Localization of plasma membrane t-SNAREs syntaxin 2 and 3 in intracellular compartments

BackgroundMembrane fusion requires the formation of a complex between a vesicle protein (v-SNARE) and the target membrane proteins (t-SNAREs). Syntaxin 2 and 3 are t-SNAREs that, according to previous over-expression studies, are predominantly localized at the plasma membrane. In the present study we investigated localization of the endogenous syntaxin 2 and 3.ResultsEndogenous syntaxin 2 and 3 were found in NRK cells in intracellular vesicular structures in addition to regions of the plasma membrane. Treatment of these cells with N-ethylmaleimide (NEM), which is known to inactivate membrane fusion, caused syntaxin 3 to accumulate in the trans-Golgi network and syntaxin 2 in perinuclear membrane vesicles. Kinetic analysis in the presence of NEM indicated that this redistribution of syntaxin 2 and 3 takes place via actin containing structures.ConclusionOur data suggest that syntaxin 2 cycles between the plasma membrane and the perinuclear compartment whereas syntaxin 3 cycles between the plasma membrane and the trans-Golgi network. It is possible that this cycling has an important role in the regulation of t-SNARE function.

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

[2]  J. Partanen,et al.  The three mouse actin-depolymerizing factor/cofilins evolved to fulfill cell-type-specific requirements for actin dynamics. , 2002, Molecular biology of the cell.

[3]  T. Galli,et al.  A novel tetanus neurotoxin-insensitive vesicle-associated membrane protein in SNARE complexes of the apical plasma membrane of epithelial cells. , 1998, Molecular biology of the cell.

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

[5]  S. Grinstein,et al.  Characterization and subcellular localization of target membrane soluble NSF attachment protein receptors (t-SNAREs) in macrophages. Syntaxins 2, 3, and 4 are present on phagosomal membranes. , 1996, Journal of immunology.

[6]  T. Galli,et al.  Interaction of Munc-18-2 with syntaxin 3 controls the association of apical SNAREs in epithelial cells. , 1998, Journal of cell science.

[7]  T. Weimbs,et al.  Intracellular redirection of plasma membrane trafficking after loss of epithelial cell polarity. , 2000, Molecular biology of the cell.

[8]  The specificity of SNARE-dependent fusion is encoded in the SNARE motif. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[9]  S. Pfeffer,et al.  Rab GTPases: specifying and deciphering organelle identity and function. , 2001, Trends in cell biology.

[10]  J. Rothman,et al.  Purification of an N-ethylmaleimide-sensitive protein catalyzing vesicular transport. , 1988, Proceedings of the National Academy of Sciences of the United States of America.

[11]  W. Antonin,et al.  Identification of SNAREs Involved in Regulated Exocytosis in the Pancreatic Acinar Cell* , 1999, The Journal of Biological Chemistry.

[12]  D. Dauzonne,et al.  Actin filaments and myosin I alpha cooperate with microtubules for the movement of lysosomes. , 2001, Molecular biology of the cell.

[13]  M. Bennett,et al.  Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells. , 1996, Molecular biology of the cell.

[14]  J. Rothman,et al.  Compartmental specificity of cellular membrane fusion encoded in SNARE proteins , 2000, Nature.

[15]  R. Watson,et al.  Transmembrane domain length determines intracellular membrane compartment localization of syntaxins 3, 4, and 5. , 2001, American journal of physiology. Cell physiology.

[16]  J. Bamburg.  Proteins of the ADF/cofilin family: essential regulators of actin dynamics. , 1999, Annual review of cell and developmental biology.

[17]  E. Kuismanen,et al.  Inhibition of the membrane fusion machinery prevents exit from the TGN and proteolytic processing by furin , 2001, FEBS letters.

[18]  A. Durrbach,et al.  Actin filaments facilitate two steps of endocytosis. , 1996, Journal of cell science.

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

[20]  M. Farquhar,et al.  Immunoperoxidase methods for the localization of antigens in cultured cells and tissue sections by electron microscopy. , 1989, Methods in cell biology.

[21]  V. Olkkonen,et al.  Endogenous plasma membrane t‐SNARE syntaxin 4 is present in rab11 positive endosomal membranes and associates with cortical actin cytoskeleton , 2002, FEBS Letters.

[22]  F. Barr,et al.  Membrane Fusion: Caught in a Trap , 2004, Current Biology.

[23]  Helen L. Yin,et al.  The Actin Cytoskeleton Is Required for Receptor-mediated Endocytosis in Mammalian Cells* , 1997, The Journal of Biological Chemistry.

[24]  P. Detmers,et al.  7-Chloro-4-nitrobenzeno-2-oxa-1,3-diazole actin as a probe for actin polymerization. , 1981, The Journal of biological chemistry.

[25]  A. Brunger,et al.  Conserved structural features of the synaptic fusion complex: SNARE proteins reclassified as Q- and R-SNAREs. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. Llorens,et al.  Syntaxin 1A and 1B display distinct distribution patterns in the rat peripheral nervous system , 1999, Neuroscience.

[27]  J. Bonifacino,et al.  The Mechanisms of Vesicle Budding and Fusion , 2004, Cell.

[28]  T. Südhof,et al.  Selective Interaction of Complexin with the Neuronal SNARE Complex , 2000, The Journal of Biological Chemistry.

[29]  K. Sakimura,et al.  Identification of four different forms of syntaxin 3. , 1995, Biochemical and biophysical research communications.

[30]  Reinhard Jahn,et al.  Crystal structure of a SNARE complex involved in synaptic exocytosis at 2.4 Å resolution , 1998, Nature.

[31]  V. Olkkonen,et al.  Syntaxin 2 splice variants exhibit differential expression patterns, biochemical properties and subcellular localizations. , 1999, Journal of cell science.

[32]  J. Rothman,et al.  A multisubunit particle implicated in membrane fusion , 1992, The Journal of cell biology.