Nesca, a novel neuronal adapter protein, links the molecular motor kinesin with the pre-synaptic membrane protein, syntaxin-1, in hippocampal neurons

Vesicular transport in neurons plays a vital role in neuronal function and survival. Nesca is a novel protein that we previously identified and herein describe its pattern of expression, subcellular localization and protein-protein interactions both in vitro and in vivo. Specifically, a large proportion of Nesca is in tight association with both actin and microtubule cytoskeletal proteins. Nesca binds to F-actin, microtubules, βIII and acetylated α-tubulin, but not neurofilaments or the actin-binding protein drebrin, in in vitro-binding assays. Nesca co-immunoprecipitates with kinesin heavy chain (KIF5B) and kinesin light-chain motors as well as with the synaptic membrane precursor protein, syntaxin-1, and is a constituent of the post-synaptic density. Moreover, in vitro-binding assays indicate that Nesca directly binds KIF5B, kinesin light-chain and syntaxin-1. In contrast, Nesca does not co-immunoprecipitate with the kinesin motors KIF1B, KIF3A nor does it bind syntaxin-4 or the synaptosome-associated protein 25 kDa (SNAP-25) in vitro. Nesca expression in neurons is highly punctuate, co-stains with syntaxin-1, and is found in fractions containing markers of early endosomes and Golgi suggesting that it is involved in vesicular transport. Collectively, these data suggest that Nesca functions as an adapter involved in neuronal vesicular transport including vesicles containing soluble N-ethylmaleimide sensitive factor attachment protein receptors that are essential to exocytosis.     

Short-range axonal/dendritic transport by myosin-V: A model for vesicle delivery to the synapse

Myosin-V is a versatile motor involved in short-range axonal/dendritic transport of vesicles in the actin-rich cortex and synaptic regions of nerve cells. It binds to several different kinds of neuronal vesicles by its globular tail domain but the mechanism by which it is recruited to these vesicles is not known. In this study, we used an in vitro motility assay derived from axoplasm of the squid giant axon to study the effects of the globular tail domain on the transport of neuronal vesicles. We found that the globular tail fragment of myosin-V inhibited actin-based vesicle transport by displacing native myosin-V and binding to vesicles. The globular tail domain pulled down kinesin, a known binding partner of myosin-V, in affinity isolation experiments. These data confirmed earlier evidence that kinesin and myosin-V interact to form a hetero-motor complex. The formation of a kinesin/myosin-V hetero-motor complex on vesicles is thought to facilitate the coordination of long-range movement on microtubules and short-range movement on actin filaments. The direct interaction of motors from both filament systems may represent the mechanism by which the transition of vesicles from microtubules to actin filaments is regulated. These results are the first demonstration that the recombinant tail of myosin-V inhibits vesicle transport in an in vitro motility assay. Future experiments are designed to determine the functional significance of the interaction between myosin-V and kinesin and to identify other proteins that bind to the globular tail domain of myosin-V.     

The microtubule associated protein syntabulin is required for glucose-stimulated and cAMP-potentiated insulin secretion

Syntabulin is a microtubule-associated protein that mediates anterograde transport of vesicles to neuronal processes. Here, we found that syntabulin was expressed in mouse pancreas and insulin-secreting β-cells, and that it partially co-localized with microtubule and insulin-containing granules. The association of syntabulin with these organelles increased upon glucose stimulation. Knock-down of syntabulin by shRNA reduced both basal and glucose-stimulated insulin secretion, and diminished cAMP-Epac2 and cAMP-PKA potentiated insulin secretion. Additionally, syntabulin was preferentially phosphorylated by the Epac2 agonist 8-pCPT-2′-O-Me-cAMP, suggesting that syntabulin could be a novel effector of Epac2 and play a critical role in cAMP-enhanced insulin secretion.     

Point Mutation in Syntaxin-1A Causes Abnormal Vesicle Recycling,Behaviors, and Short Term Plasticity

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.     

The X-ray crystal structure of neuronal Sec1 from squid sheds new light on the role of this protein in exocytosis

BACKGROUND: Sec1-like molecules have been implicated in a variety of eukaryotic vesicle transport processes including neurotransmitter release by exocytosis. They regulate vesicle transport by binding to a t-SNARE from the syntaxin family. This process is thought to prevent SNARE complex formation, a protein complex required for membrane fusion. Whereas Sec1 molecules are essential for neurotransmitter release and other secretory events, their interaction with syntaxin molecules seems to represent a negative regulatory step in secretion. RESULTS: Here we report the X-ray crystal structure of a neuronal Sec1 homologue from squid, s-Sec1, at 2.4 A resolution. Neuronal s-Sec1 is a modular protein that folds into a V-shaped three-domain assembly. Peptide and mutagenesis studies are discussed with respect to the mechanism of Sec1 regulation. Comparison of the structure of squid s-Sec1 with the previously determined structure of rat neuronal Sec1 (n-Sec1) bound to syntaxin-1a indicates conformational rearrangements in domain III induced by syntaxin binding. CONCLUSIONS: The crystal structure of s-Sec1 provides the molecular scaffold for a number of molecular interactions that have been reported to affect Sec1 function. The structural differences observed between s-Sec1 and the structure of a rat n-Sec1-syntaxin-1a complex suggest that local conformational changes are sufficient to release syntaxin-1a from neuronal Sec1, an active process that is thought to involve additional effector molecule(s).     

Interaction of Huntingtin-associated protein-1 with kinesin light chain: implications in intracellular trafficking in neurons

Huntingtin-associated protein-1 (HAP1) was initially identified as an interacting partner of huntingtin, the Huntington disease protein. Unlike huntingtin that is ubiquitously expressed throughout the brain and body, HAP1 is enriched in neurons, suggesting that its dysfunction could contribute to Huntington disease neuropathology. Growing evidence has demonstrated that HAP1 and huntingtin are anterogradely transported in axons and that the abnormal interaction between mutant huntingtin and HAP1 may impair axonal transport. However, the exact role of HAP1 in anterograde transport remains unclear. Here we report that HAP1 interacts with kinesin light chain, a subunit of the kinesin motor complex that drives anterograde transport along microtubules in neuronal processes. The interaction of HAP1 with kinesin light chain is demonstrated via a yeast two-hybrid assay, glutathione S-transferase pull down, and coimmunoprecipitation. Furthermore, HAP1 is colocalized with kinesin in growth cones of neuronal cells. We also demonstrated that knocking down HAP1 via small interfering RNA suppresses neurite outgrowth of PC12 cells. Analysis of live neuronal cells with fluorescence microscopy and fluorescence recovery after photobleaching demonstrates that suppressing the expression of HAP1 or deleting the HAP1 gene inhibits the kinesin-dependent transport of amyloid precursor protein vesicles. These studies provide a molecular basis for the participation of HAP1 in anterograde transport in neuronal cells.     

Syntabulin-mediated anterograde transport of mitochondria along neuronal processes

In neurons, proper distribution of mitochondria in axons and at synapses is critical for neurotransmission, synaptic plasticity, and axonal outgrowth. However, mechanisms underlying mitochondrial trafficking throughout the long neuronal processes have remained elusive. Here, we report that syntabulin plays a critical role in mitochondrial trafficking in neurons. Syntabulin is a peripheral membrane-associated protein that targets to mitochondria through its carboxyl-terminal tail. Using real-time imaging in living cultured neurons, we demonstrate that a significant fraction of syntabulin colocalizes and co-migrates with mitochondria along neuronal processes. Knockdown of syntabulin expression with targeted small interfering RNA or interference with the syntabulin-kinesin-1 heavy chain interaction reduces mitochondrial density within axonal processes by impairing anterograde movement of mitochondria. These findings collectively suggest that syntabulin acts as a linker molecule that is capable of attaching mitochondrial organelles to the microtubule-based motor kinesin-1, and in turn, contributes to anterograde trafficking of mitochondria to neuronal processes.     

Microtubule-dependent movement of late endocytic vesicles in vitro: requirements for Dynein and Kinesin

Our previous studies demonstrated that fluorescent early endocytic vesicles prepared from rat liver after injection of Texas red asialoorosomucoid contain asialoglycoprotein and its receptor and move and undergo fission along microtubules using kinesin I and KIFC2, with Rab4 regulating KIFC2 activity (J. Cell Sci. 116, 2749, 2003). In the current study, procedures to prepare fluorescent late endocytic vesicles were devised. In addition, flow cytometry was utilized to prepare highly purified fluorescent endocytic vesicles, permitting validation of microscopy-based experiments as well as direct biochemical analysis. These studies revealed that late vesicles bound to and moved along microtubules, but in contrast to early vesicles, did not undergo fission. As compared with early vesicles, late vesicles had reduced association with receptor, Rab4, and kinesin I but were highly associated with dynein, Rab7, dynactin, and KIF3A. Dynein and KIF3A antibodies inhibited late vesicle motility, whereas kinesin I and KIFC2 antibodies had no effect. Dynamitin antibodies prevented the association of late vesicles with microtubules. These results indicate that acquisition and exchange of specific motor and regulatory proteins characterizes and may regulate the transition of early to late endocytic vesicles. Flow cytometric purification should ultimately facilitate detailed proteomic analysis and mapping of endocytic vesicle-associated proteins.     

Alignment, zippering and splaying of microtubules during axogenesis

Neurodegenerative diseases may result in part from defects in motor-driven vesicle transport in neuronal cells. Myosin-V, an actin-based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin-V interacts with the microtubule-based motor, kinesin, to form a 'hetero-motor' complex on vesicles. The complex of these two motors, one microtubule-based and the other actin-based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin-V to kinesin and to neuronal vesicles, we used a GST-tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin-V presumably by binding to vesicles and competing away the native myosin-V motors. The GST-MyoV-tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin-V. These data show that the headless myosin-V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin-V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Symposium 4: Dynamics of the Neuronal Cytoskeleton. Actin‐based vesicle transport in nerve cell extracts: implications for neurodegenerative diseases

Neurodegenerative diseases may result in part from defects in motor‐driven vesicle transport in neuronal cells. Myosin‐V, an actin‐based motor that is highly enriched in the brain, mediates the movement of vesicles on cortical actin filaments. Recent evidence suggests that the globular tail of myosin‐V interacts with the microtubule‐based motor, kinesin, to form a ‘hetero‐motor’ complex on vesicles. The complex of these two motors, one microtubule‐based and the other actin‐based, facilitates the movement of vesicles from microtubules to actin filaments. Based on our studies of vesicle transport by these two motors in extracts of squid neurons, we hypothesize that one of the functions of the tail–tail interaction is to provide feedback between the two proteins to allow seamless transition of vesicles from microtubules to actin filaments. To study the interactions of the globular tail domain of myosin‐V to kinesin and to neuronal vesicles, we used a GST‐tagged globular tail fragment in motility assays. The MyoV tail fragment inhibited vesicle transport by 81–91% and thereby exhibited a dominant negative effect. These data show that the recombinant protein blocked the activity of native myosin‐V presumably by binding to vesicles and competing away the native myosin‐V motors. The GST‐MyoV‐tail fragment pulled down kinesin by immunoprecipitation from squid brain homogenates and therefore it exhibited binding properties of native myosin‐V. These data show that the headless myosin‐V fragment is an effective inhibitor of vesicle transport in cell extracts. These studies support the hypothesis that tail–tail interactions may be a mechanism for feedback between myosin‐V and kinesin to allow transition of vesicles from microtubules to actin filaments. Acknowledgements: Supported by NSF grant MCB9974709.     

Syntaxin isoform specificity in the regulation of renal H+-ATPase exocytosis

Intercalated and inner medullary collecting duct (IMCD) cells of the kidney mediate the transport of H+ by a plasma membrane H+-ATPase. The rate of H+ transport in these cells is regulated by exocytic insertion of H+-ATPase-laden vesicles into the apical membrane. We have shown that the exocytic insertion of proton pumps (H+-ATPase) into the apical membrane of rat IMCD cells, in culture, involves SNARE proteins (syntaxin (synt), SNAP-23, and VAMP). The membrane fusion complex observed in IMCD cells with the induction of proton pump exocytosis not only included these SNAREs but also the H+-ATPase. Based on these observations, we suggested that the targeting of these vesicles to the apical membrane is mediated by an interaction between the H+-ATPase and a specific t-SNARE. To evaluate this hypothesis, we utilized a "pull-down" assay in which we identified, by Western analysis, the proteins in a rat kidney medullary homogenate that complexed with glutathione S-transferase (GST) fusion syntaxin isoforms attached to Sepharose 4B-glutathione beads. The syntaxin isoforms employed were 1A, 1B, 2, 4, 5, and also 1A that was truncated to exclude the H3 SNARE binding domain (synt-1ADeltaH3). All full-length syntaxin isoforms formed complexes with SNAP-23 and VAMP. Neither GST nor synt-1ADeltaH3 formed complexes with these SNAREs. H+-ATPase (subunits E, a, and c) bound to syntaxin-1A and to a lesser extent to synt-1B but not to synt-1ADeltaH3 or synt-2, -4, and -5. In cultured IMCD cells transfected to express syntaxin truncated for the membrane binding domain (synt-DeltaC), expression of synt-1ADeltaC, but not synt-4DeltaC, inhibited H+-ATPase exocytosis. In conclusion, because all full-length syntaxins examined bound VAMP-2 and SNAP-23, but only non-H3-truncated syntaxin-1 bound H+-ATPase, and synt-1ADeltaC expression by intact IMCD cells inhibited H+-ATPase exocytosis, it is likely that the H+-ATPase binds directly to the H3 domain of syntaxin-1 and not through VAMP-2 or SNAP-23. Interaction between the syntaxin-1A and H+-ATPase is important in the targeted exocytosis of the proton pump to the apical membrane of intercalated cells.     

Molecular motor function in axonal transport in vivo probed by genetic and computational analysis in Drosophila

Bidirectional axonal transport driven by kinesin and dynein along microtubules is critical to neuronal viability and function. To evaluate axonal transport mechanisms, we developed a high-resolution imaging system to track the movement of amyloid precursor protein (APP) vesicles in Drosophila segmental nerve axons. Computational analyses of a large number of moving vesicles in defined genetic backgrounds with partial reduction or overexpression of motor proteins enabled us to test with high precision existing and new models of motor activity and coordination in vivo. We discovered several previously unknown features of vesicle movement, including a surprising dependence of anterograde APP vesicle movement velocity on the amount of kinesin-1. This finding is largely incompatible with the biophysical properties of kinesin-1 derived from in vitro analyses. Our data also suggest kinesin-1 and cytoplasmic dynein motors assemble in stable mixtures on APP vesicles and their direction and velocity are controlled at least in part by dynein intermediate chain.     

Differential expression of syntaxin-1 and synaptophysin in the developing and adult human retina

Synaptophysin and syntaxin-1 are membrane proteins that associate with synaptic vesicles and presynaptic active zones at nerve endings, respectively. The former is known to be a good marker of synaptogenesis; this aspect, however, is not clear with syntaxin-1. In this study, the expression of both proteins was examined in the developing human retina and compared with their distribution in postnatal to adult retinas, by immunohistochemistry. In the inner plexiform layer, both were expressed simultaneously at 11–12 weeks of gestation, when synaptogenesis reportedly begins in the central retina. In the outer plexiform layer, however, the immunoreactivities were prominent by 16 weeks of gestation. Their expression in both plexiform layers followed a centre-to-periphery gradient. The immunoreactivities for both proteins were found in the immature photoreceptor, amacrine and ganglion cells; however, synaptophysin was differentially localized in bipolar cells and their axons, and syntaxin was present in some horizontal cells. In postnatal-to-adult retinas, synaptophysin immunoreactivity was prominent in photoreceptor terminals lying in the outer plexiform layer; on the contrary, syntaxin-1 was present in a thin immunoreactive band in this layer. In the inner plexiform layer, however, both were homogeneously distributed. Our study suggests that (i) syntaxin-1 appears in parallel with synapse formation; (ii) synaptogenesis in the human retina might follow a centre-to-periphery gradient; (iii) syntaxin-1 is likely to be absent from ribbon synapses of the outer plexiform layer, but may occur at presynaptic terminals of photoreceptor and horizontal cells, as is apparent from its localization in these cells, which is hitherto unreported for any vertebrate retina.     

Structural analysis of the neuronal SNARE protein syntaxin-1A

Intracellular trafficking depends on the docking and fusion of transport vesicles with cellular membranes. Central to docking and fusion is the pairing of SNARE proteins (soluble NSF attachment protein receptors) associated with the vesicle and target membranes (v- and t-SNAREs, respectively). Here, the X-ray structure of an N-terminal conserved domain of the neuronal t-SNARE syntaxin-1A was determined to a resolution of 1.9 A using multiwavelength anomalous diffraction. This X-ray structure, which is in general agreement with an NMR structure of a similar fragment, provides new insight into the interaction surface between the N-terminal domain and the remainder of the protein. In vitro characterization of the intact cytoplasmic domain of syntaxin revealed that it forms dimers, and probably tetramers, at low micromolar concentrations, with concomitant structural changes that can be detected by limited proteolysis. These observations suggest that the promiscuity characteristic of pairing between v-SNAREs and t-SNAREs extends to the formation of homo-oligomeric t-SNARE complexes as well. They also suggest a potential role for the neuronal Sec1 protein (nSec1) in preventing the formation of syntaxin multimers.     

Motor protein independent binding of endocytic carrier vesicles to microtubules in vitro

We have established an in vitro assay to characterize the binding of endocytic carrier vesicles to microtubules. Magnetic beads coated with microtubules were used as an affinity matrix. A fraction from nocodazole-treated cells enriched in endocytic carrier vesicles, labeled with internalized horseradish peroxidase, was used in the binding experiments. Binding of the endocytic carrier vesicles to microtubules in vitro was cytosol-dependent. This activity of cytosolic factors was saturable, heat-sensitive, and insensitive to N-ethyl-maleimide. Binding was sensitive to GTP and ATP. Addition of neuronal microtubule-associated proteins completely abolished binding of the endocytic organelles to microtubules. This binding was independent of the cytosolic microtubule-based motor proteins kinesin and cytoplasmic dynein, since cytosol depleted of these proteins remained fully active. Microtubule-binding proteins from HeLa cells, however, stimulated the interaction of endocytic carrier vesicles with microtubules. Trypsinized vesicles could no longer bind to microtubules in the presence of cytosol. These results suggest that cytosolic microtubule-binding proteins, other than the known microtubule-based motor proteins, as well as membrane proteins are involved in the nucleotide-dependent interaction of endocytic carrier vesicles with microtubules.     

The phosphorylation of kinesin regulates its binding to synaptic vesicles.

Membrane organella are transported bidirectionally in cells, and the axonal transport system has provided an ideal model system for studying this bidirectional transport. Kinesin and cytoplasmic dynein were identified as candidates for the motor molecules of fast axonal transport, which transport organella along microtubules anterogradely and retrogradely. However, the mechanism that controls this bidirectional transport is unknown. Our previous work revealed that kinesin in axons was associated abundantly with anterogradely transported membranous organella, most of which are believed to be precursors of synaptic vesicles and axonal plasma membranes, while the fractions bound to retrogradely transported ones were very small (Hirokawa, N., Sato-Yoshitake, R., Kobayashi, N., Pfister, K. K., Bloom, G. S., and Brady, S. T. (1991) J. Cell Biol. 114, 295-302). Here we demonstrated in vitro that the binding of kinesin to synaptic vesicles was concentration-dependent and saturable and could be released by high salt concentration. When kinesin was phosphorylated by cAMP-dependent protein kinase, its binding to symaptic vesicles was significantly reduced. By motility assay and by statistical analysis using electron microscopy, we further revealed that synaptic vesicles preincubated with phosphorylated kinesin associated less frequently with microtubules than synaptic vesicles preincubated with unphosphorylated kinesin. The phosphorylation of kinesin should therefore play an essential role in regulating the direction of fast axonal transport by inhibiting its binding to membrane organella, thus releasing it from membrane organella at nerve terminals.     

Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release. Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase. Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.     

Single Molecule Imaging Reveals Differences in Microtubule Track Selection Between Kinesin Motors

Cells generate diverse microtubule populations by polymerization of a common α/β-tubulin building block. How microtubule associated proteins translate microtubule heterogeneity into specific cellular functions is not clear. We evaluated the ability of kinesin motors involved in vesicle transport to read microtubule heterogeneity by using single molecule imaging in live cells. We show that individual Kinesin-1 motors move preferentially on a subset of microtubules in COS cells, identified as the stable microtubules marked by post-translational modifications. In contrast, individual Kinesin-2 (KIF17) and Kinesin-3 (KIF1A) motors do not select subsets of microtubules. Surprisingly, KIF17 and KIF1A motors that overtake the plus ends of growing microtubules do not fall off but rather track with the growing tip. Selection of microtubule tracks restricts Kinesin-1 transport of VSVG vesicles to stable microtubules in COS cells whereas KIF17 transport of Kv1.5 vesicles is not restricted to specific microtubules in HL-1 myocytes. These results indicate that kinesin families can be distinguished by their ability to recognize microtubule heterogeneity. Furthermore, this property enables kinesin motors to segregate membrane trafficking events between stable and dynamic microtubule populations.     

Structural basis for the Golgi membrane recruitment of Sly1p by Sed5p

Cytosolic Sec1/munc18-like proteins (SM proteins) are recruited to membrane fusion sites by interaction with syntaxin-type SNARE proteins, constituting indispensable positive regulators of intracellular membrane fusion. Here we present the crystal structure of the yeast SM protein Sly1p in complex with a short N-terminal peptide derived from the Golgi-resident syntaxin Sed5p. Sly1p folds, similarly to neuronal Sec1, into a three-domain arch-shaped assembly, and Sed5p interacts in a helical conformation predominantly with domain I of Sly1p on the opposite site of the nSec1/syntaxin-1-binding site. Sequence conservation of the major interactions suggests that homologues of Sly1p as well as the paralogous Vps45p group bind their respective syntaxins in the same way. Furthermore, we present indirect evidence that nSec1 might be able to contact syntaxin 1 in a similar fashion. The observed Sly1p-Sed5p interaction mode therefore indicates how SM proteins can stay associated with the assembling fusion machinery in order to participate in late fusion steps.     

Theoretical analysis of lipid transport in sciatic nerve.

We modify our previous mathematical model of axonal transport to analyze data on the fast transport of lipids in rat sciatic nerve given in Toews et al. (J. Neurochem. 40, 555-562 (1983)). The theoretical model accounts well for the shapes of the profiles of phosphatidylcholine, phosphatidylethanolamine, cholesterol and diphosphatidylglycerol. The parameters obtained support the qualitative conclusions of Toews et al. and provide quantitative estimates of the underlying processes, e.g., rates of vesicle and mitochondria translocation, rate constants for association and dissociation between vesicles, kinesin and microtubules, rates of deposition and rates of loss of each class of lipid from the nerve by leakage or via removal by the retrograde transport system. The analysis suggests that two classes of vesicles moving at different speeds may be involved in the transport of phosphatidylcholine and phosphatidylethanolamine.