MICAL-L1 is required for cargo protein delivery to the cell surface.

Secreted proteins are transported along intracellular route from the endoplasmic reticulum through the Golgi before reaching the plasma membrane. Small GTPase Rab and their effectors play a key role in membrane trafficking. Using confocal microscopy, we showed that MICAL-L1 was associated with tubulo-vesicular structures and exhibited a significant colocalization with markers of the Golgi apparatus and recycling endosomes. Super resolution STORM microscopy suggested at the molecular level, a very close association of MICAL-L1 and microdomains in the Golgi cisternae. Using a synchronized secretion assay, we report that the shRNA-mediated depletion of MICAL-L1 impaired the delivery of a subset of cargo proteins to the cell surface. The process of membrane tubulation was monitored in vitro, and we observe that recombinant MICAL-L1-RBD domain may contribute to promote PACSINs-mediated membrane tubulation. Interestingly, two hydrophobic residues at the C-terminus of MICAL-L1 appeared to be important for phosphatidic acid binding, and for association with membrane tubules. Our results reveal a new role for MICAL-L1 in cargo delivery to the plasma membrane.

Role of tetanus neurotoxin insensitive vesicle-associated membrane protein (TI-VAMP) in vesicular transport mediating neurite outgrowth.

How vesicular transport participates in neurite outgrowth is still poorly understood. Neurite outgrowth is not sensitive to tetanus neurotoxin thus does not involve synaptobrevin-mediated vesicular transport to the plasma membrane of neurons. Tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP) is a vesicle-SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment protein [SNAP] receptor), involved in transport to the apical plasma membrane in epithelial cells, a tetanus neurotoxin-resistant pathway. Here we show that TI-VAMP is essential for vesicular transport-mediating neurite outgrowth in staurosporine-differentiated PC12 cells. The NH(2)-terminal domain, which precedes the SNARE motif of TI-VAMP, inhibits the association of TI-VAMP with synaptosome-associated protein of 25 kD (SNAP25). Expression of this domain inhibits neurite outgrowth as potently as Botulinum neurotoxin E, which cleaves SNAP25. In contrast, expression of the NH(2)-terminal deletion mutant of TI-VAMP increases SNARE complex formation and strongly stimulates neurite outgrowth. These results provide the first functional evidence for the role of TI-VAMP in neurite outgrowth and point to its NH(2)-terminal domain as a key regulator in this process.

A small rab GTPase is distributed in cytoplasmic vesicles in non polarized cells but colocalizes with the tight junction marker ZO-1 in polarized epithelial cells.

Small rab/Ypt1/Sec4 GTPase family have been involved in the regulation of membrane traffic along the biosynthetic and endocytic pathways in eucaryotic cells. Polarized epithelial cells have morphologically and functionally distinct apical and basolateral surfaces separated by tight junctions. The establishment and maintenance of these structures require delivery of membrane proteins and lipids to these domains. In this work, we have isolated a cDNA clone from a human intestinal cDNA library encoding a small GTPase, rab13, closely related to the yeast Sec4 protein. Confocal microscopy analysis on polarized Caco-2 cells shows that rab13 protein colocalized with the tight junction marker ZO-1. Cryostat sections of tissues confirm that rab13 localized to the junctional complex region of a variety of epithelia, including intestine, kidney, liver, and of endothelial cells. This localization requires assembly and integrity of the tight junctions. Disruption of tight junctions by incubation in low Ca2+ media induces the redistribution of rab13. In cells devoid of tight junctions, rab13 was found associated with vesicles dispersed throughout the cytoplasm. Cell-cell contacts initiated by E-cadherin in transfected L cells do not recruit rab13 to the resulting adherens-like junction complexes. The participation of rab13 in polarized transport, in the assembly and/or the activity of tight junctions is discussed.

The product of rab2, a small GTP binding protein, increases neuronal adhesion, and neurite growth in vitro.

The rab genes code for small GTP binding proteins that share with p21ras the ability to bind and hydrolyze GTP. They present significant sequence homologies with the products of YPT1 and SEC4, two small GTP binding proteins involved in the regulation of secretion in the yeast. Several rab genes are expressed in the developing and adult mouse brain. To test directly the possible involvement of these genes in neuronal differentiation, purified rab proteins produced in E. coli were introduced into neurons dissociated from E15 rat midbrain. The most striking effects were obtained with rab2 protein (rab2p). Compared with untreated cells, neurons loaded with rab2p presented an enhanced adhesion to the culture substratum. This phenomenon was visible 3 hr after seeding and was followed within 24 hr by a dramatic increase in neurite growth. Loading the same population of neurons with the products of four other rab genes either decreased neuronal adhesion and neurite growth or had no effect. These experiments suggest that the expression of rab2p plays an important role in neuronal differentiation.

A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells.

The importance of soluble N-ethyl maleimide (NEM)-sensitive fusion protein (NSF) attachment protein (SNAP) receptors (SNAREs) in synaptic vesicle exocytosis is well established because it has been demonstrated that clostridial neurotoxins (NTs) proteolyze the vesicle SNAREs (v-SNAREs) vesicle-associated membrane protein (VAMP)/brevins and their partners, the target SNAREs (t-SNAREs) syntaxin 1 and SNAP25. Yet, several exocytotic events, including apical exocytosis in epithelial cells, are insensitive to numerous clostridial NTs, suggesting the presence of SNARE-independent mechanisms of exocytosis. In this study we found that syntaxin 3, SNAP23, and a newly identified VAMP/brevin, tetanus neurotoxin (TeNT)-insensitive VAMP (TI-VAMP), are insensitive to clostridial NTs. In epithelial cells, TI-VAMP-containing vesicles were concentrated in the apical domain, and the protein was detected at the apical plasma membrane by immunogold labeling on ultrathin cryosections. Syntaxin 3 and SNAP23 were codistributed at the apical plasma membrane where they formed NEM-dependent SNARE complexes with TI-VAMP and cellubrevin. We suggest that TI-VAMP, SNAP23, and syntaxin 3 can participate in exocytotic processes at the apical plasma membrane of epithelial cells and, more generally, domain-specific exocytosis in clostridial NT-resistant pathways.

Expression of the ras-related ralA, rho12 and rab genes in adult mouse tissues.

The expression of recently isolated mammalian ras-related ral, rho, and rab genes was examined in adult mouse tissues. Most of the genes studied were transcribed into two major messenger RNAs. The transcription of each gene appeared to be regulated in a complex manner with a tissue-specific modulation of the two transcripts. One member of the rab gene family, rab3, showed an RNA expression restricted to brain-tissues. Ral, rho, and three other rab genes had a more ubiquitous expression in murine tissues. However, the expression level of each gene showed a high degree of variation depending upon the organ. Among all the members of the enlarged ras gene family examined so far, the rab3 gene is the first example showing an expression restricted to a distinct organ.

Subcellular localization of tetanus neurotoxin-insensitive vesicle-associated membrane protein (VAMP)/VAMP7 in neuronal cells: evidence for a novel membrane compartment.

The clostridial neurotoxin-insensitive soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptors, tetanus neurotoxin-insensitive (TI)-vesicle-associated membrane protein (VAMP)/VAMP7, SNAP23, and syntaxin 3 have recently been implicated in transport of exocytotic vesicles to the apical plasma membrane of epithelial cells. This pathway had been shown previously to be insensitive to tetanus neurotoxin and botulinum neurotoxin F. TI-VAMP/VAMP7 is also a good candidate to be implicated in an exocytotic pathway involved in neurite outgrowth because tetanus neurotoxin does not inhibit this process in conditions in which it abolishes neurotransmitter release. We have now found that TI-VAMP/VAMP7 has a widespread distribution in the adult rat brain in which its localization strikingly differs from that of nerve terminal markers. TI-VAMP/VAMP7 does not enrich in synaptic vesicles nor in large dense-core granules but is associated with light membranes. In hippocampal neurons developing in vitro, TI-VAMP/VAMP7 localizes to vesicles in the axonal and dendritic outgrowths and concentrates into the leading edge of the growth cone, a region devoid of synaptobrevin 2, before synaptogenesis. After the onset of synaptogenesis, TI-VAMP/VAMP7 is found predominantly in the somatodendritic domain. In PC12 cells, TI-VAMP/VAMP7 does not colocalize with synaptobrevin 2, chromogranin B, or several markers of endocytic compartments. At the electron microscopic level, TI-VAMP/VAMP7 is mainly associated with tubules and vesicles. Altogether, these results suggest that TI-VAMP/VAMP7 defines a novel membrane compartment in neurite outgrowths and in the somatodendritic domain.

Differentiation of the epithelial apical junctional complex during mouse preimplantation development: a role for rab13 in the early maturation of the tight junction.

We have investigated the mechanisms by which the epithelial apicolateral junctional complex (AJC) is generated during trophectoderm differentiation in the mouse blastocyst using molecular, structural and functional analyses. The mature AJC comprises an apical tight junction (TJ), responsible for intercellular sealing and blastocoel formation, and subjacent zonula adherens E-cadherin/catenin adhesion complex which also extends along lateral membrane contact sites. Dual labelling confocal microscopy revealed that the AJC derived from a single 'intermediate' complex formed following embryo compaction at the 8-cell stage in which the TJ-associated peripheral membrane protein, ZO-1alpha- isoform, was co-localized with both alpha- and beta-catenin. However, following assembly of the TJ transmembrane protein, occludin, from the early 32-cell stage when blastocoel formation begins, ZO-1alpha- and other TJ proteins (ZO-1alpha+ isoform, occludin, cingulin) co-localized in an apical TJ which was separate from a subjacent E-cadherin/catenin zonula adherens complex. Thin-section electron microscopy confirmed that a single zonula adherens-like junctional complex present at the AJC site following compaction matured into a dual TJ and zonula adherens complex at the blastocyst stage. Embryo incubation in the tracer FITC-dextran 4 kDa showed that a functional TJ seal was established coincident with blastocoel formation. We also found that rab13, a small GTPase previously localized to the TJ, is expressed at all stages of preimplantation development and relocates from the cytoplasm to the site of AJC biogenesis from compaction onwards with rab13 and ZO-1alpha- co-localizing precisely. Our data indicate that the segregation of the two elements of the AJC occurs late in trophectoderm differentiation and likely has functional importance in blastocyst formation. Moreover, we propose a role for rab13 in the specification of the AJC site and the formation and segregation of the TJ.

Isoprenylation of Rab proteins possessing a C-terminal CaaX motif.

Rab proteins are small GTPases highly related to the yeast Ypt1 and Sec4 proteins involved in secretion. The Rab proteins were found associated with membranes of different compartments along the secretory and endocytic pathways. They share distinct C-terminal cysteine motifs required for membrane association. Unlike the other Rab proteins, Rab8, Rab11 and Rab13 terminate with a C-terminal CaaX motif similar to those of Ras/Rho proteins. This report demonstrates that Rab8 and Rab13 proteins are isoprenylated in vivo and geranylgeranylated in vitro. Rab11 associates in vitro geranylgeranylpyrophosphate and farnesylpyrophosphate. Our study shows that the CaaX motif is required for isoprenylation.

Biochemical properties of the YPT-related rab1B protein. Comparison with rab1A.

We recently identified a novel rat cDNA: rab1B, closely related to the rab1A cDNA and to the yeast YPT1 gene. The rab1B cDNA encodes a 202 amino acid protein (22.1 kDa) that was produced in Escherichia coli under the control of the phi 10 promoter for the T7 RNA polymerase. The rab1B protein was purified in large amounts to near homogeneity in a simplified procedure. We studied the biochemical properties of rab1B and rab1A proteins. They both bind specifically GTP and GDP and possess intrinsic GTPase activities. The rab1B Lys21----Met mutant protein does not bind GTP, whereas the Ala65----Thr mutant has a reduced GTPase activity and is competent for autophosphorylation in the presence of GTP.

Developmental changes in the localization of the synaptic vesicle protein rab3A in rat brain.

Rab3A is a protein associated with the membrane of synaptic vesicles and is involved in the control of the targeting or docking of these vesicles at the presynaptic membrane for the release of neurotransmitters. Here, we have examined the expression and localization of this protein during the development of the rat brain. Relative to total protein, the concentration of rab3A greatly increased during brain development. Both the intracellular localization of the protein and its cerebral distribution showed an age-dependent shift. In contrast to other synaptic vesicle proteins, rab3A was heavily concentrated in cell bodies when immature neurons were migrating and during early differentiation. Later, the protein disappeared from perikarya and had a diffuse distribution in the neuropil, indicating a redistribution to nerve terminals, its exclusive localization in the adult. In the developing somatosensory cortex, rab3A delimited the modular organization of the barrels well after the afferents have arrived but just around the time that mature synaptic activity has been observed. In the hippocampus, rab3A defined a novel "blob-like" organization of the mossy fibre terminals and its appearance in terminal fields closely preceded the known onset of long-term potentiation. The appearance of rab3A in specific terminal fields during the period of increased physiological activity suggests that this small GTP-binding protein may be an important late element in the establishment of the mature characteristics of the presynaptic terminal.

Localization of the ras-like rab3A protein in the adult rat brain.

Rab3A is a small GTP-binding synaptic vesicle protein, shown to dissociate from synaptic vesicle membranes upon depolarization-induced exocytosis. Using an antiserum raised against rab3A, we found that the antigen was localized to the neuropil of specific brain regions, but was not present in major fiber tracts or most cell bodies. For example, the neuropil of several thalamic nuclei (i.e., dorsal lateral geniculate nucleus, lateral posterior nucleus, ventroposterior nucleus), cerebral cortex, upper layers of the superior colliculus and matrix zones of the neostriatum, were strongly immunoreactive, while the anterior commissure, corpus callosum, optic tract and internal capsule were devoid of staining. The hippocampus, regions of cerebral cortex and the cerebellum exhibited striking laminar distributions of rab3A immunoreactivity. In the hippocampus, dark staining was observed in the stratum oriens, stratum radiatum and molecular layer of the dentate gyrus, while the pyramidal, stratum lacunosum moleculare and dentate granule layers were not stained. In cerebellum the molecular layer and to a lesser extent, the underlying granule cell layer showed enhanced immunoreactivity. Seven days after excitotoxic lesions of the cerebral cortex, rab3A immunoreactivity was diminished in the mirror locus in the contralateral cortical hemisphere and in certain thalamic nuclei ipsilateral to the injection site. These results show that rab3A is localized to a number of specific regions. Its absence from other areas suggests that this synaptic vesicle protein is not universal to all neuronal terminals and pathways. In addition, our lesion studies indicate that for some brain regions, much of the antigen originates in cortical neurons and is distributed within specific axonal projections.

Nucleotide sequence of the chicken proto-oncogene c-erbA corresponding to domain 1 of v-erbA.

The nucleotide sequence of the chicken proto-oncogene c-erbA, the cellular counterpart of the viral oncogene v-erbA domain 1, has been determined. The c-erbA gene has an exon-intron structure characteristic of the eukaryotic split genes. The c-erbA domain 1 is composed of at least eight exons and seven introns. Analysis of the sequence data reveals a long open reading frame of 239 amino acid residues that share highly significant homology with the viral protein. Our results show that the viral erbA oncogene is a truncated form of its cellular homologue. In fact the cellular gene is 42 nucleotides longer at its 5' coding extremity. The presence of a gag-specific (17/20) nucleotide stretch in this region favours the hypothesis of a homologous recombination event between the cellular erbA and the gag gene of a parental retrovirus. We have also sequenced 1400 bp of DNA lying 5' to the long open reading frame in search of the transcription initiation sites for the c-erbA messenger RNAs. Our findings and interpretations are presented here.

Identification of small GTP-binding rab proteins in human platelets: thrombin-induced phosphorylation of rab3B, rab6, and rab8 proteins.

The activation of platelets by specific agonists is a tightly regulated mechanism that leads to the secretion of the dense- and alpha-granule contents. Platelets have been shown to possess small GTP-binding proteins thought to be involved in central biological processes; however, no rab proteins, which may regulate the exocytic process at different stages, have been reported. This study has shown that rab1, rab3B, rab4, rab6, and rab8 proteins, but not rab3A protein, were present in platelets and in endothelial cells. To probe their functional significance in platelets, rab3B, rab6, and rab8 proteins were further characterized with regard to their intracellular localization and their phosphorylation properties. Whereas rab3B protein was found to be mainly cytosolic, rab6 and rab8 proteins were preferentially targeted to the plasma membrane and to the alpha granules. The activation of platelets by thrombin, a potent inducer of secretion, resulted in the phosphorylation of rab3B, rab6, and rab8 proteins, whereas no phosphorylation was observed in the presence of prostaglandin E1, which stimulates cAMP-dependent protein kinase and inhibits the secretion process. These findings provide evidence that members of the subfamily of rab proteins, rab6 and rab8, are localized in platelets to one type of specific secretory vesicle, the alpha granule, and would suggest their possible implication in the secretion process through phosphorylation mechanisms.

The chicken alpha-globin gene domain is transcribed into a 17-kilobase polycistronic RNA.

The 5' start sites and the 3' ends of giant transcripts of the approximately 20-kilobase (kb)-long chicken alpha-globin gene domain were identified by reverse transcription with specific primers and by nuclease S1 mapping using cloned and sequenced restriction fragments of the domain. A transcriptional unit of approximately 17 kb was found that includes all three embryonic and adult genes of the cluster. The largest transcript initiates 8 kb upstream of the gene, within a cluster of A + T-rich sequences placed upstream of a matrix attachment point, at one of several CAA(A)T boxes framing a cluster of four TATA boxes. The 5' ends of a group of 2.5-, 5-, and 12-kb globin transcripts accumulating in avian erythroblastosis virus-transformed cells, which transcribe globin genes abortively, map to the sequence ATATATAATAA 1 kb upstream of the embryonic pi-globin gene. This sequence might correspond to a site of RNA processing or of alternative transcription initiation. Transcription of the domain ends about 2 kb downstream of the last gene of the cluster, downstream of an enhancer and immediately upstream of a CR1 repetitive element in an A + T-rich sequence that includes a matrix attachment site. These data indicate that full-domain transcripts including embryonic as well as adult alpha-globin genes exist, and that the region transcribed is framed by A + T-rich linkers and matrix attachment points.

The human Rab genes encode a family of GTP-binding proteins related to yeast YPT1 and SEC4 products involved in secretion.

Seven cDNA clones corresponding to the rab1, rab2, rab3A, rab3B, rab4, rab5, and rab6 genes were isolated from a human pheochromocytoma cDNA library. They encode 23-25 kDa polypeptides which share approximately 30-50% homology and belong to the ras superfamily. The rab1, rab2, rab3A, and rab4 proteins are the human counterparts of the rat rab gene products that we have previously characterized. Comparison of the seven human rab proteins with the yeast YPT1 (YPT1p) and SEC4 (SEC4p) proteins reveals highly significant sequence similarities. H-rab1p shows 75% amino acid identity with YPT1p and may be therefore considered as its human counterpart. The other proteins share approximately 40% homology with YPT1p and SEC4p. The homology (approximately 30%) between these rab proteins and p21ras is restricted to the four conserved domains involved in the GTP/GDP binding. Human rab proteins were produced in Escherichia coli. Large amounts of rab proteins in soluble form can be extracted and purified without the use of detergents. All six proteins bind GTP and exhibit GTPase activities. A possible involvement of the rab proteins in secretion is discussed.

Expression of the small GTP-binding protein Rab3A in the adult rat brain.

The ras-related rab genes code for small GTP-binding proteins that are thought to control intracellular membrane trafficking. Some of the rab proteins are localized to the membrane of specific subcellular organelles, and rab3A is associated with small synaptic vesicles. We have studied rab3A mRNA expression in the adult rat brain by in situ hybridization. In the forebrain, rab3A mRNAs were mostly detected in neocortical and limbic areas such as hippocampus, enthorinal cortex, or rhinencephalic nuclei, while no significant labeling was observed in the striatum, in the hypothalamus, or in several thalamic nuclei. Rab3A expression does not directly correspond to any known neurotransmitter expression pattern nor is it completely superimposable to the pattern of expression of other synaptic vesicle proteins. These results show that rab3A is expressed and thus exerts its function in a subset of neurons in the brain.