Identification of a lysosome membrane protein which could mediate ATP-dependent stable association of lysosomes to microtubules.
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.
Mithieux
G
G
Institut National de la Santé et de la Recherche Médicale, Unité 197, Faculté de Médecine Alexis Carrel, Lyon, France.
Rousset
B
B
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
D007457:Iodine Radioisotopes; D008565:Membrane Proteins; D014404:Tubulin; D000255:Adenosine Triphosphate