Interaction of membranes of the Golgi complex with microtubules in vitro.
We have developed an in vitro assay for characterizing the binding of elements of the Golgi complex to microtubules. The binding assay comprises three distinct components, Golgi elements purified from Vero cells by subcellular fractionation, taxol-polymerized tubulin from bovine brain coupled to magnetic beads and cytosol from HeLa cells. Binding of Golgi elements to microtubules is quantitated by measuring the activity of the Golgi marker enzyme, galactosyltransferase, associated with the microtubule-coated beads retrieved with a magnet. In the presence of cytosol, 35 to 45% of the total input of galactosyltransferase activity (Golgi elements) bind to microtubules; only 3% of the Golgi elements bind to microtubules, however, in the absence of cytosolic factors. This binding is saturable at a cytosol concentration of approximately 5 mg/ml or at a high input of Golgi elements. Cytosol-stimulated binding of Golgi elements to microtubules is decreased to less than 15% when cytosol is pretreated with 2 mM N-ethylmaleimide (NEM) and it is abolished when cytosolic proteins are inactivated by heat or when microtubules have been coated with heat-stable microtubule-associated proteins (MAPs). Trypsinization of the membranes of the Golgi elements abolishes their ability to bind to microtubules. Furthermore, inactivation of cytoplasmic dynein by UV/vanadate treatment does not affect the binding. This suggests that the interaction of Golgi elements with microtubules depends on NEM-sensitive cytosolic factors and membrane-associated receptors, but not on the microtubule-based motor protein cytoplasmic dynein.
Karecla
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European Molecular Biology Laboratory, Heidelberg, Germany.
Kreis
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D000818:Animals; D001665:Binding Sites / Q000502:physiology; D003600:Cytosol / Q000378:metabolism*; D004398:Dyneins / Q000032:analysis; D005033:Ethylmaleimide / Q000494:pharmacology; D006056:Golgi Apparatus / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008870:Microtubules / Q000378:metabolism*; D009711:Nucleotides / Q000378:metabolism; D014709:Vero Cells
D008869:Microtubule-Associated Proteins; D009711:Nucleotides; D004398:Dyneins; D005033:Ethylmaleimide