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B L Tang

Publications and source records attributed to B L Tang.

At least 19 recordsLinked to original sources
PubMed2001

The syntaxins.

SUMMARY: The SNARE hypothesis predicts that a family of SNAP receptors are localized to and function in diverse intracellular membrane compartments where membrane fusion processes take place. Syntaxins, the prototype family of SNARE proteins, have a carboxy-terminal tail-anchor and multiple coiled-coil domains. There are 15 members of the syntaxin family in the human genome and 7 syntaxin-like genes in the yeast Saccharomyces cerevisiae. In conjunction with other SNAREs and with the cytoplasmic NSF and SNAP proteins, syntaxins mediate vesicle fusion in diverse vesicular transport processes along the exocytic and the endocytic pathway. They are crucial components that both drive and provide specificity to the myriad vesicular fusion processes that characterize the eukaryotic cell.

Evolution, Molecular↗
PubMed2001

A membrane protein enriched in endoplasmic reticulum exit sites interacts with COPII.

Although all mammalian COPII components have now been cloned, little is known of their interactions with other regulatory proteins involved in exit from the endoplasmic reticulum (ER). We report here that a mammalian protein (Yip1A) that is about 31% identical to S. cerevisiae and which interacts with and modulates COPII-mediated ER-Golgi transport. Yip1A transcripts are ubiquitously expressed. Transcripts of a related mammalian homologue, Yip1B, are found specifically in the heart. Indirect immunofluorescence microscopy revealed that Yip1A is localized to vesicular structures that are concentrated at the perinuclear region. The structures marked by Yip1A co-localized with Sec31A and Sec13, components of the COPII coat protein complex. Immunoelectron microscopy also showed that Yip1A co-localizes with Sec13 at ER exit sites. Overexpression of the hydrophilic N terminus of Yip1A arrests ER-Golgi transport of the vesicular stomatitis G protein and causes fragmentation and dispersion of the Golgi apparatus. A glutathione S-transferase fusion protein with the hydrophilic N terminus of Yip1A (GST-Yip1A) is able to bind to and deplete vital components from rat liver cytosol that is essential for in vitro vesicular stomatitis G transport. Peptide sequence analysis of cytosolic proteins that are specifically bound to GST-Yip1A revealed, among other proteins, mammalian COPII components Sec23 and Sec24. A highly conserved domain at the N terminus of Yip1A is required for Sec23/Sec24 interaction. Our results suggest that Yip1A is involved in the regulation of ER-Golgi traffic at the level of ER exit sites.

Amino Acid Sequence↗
PubMed2001

ADAMTS: a novel family of extracellular matrix proteases.

ADAMTS (a disintegrin and metalloprotease with thrombospondin motifs) is a novel family of extracellular proteases found in both mammals and invertebrates. Members of the family may be distinguished from the ADAM (a disintegrin and metalloprotease) family members based on the multiple copies of thrombospondin 1-like repeats they carry. With at least nine members in mammals alone, the ADAMTS family members are predicted by their structural domains to be extracellular matrix (ECM) proteins with a wide range of activities and functions distinct from members of the ADAM family that are largely anchored on the cell surface. ADAMTS2 is a procollagen N-proteinase, and the mutations of its gene are responsible for Human Ehlers-Danlos syndrome type VII C and bovine dermatosparaxis. ADAMTS4 and ADAMTS5 are aggrecanases implicated in the degradation of cartilage aggrecan in arthritic diseases. Other members of the ADAMTS family have also been implicated in roles during embryonic development and angiogenesis. Current and future studies on this emerging group of ECM proteases may provide important insights into developmental or pathological processes involving ECM remodeling.

ADAM Proteins↗
PubMed2001

Evidence for prebudding arrest of ER export in animal cell mitosis and its role in generating Golgi partitioning intermediates.

During mitosis the interconnected Golgi complex of animal cells breaks down to produce both finely dispersed elements and discrete vesiculotubular structures. The endoplasmic reticulum (ER) plays a controversial role in generating these partitioning intermediates and here we highlight the importance of mitotic ER export arrest in this process. We show that experimental inhibition of ER export (by microinjecting dominant negative Sar1 mutant proteins) is sufficient to induce and maintain transformation of Golgi cisternae to vesiculotubular remnants during interphase and telophase, respectively. We also show that buds on the ER, ER exit sites and COPII vesicles are markedly depleted in mitotic cells and COPII components Sec23p, Sec24p, Sec13p and Sec31p redistribute into the cytosol, indicating ER export is inhibited at an early stage. Finally, we find a markedly uneven distribution of Golgi residents over residual exit sites of metaphase cells, consistent with tubulovesicular Golgi remnants arising by fragmentation rather than redistribution via the ER. Together, these results suggest selective recycling of Golgi residents, combined with prebudding cessation of ER export, induces transformation of Golgi cisternae to vesiculotubular remnants in mitotic cells. The vesiculotubular Golgi remnants, containing populations of slow or nonrecycling Golgi components, arise by fragmentation of a depleted Golgi ribbon independently from the ER.

COP-Coated Vesicles↗
PubMed2001

Protein trafficking mechanisms associated with neurite outgrowth and polarized sorting in neurons.

Neuronal differentiation in vitro and in vivo involves coordinated changes in the cellular cytoskeleton and protein trafficking processes. I review here recent progress in our understanding of the membrane trafficking aspects of neurite outgrowth of neurons in culture and selective microtubule-based polarized sorting in fully polarized neurons, focusing on the involvement of some key molecules. Early neurite outgrowth appears to involve the protein trafficking machineries that are responsible for constitutive trans-Golgi network (TGN) to plasma membrane exocytosis, utilizing transport carrier generation mechanisms, SNARE proteins, Rab proteins and tethering mechanisms that are also found in non-neuronal cells. This vectorial TGN-plasma membrane traffic is directed towards several neurites, but can be switch to concentrate on the growth of a single axon. In a mature neuron, polarized targeting to the specific axonal and dendritic domains appears to involve selective microtubule-based mechanisms, utilizing motor proteins capable of distinguishing microtubule tracks to different destinations. The apparent gaps in our knowledge of these related protein transport processes will be highlighted.

Animals↗
PubMed2001

Cellular COPII proteins are involved in production of the vesicles that form the poliovirus replication complex.

Poliovirus (PV) replicates its genome in association with membranous vesicles in the cytoplasm of infected cells. To elucidate the origin and mode of formation of PV vesicles, immunofluorescence labeling with antibodies against the viral vesicle marker proteins 2B and 2BC, as well as cellular markers of the endoplasmic reticulum (ER), anterograde transport vesicles, and the Golgi complex, was performed in BT7-H cells. Optical sections obtained by confocal laser scanning microscopy were subjected to a deconvolution process to enhance resolution and signal-to-noise ratio and to allow for a three-dimensional representation of labeled membrane structures. The mode of formation of the PV vesicles was, on morphological grounds, similar to the formation of anterograde membrane traffic vesicles in uninfected cells. ER-resident membrane markers were excluded from both types of vesicles, and the COPII components Sec13 and Sec31 were both found to be colocalized on the vesicular surface, indicating the presence of a functional COPII coat. PV vesicle formation during early time points of infection did not involve the Golgi complex. The expression of PV protein 2BC or the entire P2 and P3 genomic region led to the production of vesicles carrying a COPII coat and showing the same mode of formation as vesicles produced after PV infection. These results indicate that PV vesicles are formed at the ER by the cellular COPII budding mechanism and thus are homologous to the vesicles of the anterograde membrane transport pathway.

Animals↗
PubMed2000

Mammalian homologues of yeast sec31p. An ubiquitously expressed form is localized to endoplasmic reticulum (ER) exit sites and is essential for ER-Golgi transport.

The yeast coat protein II (COPII) is responsible for vesicle budding from the endoplasmic reticulum (ER). Mammalian functional homologues for all yeast COPII components, except for Sec31p, have been reported. We have cloned a mammalian cDNA whose product (Sec31A) is about 26% identical to Saccharomyces cerevisiae Sec31p. Data base searches also revealed another partial sequence encoding a polypeptide (Sec31B) that is 40% identical to Sec31A. Northern analysis revealed that Sec31A transcripts are ubiquitously and abundantly expressed, while Sec31B transcripts are particularly enriched in the testis and thymus, but present in very low levels in other tissues. Sec31A is localized to vesicular structures that scatter throughout the cell but are concentrated at the perinuclear region. The structures marked by Sec31A contain Sec13, a component of COPII that is well characterized to mark the ER exit sites. Immunoelectron microscopy revealed that Sec31A colocalizes with Sec13 in structures with extensive vesicular-tubular profiles. Antibodies raised against a C-terminal portion of Sec31A co-precipitate Sec13 and inhibit ER-Golgi transport of temperature-arrested vesicular stomatitis G protein in a semi-intact cell assay. Cytosol immunodepleted of Sec31A failed to support vesicular stomatitis G protein transport, which can be rescued by a high molecular weight fraction of the cytosol containing both Sec31A and Sec13. We conclude that Sec31A represents a functional mammalian homologue of yeast Sec31p.

Amino Acid Sequence↗
PubMed1999

A family of mammalian proteins homologous to yeast Sec24p.

The Sec23p/Sec24p complex is a component of yeast coat protein II (COPII), the coat protein complex responsible for vesicle budding from the endoplasmic reticulum (ER). Database searches and molecular cloning reveal that four different mammalian Sec24p-like proteins exist, all with about 20% amino acid identity with the yeast Sec24p. Sec24A and Sec24B share about 50% amino acid identity. Sec24D is cloned by screening a human pancreas of cDNA library with an expressed sequence tag (EST) fragment that is homologous to, but distinct from, Sec24A and Sec24B. Sec24D shares about 50% amino acid identity with the gene product of KIAA0079, which we have designated as Sec24C. These mammalian Sec24s appear to form two subclasses based on homology. Sec24A/B and Sec24C/D share about 20% identity with each other and with the yeast Sec24p. The Sec24 sequences also share weak but significant homology to the mammalian Sec23A and Sec23B. Northern blot analysis revealed that Sec24C is ubiquitously expressed. Although Sec24D transcripts are detectable in all tissues examined, they are selectively enriched in certain tissues, particularly placenta and pancreas. myc-tagged Sec24C and sec24D colocalized with Sec13, another COPII component. This colocalization suggests that Sec24C and Sec24D are indeed associated with COPII structures on membranes of the ER-Golgi boundary. The existence of at least four forms of Sec24 in mammalian cells suggest that multiple forms of COPII complex may be involved in ER export.

Amino Acid Sequence↗
PubMed1999

Morphological and functional association of Sec22b/ERS-24 with the pre-Golgi intermediate compartment.

Yeast Sec22p participates in both anterograde and retrograde vesicular transport between the endoplasmic reticulum (ER) and the Golgi apparatus by functioning as a v-SNARE (soluble N-ethylmaleimide-sensitive factor [NSF] attachment protein receptor) of transport vesicles. Three mammalian proteins homologous to Sec22p have been identified and are referred to as Sec22a, Sec22b/ERS-24, and Sec22c, respectively. The existence of three homologous proteins in mammalian cells calls for detailed cell biological and functional examinations of each individual protein. The epitope-tagged forms of all three proteins have been shown to be primarily associated with the ER, although functional examination has not been carefully performed for any one of them. In this study, using antibodies specific for Sec22b/ERS-24, it is revealed that endogenous Sec22b/ERS-24 is associated with vesicular structures in both the perinuclear Golgi and peripheral regions. Colabeling experiments for Sec22b/ERS-24 with Golgi mannosidase II, the KDEL receptor, and the envelope glycoprotein G (VSVG) of vesicular stomatitis virus (VSV) en route from the ER to the Golgi under normal, brefeldin A, or nocodazole-treated cells suggest that Sec22b/ERS-24 is enriched in the pre-Golgi intermediate compartment (IC). In a well-established semi-intact cell system that reconstitutes transport from the ER to the Golgi, transport of VSVG is inhibited by antibodies against Sec22b/ERS-24. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle/transport intermediate docking but before the actual fusion event. Antibodies against Sec22b/ERS-24 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. Transport of VSVG accumulated in pre-Golgi IC by incubation at 15 degreesC is also inhibited by Sec22b/ERS-24 antibodies. Morphologically, VSVG is transported from the ER to the Golgi apparatus via vesicular intermediates that scatter in the peripheral as well as the Golgi regions. In the presence of antibodies against Sec22b/ERS-24, VSVG is seen to accumulate in these intermediates, suggesting that Sec22b/ERS-24 functions at the level of the IC in ER-Golgi transport.

Animals↗
PubMed1999

Localization and recycling of gp27 (hp24gamma3): complex formation with other p24 family members.

We report here the characterization of gp27 (hp24gamma3), a glycoprotein of the p24 family of small and abundant transmembrane proteins of the secretory pathway. Immunoelectron and confocal scanning microscopy show that at steady state, gp27 localizes to the cis side of the Golgi apparatus. In addition, some gp27 was detected in COPI- and COPII-coated structures throughout the cytoplasm. This indicated cycling that was confirmed in three ways. First, 15 degrees C temperature treatment resulted in accumulation of gp27 in pre-Golgi structures colocalizing with anterograde cargo. Second, treatment with brefeldin A caused gp27 to relocate into peripheral structures positive for both KDEL receptor and COPII. Third, microinjection of a dominant negative mutant of Sar1p trapped gp27 in the endoplasmic reticulum (ER) by blocking ER export. Together, this shows that gp27 cycles extensively in the early secretory pathway. Immunoprecipitation and coexpression studies further revealed that a significant fraction of gp27 existed in a hetero-oligomeric complex. Three members of the p24 family, GMP25 (hp24alpha2), p24 (hp24beta1), and p23 (hp24delta1), coprecipitated in what appeared to be stochiometric amounts. This heterocomplex was specific. Immunoprecipitation of p26 (hp24gamma4) failed to coprecipitate GMP25, p24, or p23. Also, very little p26 was found coprecipitating with gp27. A functional requirement for complex formation was suggested at the level of ER export. Transiently expressed gp27 failed to leave the ER unless other p24 family proteins were coexpressed. Comparison of attached oligosaccharides showed that gp27 and GMP25 recycled differentially. Only a very minor portion of GMP25 displayed complex oligosaccharides. In contrast, all of gp27 showed modifications by medial and trans enzymes at steady state. We conclude from these data that a portion of gp27 exists as hetero-oligomeric complexes with GMP25, p24, and p23 and that these complexes are in dynamic equilibrium with individual p24 proteins to allow for differential recycling and distributions.

Amino Acid Sequence↗
PubMed1999

Forward and retrograde trafficking in mitotic animal cells. ER-Golgi transport arrest restricts protein export from the ER into COPII-coated structures.

Protein transport arrest occurs between the ER and Golgi stack of mitotic animal cells, but the location of this block is unknown. In this report we use the recycling intermediate compartment protein ERGIC 53/p58 and the plasma membrane protein CD8 to establish the site of transport arrest. Recycled ERGIC 53/p58 and newly synthesised CD8 accumulate in ER cisternae but not in COPII-coated export structures or more distal sites. During mitosis the tubulovesicular ER-related export sites were depleted of the COPII component Sec13p, as shown by immunoelectron microscopy, indicating that COPII budding structures are the target for mitotic inhibition. The extent of recycling of Golgi stack residents was also investigated. In this study we used oligosaccharide modifications on CD8 trapped in the ER of mitotic cells as a sensitive assay for recycling of Golgi stack enzymes. We find that modifications conferred by the Golgi stack-resident GalNac transferase do occur on newly synthesised CD8, but these modifications are entirely due to newly synthesised transferase rather than to enzyme recycled from the Golgi stack. Taken together our findings establish for the first time that the site of ER-Golgi transport arrest of mitotic cells is COPII budding structures, and they clearly speak against a role for recycling in partitioning of Golgi stack proteins via translocation to the ER.

Animals↗
PubMed1998

A 29-kilodalton Golgi soluble N-ethylmaleimide-sensitive factor attachment protein receptor (Vti1-rp2) implicated in protein trafficking in the secretory pathway.

Expressed sequence tags coding for a potential SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) were revealed during data base searches. The deduced amino acid sequence of the complete coding region predicts a 217-residue protein with a COOH-terminal hydrophobic membrane anchor. Affinity-purified antibodies raised against the cytoplasmic region of this protein specifically detect a 29-kilodalton integral membrane protein enriched in the Golgi membrane. Indirect immunofluorescence microscopy reveals that this protein is mainly associated with the Golgi apparatus. When detergent extracts of the Golgi membrane are incubated with immobilized glutathione S-transferase alpha soluble N-ethylmaleimide-sensitive factor attachment protein (GST-alpha-SNAP), this protein was specifically retained. This protein has been independently identified and termed Vti1-rp2, and it is homologous to Vti1p, a yeast Golgi SNARE. We further show that Vti1-rp2 can be qualitatively coimmunoprecipitated with Golgi syntaxin 5 and syntaxin 6, suggesting that Vti1-rp2 exists in at least two distinct Golgi SNARE complexes. In cells microinjected with antibodies against Vti1-rp2, transport of the envelope protein (G-protein) of vesicular stomatitis virus from the endoplasmic reticulum to the plasma membrane was specifically arrested at the Golgi apparatus, providing further evidence for functional importance of Vti1-rp2 in protein trafficking in the secretory pathway.

Amino Acid Sequence↗
PubMed1998

Syntaxin 11: a member of the syntaxin family without a carboxyl terminal transmembrane domain.

We have cloned a novel syntaxin-like molecule, designated human syntaxin 11 (hsyn11). The open reading frame encodes a polypeptide of 287 amino acids with potential coiled-coil domains. hsyn11 has extensive homology to members of the syntaxin family, particularly syntaxin 1 and syntaxin 2. Unlike other members of the syntaxin family, however, hsyn11 has a short cysteinerich carboxyl-terminal tail but not a typical hydrophobic domain which may serve as a membrane anchor. Northern blot analysis revealed two transcripts of approximately 0.8 kb and approximately 1.7 kb in length that are particularly abundant in heart and placenta, although lower levels were also detectable in other tissues except in the brain. Consistent with the lack of a distinct membrane anchorage sequence in hsyn11, indirect immunofluorescence microscopy of transiently expressed N-terminally myc-tagged hsyn11 revealed a diffuse, cytoplasmic labeling.

Amino Acid Sequence↗
PubMed1998

Syntaxin 12, a member of the syntaxin family localized to the endosome.

We have cloned a new member of the syntaxin family of proteins. The open reading frame encodes a polypeptide of 272 amino acids with potential coiled-coil domains and a C-terminal hydrophobic tail. Northern blot analysis showed that the transcript is fairly ubiquitous. A soluble recombinant form of the polypeptide without the hydrophobic region binds to alpha-SNAP (soluble N-ethylmaleimide-sensitive factor attachment protein) and syndet/SNAP-23 in vitro. Polyclonal antibody raised against the recombinant protein recognized a 39-kDa protein in the membrane fraction of cell lysates. Indirect immunofluorescence studies using the polyclonal antibody showed that the protein is localized to intracellular membrane structures. Selective permeabilization studies with digitonin and saponin indicate that the epitope(s) recognized by the antibody is expose to the cytoplasm, consistent with the predicted orientation characteristic of SNAP receptor molecules. Morphological alterations of the staining pattern of the protein with brefeldin A and wortmannin treatment indicate that the protein is localize to the endosome. The cDNA we have cloned apparently corresponded to three previously described expressed sequence tags named as syntaxins 12, 13, and 14, respectively. We therefore propose to retain the name syntaxin 12 for this protein.

Amino Acid Sequence↗
PubMed1998

Hsec22c: a homolog of yeast Sec22p and mammalian rsec22a and msec22b/ERS-24.

We have cloned a new member of a family of mammalian proteins homologous to Sec22p, a v-SNARE in Saccharomyces cerevisiae required for transport between the endoplasmic reticulum (ER) and the Golgi apparatus. The open reading frame encodes a polypeptide of 250 amino acids which is homologous to, but obviously different from, the recently reported mammalian Sec22p homologs rat sec22a, mouse sec22b, and hamster ERS-24. Northern blot analysis revealed two transcripts of about 1 and 5 kb respectively which are ubiquitously expressed. myc-epitope tagged sec22c is localized to the ER. Overexpression of the myc-tagged protein resulted in an anomalous staining pattern of SNARE molecules participating in ER-Golgi transport such as syntaxin 5 and mammalian bet1, but not the endosomal SNARE syntaxin 7. The presence of multiple forms of sec22 protein in the mammalian early secretory pathway is in-line with task specification in a highly elaborate transport machinery.

Amino Acid Sequence↗
PubMed1998

Molecular cloning and localization of human syntaxin 16, a member of the syntaxin family of SNARE proteins.

We have cloned a new member of the syntaxin family of proteins, designated human syntaxin 16 (hsyn16). The open reading frame encodes a polypeptide of 307 amino acids with potential coiled-coil domains and a carboxy-terminal hydrophobic tail, which is characteristic of other members of the syntaxin family. The encoded polypeptide bears sequence homology to known syntaxin molecules. Northern blot analysis revealed a single transcript that is fairly ubiquitous, being slightly more enriched in heart and pancreas. Indirect immunofluorescence localised myc-tagged hsyn16 (myc-hsyn16) to the Golgi apparatus, colocalizing well with lens culinaris agglutinin, an established Golgi marker, as well as with other Golgi SNAREs such as GS28 and syntaxin 5. Myc-hsyn16 is redistributed to the endoplasmic reticulum upon brefeldin A treatment, indicating that it is localised to the Golgi stack. The ubiquitous expression and Golgi localization of hsy16 suggest that it is involved in a vesicular transport step within the organelle.

Amino Acid Sequence↗