RESUMEN
Tea2 is a kinesin family member from Schizosaccharomyces pombe that is targeted to microtubule tips and cell ends in a process that depends on Mal3. Constructs of Tea2 containing the motor domain only or the motor domain plus the N-terminal extension are monomeric, whereas a construct including the first predicted coiled coil region is dimeric. These constructs have a low basal rate of ATP hydrolysis of <0.1 s(-1), but microtubules stimulate the rate of ATP hydrolysis to a maximum of approximately 15 s(-1). Hydrodynamic analysis of Mal3 indicates that it is dimeric. Mal3 is known to associate with Tea2, and analysis with the above Tea2 constructs indicates that the principal site of interaction of Mal3 with Tea2 is the N-terminal extension, although a weaker interaction is also observed with the motor domain alone. In parallel to the binding studies, Mal3 strongly stimulates the ATPase of constructs containing the N-terminal extension by decreasing the K0.5(MT) for stimulation by microtubules but only weakly stimulates motor domains without the N-terminal extension. Mal3 reduces the K0.5(MT) values without affecting the k(cat) value at saturating microtubule level. Binding of Mal3 to microtubules induces an increase in the binding of Tea2 and a reciprocal stimulation of Mal3 binding by Tea2 is also observed. Tea2 is a plus end directed motor that drives sliding of axonemes when adsorbed to a glass surface. The sliding rate is initially unaffected by Mal3, but axonemes stop moving on continued exposure to Mal3.
Asunto(s)
Adenosina Trifosfatasas/química , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/fisiología , Microtúbulos/ultraestructura , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/fisiología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/química , Sitios de Unión , Dimerización , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Hidrólisis , Cinética , Proteínas Asociadas a Microtúbulos/metabolismo , Unión Proteica , Estructura Terciaria de Proteína , Transporte de Proteínas , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Temperatura , Factores de TiempoRESUMEN
Kinesins are microtubule-based motor proteins that transport cargo to specific locations within the cell. However, the mechanisms by which cargoes are directed to specific cellular locations have remained elusive. Here, we investigated the in vivo movement of the Schizosaccharomyces pombe kinesin Tea2 to establish how it is targeted to microtubule tips and cell ends. Tea2 is loaded onto microtubules in the middle of the cell, in close proximity to the nucleus, and then travels using its intrinsic motor activity primarily at the tips of polymerizing microtubules. The microtubule-associated protein Mal3, an EB1 homologue, is required for loading and/or processivity of Tea2 and this function can be substituted by human EB1. In addition, the cell-end marker Tea1 is required to anchor Tea2 to cell ends. Movement of Tea1 and the CLIP170 homologue Tip1 to cell ends is abolished in Tea2 rigor (ATPase) mutants. We propose that microtubule-based transport from the vicinity of the nucleus to cell ends can be precisely regulated, with Mal3 required for loading/processivity, Tea2 for movement and Tea1 for cell-end anchoring.