A 29-Kilodalton Golgi SolubleN-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 α soluble N-ethylmaleimide-sensitive factor attachment protein (GST-α-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.

[1]  G. Warren,et al.  Antibodies to the Golgi complex and the rough endoplasmic reticulum , 1982, The Journal of cell biology.

[2]  S. Whiteheart,et al.  SNAPs and NSF: general members of the fusion apparatus. , 1995, Trends in cell biology.

[3]  Richard H Schaller.  Membrane trafficking in the presynaptic nerve terminal , 1995, Neuron.

[4]  George Palade,et al.  Intracellular Aspects of the Process of Protein Synthesis , 1975, Science.

[5]  R. Scheller,et al.  Protein Interactions Regulating Vesicle Transport between the Endoplasmic Reticulum and Golgi Apparatus in Mammalian Cells , 1997, Cell.

[6]  H. Pelham,et al.  SED5 encodes a 39-kD integral membrane protein required for vesicular transport between the ER and the Golgi complex , 1992, The Journal of cell biology.

[7]  R. Scheller,et al.  Seven Novel Mammalian SNARE Proteins Localize to Distinct Membrane Compartments* , 1998, The Journal of Biological Chemistry.

[8]  R. Schekman,et al.  Vesicle-mediated protein sorting. , 1992, Annual review of biochemistry.

[9]  C M Dobson,et al.  Protein Folding Monitored at Individual Residues During a Two-Dimensional NMR Experiment , 1996, Science.

[10]  Yue Xu,et al.  Syntaxin 7, a Novel Syntaxin Member Associated with the Early Endosomal Compartment* , 1998, The Journal of Biological Chemistry.

[11]  J. Rothman,et al.  A v-SNARE implicated in intra-Golgi transport , 1996, The Journal of cell biology.

[12]  Reinhard Jahn,et al.  Vesicle fusion from yeast to man , 1994, Nature.

[13]  J. Rothman,et al.  A rab protein is required for the assembly of SNARE complexes in the docking of transport vesicles , 1994, Cell.

[14]  W. Hong,et al.  N-Ethylmaleimide-sensitive Factor (NSF) and α-Soluble NSF Attachment Proteins (SNAP) Mediate Dissociation of GS28-Syntaxin 5 Golgi SNAP Receptors (SNARE) Complex* , 1997, The Journal of Biological Chemistry.

[15]  R. Schekman,et al.  Coat Proteins and Vesicle Budding , 1996, Science.

[16]  R. Scheller,et al.  A New Syntaxin Family Member Implicated in Targeting of Intracellular Transport Vesicles* , 1996, Journal of Biological Chemistry.

[17]  J. Rothman,et al.  Implications of the SNARE hypothesis for intracellular membrane topology and dynamics , 1994, Current Biology.

[18]  Paul Tempst,et al.  SNAP receptors implicated in vesicle targeting and fusion , 1993, Nature.

[19]  W. Balch,et al.  Syntaxin 5 regulates endoplasmic reticulum to Golgi transport. , 1994, The Journal of biological chemistry.

[20]  S. Emr,et al.  Novel syntaxin homologue, Pep12p, required for the sorting of lumenal hydrolases to the lysosome-like vacuole in yeast. , 1996, Molecular biology of the cell.

[21]  S. Wong,et al.  The mammalian ARF-like protein 1 (Arl1) is associated with the Golgi complex. , 1996, Journal of cell science.

[22]  J. Rothman,et al.  Mechanisms of intracellular protein transport , 1994, Nature.

[23]  T. Stevens,et al.  A Human Homolog Can Functionally Replace the Yeast Vesicle-associated SNARE Vti1p in Two Vesicle Transport Pathways* , 1998, The Journal of Biological Chemistry.

[24]  J. McNew,et al.  Characterization of a novel yeast SNARE protein implicated in Golgi retrograde traffic. , 1997, Molecular biology of the cell.

[25]  J. Rothman,et al.  Protein Sorting by Transport Vesicles , 1996, Science.

[26]  S. Pfeffer.  Transport vesicle docking: SNAREs and associates. , 1996, Annual review of cell and developmental biology.

[27]  B. Tang,et al.  The Mammalian Protein (rbet1) Homologous to Yeast Bet1p Is Primarily Associated with the Pre-Golgi Intermediate Compartment and Is Involved in Vesicular Transport from the Endoplasmic Reticulum to the Golgi Apparatus , 1997, The Journal of cell biology.

[28]  R. Scheller,et al.  Syntaxin 6 functions in trans-Golgi network vesicle trafficking. , 1997, Molecular biology of the cell.

[29]  R. Pepperkok,et al.  β-COP is essential for biosynthetic membrane transport from the endoplasmic reticulum to the Golgi complex in vivo , 1993, Cell.

[30]  B. Tang,et al.  Monoclonal antibody HFD9 identifies a novel 28 kDa integral membrane protein on the cis-Golgi. , 1995, Journal of cell science.

[31]  J. Dixon,et al.  Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. , 1991, Analytical biochemistry.

[32]  S. Wong,et al.  GS15, a 15-Kilodalton Golgi SolubleN-Ethylmaleimide-sensitive Factor Attachment Protein Receptor (SNARE) Homologous to rbet1* , 1997, The Journal of Biological Chemistry.

[33]  H. Pelham,et al.  Localization of Sed5, a putative vesicle targeting molecule, to the cis- Golgi network involves both its transmembrane and cytoplasmic domains , 1994, The Journal of cell biology.

[34]  J. Lippincott-Schwartz,et al.  Brefeldin A: insights into the control of membrane traffic and organelle structure , 1992, The Journal of cell biology.

[35]  T. Stevens,et al.  The Yeast v-SNARE Vti1p Mediates Two Vesicle Transport Pathways through Interactions with the t-SNAREs Sed5p and Pep12p , 1997, The Journal of cell biology.

[36]  Thomas C. Südhof,et al.  The synaptic vesicle cycle: a cascade of protein–protein interactions , 1995, Nature.

[37]  H. Pelham,et al.  Two syntaxin homologues in the TGN/endosomal system of yeast , 1998, The EMBO journal.

[38]  V. Subramaniam,et al.  A SNARE involved in protein transport through the Golgi apparatus , 1997, Nature.

[39]  P. Robbins,et al.  Isolation, characterization, and expression of cDNAs encoding murine alpha-mannosidase II, a Golgi enzyme that controls conversion of high mannose to complex N-glycans , 1991, The Journal of cell biology.

[40]  R. Scheller,et al.  The syntaxin family of vesicular transport receptors , 1993, Cell.