title: Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules.
abstract: We have previously reported that purified thyroid lysosomes bind to reconstituted microtubules to form stable complexes (Mithieux, G., Audebet, C., and Rousset, B. (1988) Biochim. Biophys. Acta 969, 121-130), a process which is inhibited by ATP (Mithieux, G., and Rousset, B. (1988) Biochim. Biophys. Acta 971, 29-37). Among detergent-solubilized lysosomal membrane protein, we identified a 50-kDa molecular component which binds to preassembled microtubules. The binding of this polypeptide to microtubules was decreased in the presence of ATP. We purified this 50-kDa protein by affinity chromatography on immobilized ATP. The 50-kDa protein bound to the ATP column was eluted by 1 mM ATP. The purified protein, labeled with 125I, exhibited the ability of interacting with microtubules. The binding process was inhibited by increasing concentrations of ATP, the half-maximal inhibitory effect being obtained at an ATP concentration of 0.35 mM. The interaction of the 50-kDa protein with microtubules is a saturable phenomenon since the binding of the 125I-labeled 50-kDa protein was inhibited by unlabeled solubilized lysosomal membrane protein containing the 50-kDa polypeptide but not by the same protein fraction from which the 50-kDa polypeptide had been removed by the ATP affinity chromatography procedure. The 50-kDa protein has the property to bind to pure tubulin coupled to an insoluble matrix. The 50-kDa protein was eluted from the tubulin affinity column by ATP. These findings support the conclusion that a protein inserted into the lysosomal membrane is able to bind directly to microtubules in a process which can be regulated by ATP. We propose that this protein could account for the association of lysosomes to microtubules demonstrated both in vitro and in intact cells.
authors.affiliation: Institut National de la Santé et de la Recherche Médicale, Unité 197, Faculté de Médecine Alexis Carrel, Lyon, France.
authors.forename: G
authors.initials: G
authors.lastname: Mithieux
authors.forename: B
authors.initials: B
authors.lastname: Rousset
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology*; D000818:Animals; D001345:Autoradiography; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007425:Intracellular Membranes / Q000032:analysis; D007457:Iodine Radioisotopes; D008247:Lysosomes / Q000032:analysis / Q000378:metabolism*; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008970:Molecular Weight; D051381:Rats; D014404:Tubulin / Q000378:metabolism
chemical_list: D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate