Intracellular Membrane Trafficking Research Articles

Mechanical Role of Actin Dynamics in the Rheology of the Golgi Complex and in Golgi-Associated Trafficking Events

In vitro studies have shown that physical parameters, such as membrane curvature, tension, and composition, influence the budding and fission of transport intermediates. Endocytosis in living cells also appears to be regulated by the mechanical load experienced by the plasma membrane. In contrast, how these parameters affect intracellular membrane trafficking in living cells is not known. To address this question, we investigate here the impact of a mechanical stress on the organization of the Golgi complex and on the formation of transport intermediates from the Golgi complex. Using confocal microscopy, we visualize the deformation of Rab6-positive Golgi membranes applied by an internalized microsphere trapped in optical tweezers and simultaneously measure the corresponding forces. Our results show that the force necessary to deform Golgi membranes drops when actin dynamics is altered and correlates with myosin II activity. We also show that the applied stress has a long-range effect on Golgi membranes, perturbs the dynamics of Golgi-associated actin, and induces a sharp decrease in the formation of Rab6-positive vesicles from the Golgi complex as well as tubulation of Golgi membranes. We suggest that acto-myosin contractility strongly contributes to the local rigidity of the Golgi complex and regulates the mechanics of the Golgi complex to control intracellular membrane trafficking.

Crystallization and preliminary X-ray analysis of RabX3, a tandem GTPase from Entamoeba histolytica.

Ras superfamily GTPases regulate signalling pathways that control multiple biological processes by modulating the GTP/GDP cycle. Various Rab GTPases, which are the key regulators of vesicular trafficking pathways, play a vital role in the survival and virulence of the enteric parasite Entamoeba histolytica. The Rab GTPases act as binary molecular switches that utilize the conformational changes associated with the GTP/GDP cycle to elicit responses from target proteins and thereby regulate a broad spectrum of cellular processes including cell proliferation, cytoskeletal assembly, nuclear transport and intracellular membrane trafficking in eukaryotes. Entamoeba histolytica RabX3 (EhRabX3) is a unique GTPase in the amoebic genome, the only member in the eukaryotic Ras superfamily that harbours tandem G-domains and shares only 8-16% sequence identity with other GTPases. Recent studies suggested that EhRabX3 binds to a single guanine nucleotide through its N-terminal G-domain (NTD), while the C-terminal G-domain (CTD) plays a potential role in binding of the nucleotide to the NTD. Thus, understanding the intermolecular regulation between the two GTPase domains is expected to reveal valuable information on the overall action of EhRabX3. To provide structural insights into the inclusive action of this unique GTPase, EhRabX3 was crystallized by successive micro-seeding using the vapour-diffusion method. A complete data set was collected to 3.3 Å resolution using a single native EhRabX3 crystal at 100 K on BM14 at the ESRF, Grenoble, France. The crystal belonged to monoclinic space group C2, with unit-cell parameters a=198.6, b=119.3, c=89.2 Å, β=103.1°. Preliminary analysis of the data using the Matthews Probability Calculator suggested the presence of four to six molecules in the asymmetric unit.

THU0467 Silencing of the MEFV Gene in Familial Mediterranean Fever and Transcriptomic Analysis in Human Monocytes: Evidence for the Involvement of Pyrin in Innate Immune Responses and Autophagy

BackgroundMutations in MEFV gene encoding pyrin [1] account for Familial Mediterranean fever FMF, a recessively inherited disease but the high number of heterozygote patients is puzzling and requires additional investigation...

AP5Z1/SPG48 frequency in autosomal recessive and sporadic spastic paraplegia.

Hereditary spastic paraplegias (HSP) constitute a rare and highly heterogeneous group of neurodegenerative disorders, defined clinically by progressive lower limb spasticity and pyramidal weakness. Autosomal recessive HSP as well as sporadic cases present a significant diagnostic challenge. Mutations in AP5Z1, a gene playing a role in intracellular membrane trafficking, have been recently reported to be associated with spastic paraplegia type 48 (SPG48). Our objective was to determine the relative frequency and clinical relevance of AP5Z1 mutations in a large cohort of 127 HSP patients. We applied a targeted next-generation sequencing approach to analyze all coding exons of the AP5Z1 gene. With the output of high-quality reads and a mean coverage of 51-fold, we demonstrated a robust detection of variants. One 43-year-old female with sporadic complicated paraplegia showed two heterozygous nonsynonymous variants of unknown significance (VUS3; p.[R292W];[(T756I)]). Thus, AP5Z1 gene mutations are rare, at least in Europeans. Due to its low frequency, systematic genetic testing for AP5Z1 mutations is not recommended until larger studies are performed to add further evidence. Our findings demonstrate that amplicon-based deep sequencing is technically feasible and allows a compact molecular characterization of multiple HSP patients with high accuracy.

Crystal structure of a Chlamydomonas reinhardtii flagellar RabGAP TBC-domain at 1.8 Å resolution.

Rab GTPases play a crucial role in the regulation of many intracellular membrane trafficking pathways including endocytosis and ciliogenesis. Rab GTPase activating proteins (RabGAPs) increase the GTP hydrolysis rate of Rab GTPases and turn them into guanine nucleotide diphosphate (GDP) bound inactive form. Here, we determined the crystal structure of the putative catalytic domain of a RabGAP (which we name CrfRabGAP) that is found in the flagellar proteome of the unicellular green alga Chlamydomonas reinhardtii. BLAST searches revealed potential human orthologues of CrfRabGAP as TBC1D3 and TBC1D26. Sequence and structural comparison with other canonical RabGAPs revealed that the CrfRabGAP does not contain the canonical catalytic residues required for the activation of Rab GTPases. The function of noncanonical RabGAPs-like CrfRabGAP might be to serve as Rab effectors rather than activators.

Star players sidelined in chloride homeostasis in neurons.

Star players sidelined in chloride homeostasis in neurons.

Surfactant Uptake Dynamics in Mammalian Cells Elucidated with Quantitative Coherent Anti-Stokes Raman Scattering Microspectroscopy

The mechanism of surfactant-induced cell lysis has been studied with quantitative coherent anti-Stokes Raman scattering (CARS) microspectroscopy. The dynamics of surfactant molecules as well as intracellular biomolecules in living Chinese Hamster Lung (CHL) cells has been examined for a low surfactant concentration (0.01 w%). By using an isotope labeled surfactant having CD bonds, surfactant uptake dynamics in living cells has been traced in detail. The simultaneous CARS imaging of the cell itself and the internalized surfactant has shown that the surfactant molecules is first accumulated inside a CHL cell followed by a sudden leak of cytosolic components such as proteins to the outside of the cell. This finding indicates that surfactant uptake occurs prior to the cell lysis, contrary to what has been believed: surface adsorption of surfactant molecules has been thought to occur first with subsequent disruption of cell membranes. Quantitative CARS microspectroscopy enables us to determine the molecular concentration of the surfactant molecules accumulated in a cell. We have also investigated the effect of a drug, nocodazole, on the surfactant uptake dynamics. As a result of the inhibition of tubulin polymerization by nocodazole, the surfactant uptake rate is significantly lowered. This fact suggests that intracellular membrane trafficking contributes to the surfactant uptake mechanism.

The Functional Significance of Phospho‐Rab GTPases (LB194)

Intracellular membrane trafficking is an essential and conserved process throughout eukaryotes. To preserve organelle identity in a complex cellular system, control mechanisms must exist to oversee the production and targeting of intracellular vesicles. Key control elements are the Rab GTPases, which utilize a nucleotide‐bound cycle to act as master regulators for each discrete trafficking step. Several studies, including global proteomic screens, have identified multiple Rabs as phosphoproteins, but the functional consequence(s) of this modification are unknown. The Saccharomyces cerevisiae Rab GTPase Sec4p is negatively regulated by phosphorylation, but the molecular mechanisms and proteins responsible for this modification are unknown. Using Sec4p as a model for Rab phosphorylation and unique antibodies able to specifically detect modified Sec4p, we have shown that phosphorylation is tightly regulated in conjunction with cell cycle progression and spatial location within the cell. Currently, we are performing an overexpression screen to elucidate the kinase that modifies Sec4p as well as the biochemical pathway responsible for activating Sec4p phosphorylation. We hypothesize that Rab phosphorylation may be a novel regulatory mechanism through which additional cellular processes can impinge upon membrane trafficking.Grant Funding Source: National Institute of Health

Developing human mammary‐gland functions during the first year of lactation (II) (622.7)

Proteomic analysis of human milk provides a non‐invasive means to explore the physiology of lactating mammary gland. We applied quantitative milk proteomic analysis (TMT labeling) over one year of lactation (n =10 women with samples at 1 week, 1, 3, 6, 9 and 12 months) as probes for the developing mammary‐gland function. Proteins involved in the processes of endoplasmic reticulum‐assisted degradation and proteasomal degradation are, in general, upregulated over lactation, suggesting increased activities in protein catabolism. Rab GTPases that control intracellular membrane trafficking in various endocytosis and secretion processes are typically present at highest abundance at 6 months of lactation. The extracellular domain of intercellular adhesion molecule 1 (ICAM‐1) is upregulated over lactation. The increased proteolytic shedding of ICAM‐1 may represent a means to suppress leukocyte recruitment into milk as lactation progresses. In contrast, the shedding of E‐cadherin decreases over lactation, suggesting increasing barrier integrity at the adherens junctions in lactating human mammary epithelium. These insights combining in‐depth determination of protein content in milk with bioinformatic analysis illustrate the large potential for advancing our understanding of the lactating mammary gland.

Molecular cloning and localization of GABAA receptor‐associated protein in the rotifer Brachionus plicatilis

γ‐Aminobutyric acid receptor type A‐associated protein (GABARAP) and its homologs constitute a protein family found in many eukaryotes from yeast to human, and are known to be involved in intracellular membrane trafficking of GABAA receptors and autophagy. In this study, we cloned cDNA‐encoding GABARAP from the monogonont rotifer Brachionus plicatilis and examined for its tissue distribution at the protein level in neonates, males and females. Using reverse transcription (RT)‐PCR and rapid amplification of cDNA ends (RACE) techniques, we showed that like other GABARAPs, rotifer GABARAP was also composed of 117 amino acids and highly homologous to vertebrate GABARAP2 ortholog (74–76% identity). GABARAP was demonstrated with its specific antibody to be ubiquitously distributed, irrespective of neonates, males, and females, in the coronal area that covers brain and contains most mechano‐ and chemoreceptors. Rotifer GABARAP was also expressed in the mature eggs but not in immature eggs. Double immunostaining with mammalian anti‐GABA γ receptor antibody showed that rotifer GABARAP co‐localized with GABA receptor, suggesting the association of the two proteins. The presence of GABARAP in rotifer implies that it is highly conserved during evolution, and plays important roles in various biological processes.

A RabGAP Regulates Life-Cycle Duration via Trimeric G-protein Cascades in Dictyostelium discoideum

BackgroundThe life-cycle of cellular slime molds comprises chronobiologically regulated processes. During the growth phase, the amoeboid cells proliferate at a definite rate. Upon starvation, they synthesize cAMP as both first and second messengers in signalling pathways and form aggregates, migrating slugs, and fruiting bodies, consisting of spores and stalk cells, within 24 h. In Dictyostelium discoideum, because most growth-specific events cease during development, proliferative and heterochronic mutations are not considered to be interrelated and no genetic factor governing the entire life-cycle duration has ever been identified.Methodology/Principal FindingsUsing yeast 2-hybrid library screening, we isolated a Dictyostelium discoideum RabGAP, Dd Rbg-3, as a candidate molecule by which the Dictyostelium Gα2 subunit directs its effects. Rab GTPase-activating protein, RabGAP, acts as a negative regulator of Rab small GTPases, which orchestrate the intracellular membrane trafficking involved in cell proliferation. Deletion mutants of Dd rbg-3 exhibited an increased growth rate and a shortened developmental period, while an overexpression mutant demonstrated the opposite effects. We also show that Dd Rbg-3 interacts with 2 Gα subunits in an activity-dependent manner in vitro. Furthermore, both human and Caenorhabditis elegans rbg-3 homologs complemented the Dd rbg-3–deletion phenotype in D. discoideum, indicating that similar pathways may be generally conserved in multicellular organisms.Conclusions/SignificanceOur findings suggest that Dd Rbg-3 acts as a key element regulating the duration of D. discoideum life-span potentially via trimeric G-protein cascades.

Regulation of PIK3C3/VPS34 complexes by MTOR in nutrient stress-induced autophagy

Autophagy is a cellular defense response to stress conditions, such as nutrient starvation. The type III phosphatidylinositol (PtdIns) 3-kinase, whose catalytic subunit is PIK3C3/VPS34, plays a critical role in intracellular membrane trafficking and autophagy induction. PIK3C3 forms multiple complexes and the ATG14-containing PIK3C3 is specifically involved in autophagy induction. Mechanistic target of rapamycin (MTOR) complex 1, MTORC1, is a key cellular nutrient sensor and integrator to stimulate anabolism and inhibit catabolism. Inactivation of TORC1 by nutrient starvation plays a critical role in autophagy induction. In this report we demonstrated that MTORC1 inactivation is critical for the activation of the autophagy-specific (ATG14-containing) PIK3C3 kinase, whereas it has no effect on ATG14-free PIK3C3 complexes. MTORC1 inhibits the PtdIns 3-kinase activity of ATG14-containing PIK3C3 by phosphorylating ATG14, which is required for PIK3C3 inhibition by MTORC1 both in vitro and in vivo. Our data suggest a mechanistic link between amino acid starvation and autophagy induction via the direct activation of the autophagy-specific PIK3C3 kinase.

Tethering the assembly of SNARE complexes

The fusion of transport vesicles with their target membranes is fundamental for intracellular membrane trafficking and diverse physiological processes and is driven by the assembly of functional soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes. Prior to fusion, transport vesicles are physically linked to their target membranes by various tethering factors. Recent studies suggest that tethering factors also positively regulate the assembly of functional SNARE complexes, thereby coupling tethering with fusion events. This coupling is mediated, at least in part, by direct physical interactions between tethering factors, SNAREs, and Sec1/Munc18 (SM) proteins. In this review we summarize recent progress in understanding the roles of tethering factors in the assembly of specific and functional SNARE complexes driving membrane-fusion events.

Expanding the Regulatory Repertoire of GEFs

Small GTPases comprise a large and heterogeneous group of regulatory proteins that act throughout the cell as on/off switches, orchestrating processes as diverse as endocytosis at the plasma membrane to protein export from the nucleus. GTPases themselves are controlled by proteins called guanine nucleotide exchange factors (GEFs). Upon binding a GEF, the GTPase releases a molecule of GDP and picks up a GTP from the cytosol, inducing a conformational change in the GTPase that allows it to switch on its target protein. GTPases by themselves are relatively indiscriminate, largely relying on GEFs to determine the location and timing of their activity. Thus, understanding the factors that determine where and when a GEF binds to its GTPase is critical for understanding a wide range of transport and signaling activities in the cell. Model of the guanine nucleotide exchange factor (GEF) BRAG2 bound to membrane-anchored Arf GTPase. GTPases of the Arf class regulate most intracellular membrane trafficking, and their activity is confined to membranes by the GEFs they bind to. For one group of Arf-GEFs called cytohesins, detailed study has shown that binding specificity is controlled in part by two key factors. First, a positively charged lipid-binding pocket in the so-called PH domain attracts only a small number of specific, negatively charged phosphatidylinositides, a type of membrane lipid. Second, cytohesins in solution are auto-inhibited by an interaction between the PH domain and a catalytic domain called Sec7; membrane binding undoes that interaction, liberating the GEF to act on its target Arf. This process is further amplified by a positive feedback loop whereby freshly produced Arf-GTP binds to the PH domain. But there are other Arf-GEFs about which little is known, including BRAG2, which has critical functions in multiple processes, such as muscle fiber development and breast tumor invasion. In a new study, Kaheina Aizel, Mahel Zeghouf, Jacqueline Cherfils, and colleagues show that the factors that control the activity of this Arf-GEF are quite different from those of the cytohesins, expanding our broader understanding of this important class of regulatory molecules. The authors began by measuring BRAG2's activity in solution, and found that, unlike cytohesin, there was no evidence of auto-inhibition. The explanation came when they determined the crystal structure; an expansion of the PH domain in BRAG2 allowed it to establish contacts elsewhere on the protein surface, and prevented it from blocking the catalytic activity of the Sec7 domain. This change in structure had an impact on BRAG2's exchange efficiency when paired with Arf1. The enlarged non-canonical PH domain increased the exchange activity of the Sec7 domain ten-fold compared to the Sec7 domain alone. And while BRAG2 could operate in solution, its efficiency was increased 160-fold when bound to a membrane. Interestingly, these effects were cumulative, so that the full, membrane-bound BRAG2 was more than 2000 times as efficient at promoting nucleotide exchange compared to the protein in solution without the PH domain. In further contrast, BRAG2 was not regulated by a positive feedback loop. Next, they turned to the amino acid sequence within the catalytic pocket. They found that a positively charged lysine seen in cytohesins had been replaced by a negatively charged glutamic acid, meaning the pocket should not attract phosphatidylinositides. BRAG2 did attract negatively charged lipids, but did so through a much larger positively charged surface around the periphery of the binding pocket. As a consequence, it was much less discriminating in its choice of lipid partners, and attracted a much wider diversity of lipids compared to cytohesins, including multiple phosphatidylinositides as well as phosphatidylserine. In sum, this study demonstrates that the regulation and lipid specificity of BRAG2 differs markedly from cytohesins, at once identifying a new regulatory scheme for GEFs and suggesting there are probably others yet to be discovered. And by shedding light on how this particular GEF is controlled, it may suggest new approaches for disrupting the activity of this protein in its role as a promoter of tumor invasion, or facilitating it in its role as a promoter of muscle repair. Aizel K, Biou V, Navaza J, Duarte LV, Campanacci V, et al. (2013) Integrated Conformational and Lipid-Sensing Regulation of Endosomal ArfGEF BRAG2. doi:10.1371/journal.pbio.1001654

Lipid Segregation and Membrane Budding Induced by the Peripheral Membrane Binding Protein Annexin A2*

The formation of dynamic membrane microdomains is an important phenomenon in many signal transduction and membrane trafficking events. It is driven by intrinsic properties of membrane lipids and integral as well as membrane-associated proteins. Here we analyzed the ability of one peripherally associated membrane protein, annexin A2 (AnxA2), to induce the formation of phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2)-rich domains in giant unilamellar vesicles (GUVs) of complex lipid composition. AnxA2 is a cytosolic protein that can bind PI(4,5)P2 and other acidic phospholipids in a Ca(2+)-dependent manner and that has been implicated in cellular membrane dynamics in endocytosis and exocytosis. We show that AnxA2 binding to GUVs induces lipid phase separation and the recruitment of PI(4,5)P2, cholesterol and glycosphingolipids into larger clusters. This property is observed for the full-length monomeric protein, a mutant derivative comprising the C-terminal protein core domain and for AnxA2 residing in a heterotetrameric complex with its intracellular binding partner S100A10. All AnxA2 derivatives inducing PI(4,5)P2 clustering are also capable of forming interconnections between PI(4,5)P2-rich microdomains of adjacent GUVs. Furthermore, they can induce membrane indentations rich in PI(4,5)P2 and inward budding of these membrane domains into the lumen of GUVs. This inward vesiculation is specific for AnxA2 and not shared with other PI(4,5)P2-binding proteins such as the pleckstrin homology (PH) domain of phospholipase Cδ1. Together our results indicate that annexins such as AnxA2 can efficiently induce membrane deformations after lipid segregation, a mechanism possibly underlying annexin functions in membrane trafficking.

Tbc1d15-17 regulates synaptic development at the Drosophila neuromuscular junction.

Members of the Tre-2/Bub2/Cdc16 (TBC) family of proteins are believed to function as GTPase-activating proteins (GAPs) for Rab GTPases, which play pivotal roles in intracellular membrane trafficking. Although membrane trafficking is fundamental to neuronal morphogenesis and function, the roles of TBC-family Rab GAPs have been poorly characterized in the nervous system. In this paper, we provide genetic evidence that Tbc1d15-17, the Drosophila homolog of mammalian Rab7-GAP TBC1d15, is required for normal presynaptic growth and postsynaptic organization at the neuromuscular junction (NMJ). A loss-of-function mutation in Tbc1d15-17 or its presynaptic knockdown leads to an increase in synaptic bouton number and NMJ length. Tbc1d15-17 mutants are also defective in the distribution of the postsynaptic scaffold Discs-large (Dlg) and in the level of the postsynaptic glutamate subunit GluRIIA. These postsynaptic phenotypes are recapitulated by postsynaptic knockdown of Tbc1d15-17. We also show that presynaptic overexpression of a constitutively active Rab7 mutant in a wild-type background causes a synaptic overgrowth phenotype resembling that of Tbc1d15-17 mutants, while a dominant-negative form of Rab7 has the opposite effect. Together, our findings establish a novel role for Tbc1d15-17 and its potential substrate Rab7 in regulating synaptic development.

Muscle-specific function of the centronuclear myopathy and Charcot–Marie–Tooth neuropathy-associated dynamin 2 is required for proper lipid metabolism, mitochondria, muscle fibers, neuromuscular junctions and peripheral nerves

The ubiquitously expressed large GTPase Dynamin 2 (DNM2) plays a critical role in the regulation of intracellular membrane trafficking through its crucial function in membrane fission, particularly in endocytosis. Autosomal-dominant mutations in DNM2 cause tissue-specific human disorders. Different sets of DNM2 mutations are linked to dominant intermediate Charcot-Marie-Tooth neuropathy type B, a motor and sensory neuropathy affecting primarily peripheral nerves, or autosomal-dominant centronuclear myopathy (CNM) presenting with primary damage in skeletal muscles. To understand the underlying disease mechanisms, it is imperative to determine to which degree the primary affected cell types require DNM2. Thus, we used cell type-specific gene ablation to examine the consequences of DNM2 loss in skeletal muscle cells, the major relevant cell type involved in CNM. We found that DNM2 function in skeletal muscle is required for proper mouse development. Skeletal muscle-specific loss of DNM2 causes a reduction in muscle mass and in the numbers of muscle fibers, altered muscle fiber size distributions, irregular neuromuscular junctions (NMJs) and isolated degenerating intramuscular peripheral nerve fibers. Intriguingly, a lack of muscle-expressed DNM2 triggers an increase of lipid droplets (LDs) and mitochondrial defects. We conclude that loss of DNM2 function in skeletal muscles initiates a chain of harmful parallel and serial events, involving dysregulation of LDs and mitochondrial defects within altered muscle fibers, defective NMJs and peripheral nerve degeneration. These findings provide the essential basis for further studies on DNM2 function and malfunction in skeletal muscles in health and disease, potentially including metabolic diseases such as diabetes.

Two Rab GTPases, EsRab-1 and EsRab-3, involved in anti-bacterial response of Chinese mitten crab Eriocheir sinensis

Rab GTPase is essential for the control of intracellular membrane trafficking in all eukaryotic cells and further affects the ability of phagocytic cells to scavenge pathogen. In the present study, the cDNAs for two crab Rab proteins (EsRab-1 and EsRab-3) were identified from the Chinese mitten crab Eriocheir sinensis by rapid amplification of cDNA ends (RACE) approaches and expressed sequence tag (EST) analysis. The full-length cDNAs of EsRab-1 and EsRab-3 were of 892 bp and 965 bp with ORFs of 615 bp and 630 bp, respectively. The cDNAs encoded two peptides of 204 and 209 amino acid residues with the conserved GTP/Mg2+ binding sites, Switch I region and Switch II region in RAB domains. The mRNA transcripts of EsRab-1 and EsRab-3 were both highest expressed in hepatopancreas, and marginally expressed in other tissues including hemocytes, muscle, gonad, gill and heart. After the crabs were challenged by bacteria Vibrio anguillarum, the expression levels of both EsRab-1 and EsRab-3 in hemocytes were significantly up-regulated, and reached the highest level at 1.5 h post-stimulation, which was 7-fold (P < 0.05) and 6-fold (P < 0.01) of blank group for EsRab-1 and EsRab-3, respectively. No significant change of mRNA expression was detected for either EsRab-1 or EsRab-3 in crabs stimulated by Pichia pastoris. These results clearly suggested the involvement of Rab proteins in crab anti-bacterial immunity.

The ALS8 protein VAPB interacts with the ER–Golgi recycling protein YIF1A and regulates membrane delivery into dendrites

The vesicle-associated membrane protein (VAMP) associated protein B (VAPB) is an integral membrane protein localized to the endoplasmic reticulum (ER). The P56S mutation in VAPB has been linked to motor neuron degeneration in amyotrophic lateral sclerosis type 8 (ALS8) and forms ER-like inclusions in various model systems. However, the role of wild-type and mutant VAPB in neurons is poorly understood. Here, we identified Yip1-interacting factor homologue A (YIF1A) as a new VAPB binding partner and important component in the early secretory pathway. YIF1A interacts with VAPB via its transmembrane regions, recycles between the ER and Golgi and is mainly localized to the ER-Golgi intermediate compartments (ERGICs) in rat hippocampal neurons. VAPB strongly affects the distribution of YIF1A and is required for intracellular membrane trafficking into dendrites and normal dendritic morphology. When VAPB-P56S is present, YIF1A is recruited to the VAPB-P56S clusters and loses its ERGIC localization. These data suggest that both VAPB and YIF1A are important for ER-to-Golgi transport and that missorting of YIF1A may contribute to VAPB-associated motor neuron disease.

The Role of the N-D1 Linker of the N-Ethylmaleimide-Sensitive Factor in the SNARE Disassembly

N-ethylmaleimide-sensitive factor (NSF) is a member of the type II AAA+ (ATPase associated with various cellular activities) family. It plays a critical role in intracellular membrane trafficking by disassembling soluble NSF attachment protein receptor (SNARE) complexes. Each NSF protomer consists of an N-terminal domain (N domain) followed by two AAA ATPase domains (D1 and D2) in tandem. The N domain is required for SNARE/α-SNAP binding and the D1 domain accounts for the majority of ATP hydrolysis. Little is known about the role of the N-D1 linker in the NSF function. This study presents detailed mutagenesis analyses of NSF N-D1 linker, dissecting its role in the SNARE disassembly, the SNARE/α-SNAP complex binding, the basal ATPase activity and the SNARE/α-SNAP stimulated ATPase activity. Our results show that the N-terminal region of the N-D1 linker associated mutants cause severe defect in SNARE complex disassembly, but little effects on the SNARE/α-SNAP complex binding, the basal and the SNARE/α-SNAP stimulated ATPase activity, suggesting this region may be involved in the motion transmission from D1 to N domain. Mutating the residues in middle and C-terminal region of the N-D1 linker increases the basal ATPase activity, indicating it may play a role in autoinhibiting NSF activity until it encounters SNARE/α-SNAP complex substrate. Moreover, mutations at the C-terminal sequence GIGG exhibit completely abolished or severely reduced activities of the substrate binding, suggesting that the flexibility of N-D1 linker is critical for the movement of the N domain that is required for the substrate binding. Taken together, these data suggest that the whole N-D1 linker is critical for the biological function of NSF to disassemble SNARE complex substrate with different regions responsible for different roles.