Abstract
Syntaxin 4 is a component of the SNARE complex that regulates membrane docking and fusion. Using a yeast two-hybrid screen, we identify a novel interaction between syntaxin 4 and cytoplasmic murine CENPF, a protein previously demonstrated to associate with the microtubule network and SNAP-25. The binding domain for syntaxin 4 in CENPF was defined by yeast two-hybrid assay and co-immunoprecipitation. Confocal analyses in cell culture reveal a high degree of colocalization between endogenously expressed proteins in interphase cells. Additionally, the endogenous SNARE proteins can be isolated as a complex with CENPF in immunoprecipitation experiments. Further analyses demonstrate that murine CENPF and syntaxin 4 colocalize with components of plasma membrane recycling: SNAP-25 and VAMP2. Depletion of endogenous CENPF disrupts GLUT4 trafficking whereas expression of a dominant-negative form of CENPF inhibits cell coupling. Taken together, these studies demonstrate that CENPF provides a direct link between proteins of the SNARE system and the microtubule network and indicate a diverse role for murine CENPF in vesicular transport.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biological Transport / physiology
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COS Cells
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Cell Membrane / genetics
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Cell Membrane / metabolism
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Chlorocebus aethiops
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Chromosomal Proteins, Non-Histone / genetics
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Chromosomal Proteins, Non-Histone / metabolism*
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Cytoplasm / genetics
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Cytoplasm / metabolism*
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Glucose Transporter Type 4 / genetics
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Glucose Transporter Type 4 / metabolism
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Mice
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Microfilament Proteins / genetics
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Microfilament Proteins / metabolism*
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Microtubules / genetics
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Microtubules / metabolism
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NIH 3T3 Cells
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Qa-SNARE Proteins / genetics
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Qa-SNARE Proteins / metabolism*
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Saccharomyces cerevisiae / genetics
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Synaptosomal-Associated Protein 25 / genetics
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Synaptosomal-Associated Protein 25 / metabolism
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Transport Vesicles / genetics
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Transport Vesicles / metabolism*
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Two-Hybrid System Techniques
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Vesicle-Associated Membrane Protein 2 / genetics
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Vesicle-Associated Membrane Protein 2 / metabolism
Substances
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Chromosomal Proteins, Non-Histone
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Glucose Transporter Type 4
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Microfilament Proteins
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Qa-SNARE Proteins
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Slc2a4 protein, mouse
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Synaptosomal-Associated Protein 25
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Vesicle-Associated Membrane Protein 2
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centromere protein F
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vesicle-associated membrane protein 2, mouse