with Ypt1p. Our findings imply that TRAPPII activates Ypt1p by an identical mechanism. This view of a multimeric membrane-tethering assembly complexed with a Rab provides a framework for understanding events preceding membrane fusion at the molecular level.

Synaptojanin 1 (SJ1) is a major presynaptic phosphatase that couples synaptic vesicle endocytosis to the dephosphorylation of PI(4,5)P2, a reaction needed for the shedding of endocytic factors from their membranes. While the role of SJ1's 5-phosphatase module in this process is well recognized, the contribution of its Sac phosphatase domain, whose preferred substrate is PI4P, remains unclear. Recently a homozygous mutation in its Sac domain was identified in early-onset parkinsonism patients. We show that mice carrying this mutation developed neurological manifestations similar to those of human patients. Synapses of these mice displayed endocytic defects and a striking accumulation of clathrin-coated intermediates, strongly implicating Sac domain's activity in endocytic protein dynamics. Mutant brains had elevated auxilin (PARK19) and parkin (PARK2) levels. Moreover, dystrophic axonal terminal changes were selectively observed in dopaminergic axons in the dorsal striatum. These results strengthen evidence for a link between synaptic endocytic dysfunction and Parkinson's disease.

Lipid transport proteins at membrane contact sites, where two organelles are closely apposed, play key roles in trafficking lipids between cellular compartments while distinct membrane compositions for each organelle are maintained. Understanding the mechanisms underlying non-vesicular lipid trafficking requires characterization of the lipid transporters residing at contact sites. Here, we show that the mammalian proteins in the lipid transfer proteins anchored at a membrane contact site (LAM) family, called GRAMD1a-c, transfer sterols with similar efficiency as the yeast orthologues, which have known roles in sterol transport. Moreover, we have determined the structure of a lipid transfer domain of the yeast LAM protein Ysp2p, both in its apo-bound and sterol-bound forms, at 2.0 Å resolution. It folds into a truncated version of the steroidogenic acute regulatory protein-related lipid transfer (StART) domain, resembling a lidded cup in overall shape. Ergosterol binds within the cup, with its 3-hydroxy group interacting with protein indirectly via a water network at the cup bottom. This ligand binding mode likely is conserved for the other LAM proteins and for StART domains transferring sterols.

Numerous genes whose mutations cause, or increase the risk of, Parkinson's disease (PD) have been identified. An inactivating mutation (R258Q) in the Sac inositol phosphatase domain of synaptojanin 1 (SJ1/PARK20), a phosphoinositide phosphatase implicated in synaptic vesicle recycling, results in PD. The gene encoding Sac2/INPP5F, another Sac domain-containing protein, is located within a PD risk locus identified by genome-wide association studies. Knock-In mice carrying the SJ1 patient mutation (SJ1RQKI) exhibit PD features, while Sac2 knockout mice (Sac2KO) do not have obvious neurologic defects. We report a "synthetic" effect of the SJ1 mutation and the KO of Sac2 in mice. Most mice with both mutations died perinatally. The occasional survivors had stunted growth, died within 3 wk, and showed abnormalities of striatal dopaminergic nerve terminals at an earlier stage than SJ1RQKI mice. The abnormal accumulation of endocytic factors observed at synapses of cultured SJ1RQKI neurons was more severe in double-mutant neurons. Our results suggest that SJ1 and Sac2 have partially overlapping functions and are consistent with a potential role of Sac2 as a PD risk gene.

Ectopic expression in fibroblasts of synapsin 1 and synaptophysin is sufficient to generate condensates of vesicles highly reminiscent of synaptic vesicle (SV) clusters and with liquid-like properties. Here we show that unlike synaptophysin, other major integral SV membrane proteins fail to form condensates with synapsin, but co-assemble into the clusters formed by synaptophysin and synapsin in this ectopic expression system. Another vesicle membrane protein, ATG9A, undergoes activity-dependent exo-endocytosis at synapses, raising questions about the relation of ATG9A traffic to the traffic of SVs. We find that both in fibroblasts and in nerve terminals ATG9A does not co-assemble into synaptophysin-positive vesicle condensates but localizes on a distinct class of vesicles that also assembles with synapsin but into a distinct phase. Our findings suggest that ATG9A undergoes differential sorting relative to SV proteins and also point to a dual role of synapsin in controlling clustering at synapses of SVs and ATG9A vesicles.

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C. elegans ortholog of the canonical microtubule end binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and Dense Core Vesicles (DCVs) prior to their axonal delivery. Using single-neuron, in vivo labelling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargo, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. In addition to its microtubule binding CH domain, mammalian EB1 interacted with mammalian KIF1A in an EBH domain dependent manner, and expression of mammalian EB1 or the EBH domain was sufficient to rescue DCV transport in ebp-1 mutants. Our results suggest a model in which kinesin-3 binding and microtubule binding by EBP-1 cooperate to transiently enrich the motor near sites of DCV biogenesis to promote motor-cargo association. In support of this model, tethering either EBP-1 or a kinesin-3 KIF1A/UNC-104 interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule associated protein and provide insight into how specific kinesin-3 cargo are delivered to the axon.

Chorea-acanthocytosis (ChAc) and McLeod syndrome are diseases with shared clinical manifestations caused by mutations in VPS13A and XK, respectively. Key features of these conditions are the degeneration of caudate neurons and the presence of abnormally shaped erythrocytes. XK belongs to a family of plasma membrane (PM) lipid scramblases whose action results in exposure of PtdSer at the cell surface. VPS13A is an endoplasmic reticulum (ER)-anchored lipid transfer protein with a putative role in the transport of lipids at contacts of the ER with other membranes. Recently VPS13A and XK were reported to interact by still unknown mechanisms. So far, however, there is no evidence for a colocalization of the two proteins at contacts of the ER with the PM, where XK resides, as VPS13A was shown to be localized at contacts between the ER and either mitochondria or lipid droplets. Here we show that VPS13A can also localize at ER-PM contacts via the binding of its PH domain to a cytosolic loop of XK, that such interaction is regulated by an intramolecular interaction within XK, and that both VPS13A and XK are highly expressed in the caudate neurons. Binding of the PH domain of VPS13A to XK is competitive with its binding to intracellular membranes that mediate other tethering functions of VPS13A. Our findings support a model according to which VPS13A-dependent lipid transfer between the ER and the PM is coupled to lipid scrambling within the PM. They raise the possibility that defective cell surface exposure of PtdSer may be responsible for neurodegeneration.

Mutations in VPS13B, a member of a protein family implicated in bulk lipid transport between adjacent membranes, cause Cohen syndrome. VPS13B is known to be concentrated in the Golgi complex, but its precise location within this organelle and thus the site(s) where it achieves lipid transport remains unclear. Here we show that VPS13B is localized at the interface between cis and trans Golgi sub-compartments and that Golgi complex re-formation after Brefeldin A (BFA) induced disruption is delayed in VPS13B KO cells. This delay is phenocopied by loss of FAM177A1, a Golgi complex protein of unknown function reported to be a VPS13B interactor and whose mutations also result in a developmental disorder. In zebrafish, the vps13b orthologue, not previously annotated in this organism, genetically interacts with fam177a1. Collectively, these findings raise the possibility that bulk lipid transport by VPS13B may play a role in expanding Golgi membranes and that VPS13B may be assisted in this function by FAM177A1.

Phosphoglycerate kinase 1 (PGK1), the first ATP producing glycolytic enzyme, has emerged as a therapeutic target for Parkinson's Disease (PD), since a potential enhancer of its activity was reported to significantly lower PD risk. We carried out a suppressor screen of hypometabolic synaptic deficits and demonstrated that PGK1 is a rate limiting enzyme in nerve terminal ATP production. Increasing PGK1 expression in mid-brain dopamine neurons protected against hydroxy-dopamine driven striatal dopamine nerve terminal dysfunction in-vivo and modest changes in PGK1 activity dramatically suppressed hypometabolic synapse dysfunction in vitro. Furthermore, PGK1 is cross-regulated by PARK7 (DJ-1), a PD associated molecular chaperone, and synaptic deficits driven by PARK20 (Synaptojanin-1) can be reversed by increasing local synaptic PGK1 activity. These data indicate that nerve terminal bioenergetic deficits may underly a spectrum of PD susceptibilities and the identification of PGK1 as the limiting enzyme in axonal glycolysis provides a mechanistic underpinning for therapeutic protection.

Axonal transport is key to neuronal function. Efficient transport requires specific motor-cargo association in the soma, yet the mechanisms regulating this early step remain poorly understood. We found that EBP-1, the C. elegans ortholog of the canonical-microtubule-end-binding protein EB1, promotes the specific association between kinesin-3/KIF1A/UNC-104 and dense core vesicles (DCVs) prior to their axonal delivery. Using single-neuron, in vivo labeling of endogenous cargo and EBs, we observed reduced axonal abundance and reduced secretion of DCV cargo, but not other KIF1A/UNC-104 cargoes, in ebp-1 mutants. This reduction could be traced back to fewer exit events from the cell body, where EBP-1 colocalized with the DCV sorting machinery at the trans Golgi, suggesting that this is the site of EBP-1 function. EBP-1 calponin homology (CH) domain was required for directing microtubule growth on the Golgi, and mammalian EB1 interacted with KIF1A in an EBH-domain-dependent manner. Loss- and gain-of-function experiments suggest a model in which both kinesin-3 binding and guidance of microtubule growth at the trans Golgi by EBP-1 promote motor-cargo association at sites of DCV biogenesis. In support of this model, tethering either EBP-1 or a kinesin-3/KIF1A/UNC-104-interacting domain from an unrelated protein to the Golgi restored the axonal abundance of DCV proteins in ebp-1 mutants. These results uncover an unexpected role for a microtubule-associated protein and provide insights into how specific kinesin-3 cargo is delivered to the axon.