A role for regulated binding of p150Glued to microtubule plus ends in organelle transport

doi:10.1083/jcb.200201029

Published online 15 July 2002. doi:10.1083/jcb.200201029

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© The Rockefeller University Press, 0021-9525/2002/7/305 $5.00
The Journal of Cell Biology, Volume 158, Number 2, July 22, 2002 305-319

Article

A role for regulated binding of p150^Glued to microtubule plus ends in organelle transport

Patricia S. Vaughan, Pedro Miura, Matthew Henderson, Belinda Byrne and Kevin T. Vaughan

Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556

Address correspondence to Kevin T. Vaughan, Department of Biological Sciences, P.O. Box 369, University of Notre Dame, Notre Dame, IN 46556. Tel.: (574) 631-3733. Fax: (574) 631-7413. E-mail: Vaughan.4{at}nd.edu

A subset of microtubule-associated proteins, including cytoplasmiclinker protein (CLIP)-170, dynactin, EB1, adenomatous polyposiscoli, cytoplasmic dynein, CLASPs, and LIS-1, has been shownrecently to target to the plus ends of microtubules. The mechanismsand functions of this binding specificity are not understood,although a role in encouraging microtubule elongation has beenproposed. To extend previous work on the role of dynactin inorganelle transport, we analyzed p150^Glued by live-cell imaging.Time-lapse analysis of p150^Glued revealed targeting to the plusends of growing microtubules, requiring the NH₂-terminal cytoskeleton-associatedprotein–glycine rich domain, but not EB1 or CLIP-170.Effectors of protein kinase A modulated microtubule bindingand suggested p150^Glued phosphorylation as a factor in plus-endbinding specificity. Using a phosphosensitive monoclonal antibody,we mapped the site of p150^Glued phosphorylation to Ser-19. Invivo and in vitro analysis of phosphorylation site mutants revealedthat p150^Glued phosphorylation mediates dynamic binding to microtubules.To address the function of dynamic binding, we imaged GFP-p150^Gluedduring the dynein-dependent transport of Golgi membranes. Live-cellanalysis revealed a transient interaction between Golgi membranesand GFP-p150^Glued–labeled microtubules just prior to transport,implicating microtubules and dynactin in a search–capturemechanism for minus-end–directed organelles.

Key Words: dynactin; p150^Glued; microtubule; phosphorylation; cytoplasmic dynein

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