A role for regulated binding of p150(Glued) to microtubule plus ends in organelle transport.
A subset of microtubule-associated proteins, including cytoplasmic linker protein (CLIP)-170, dynactin, EB1, adenomatous polyposis coli, cytoplasmic dynein, CLASPs, and LIS-1, has been shown recently to target to the plus ends of microtubules. The mechanisms and functions of this binding specificity are not understood, although a role in encouraging microtubule elongation has been proposed. To extend previous work on the role of dynactin in organelle transport, we analyzed p150(Glued) by live-cell imaging. Time-lapse analysis of p150(Glued) revealed targeting to the plus ends of growing microtubules, requiring the NH2-terminal cytoskeleton-associated protein-glycine rich domain, but not EB1 or CLIP-170. Effectors of protein kinase A modulated microtubule binding and suggested p150(Glued) phosphorylation as a factor in plus-end binding specificity. Using a phosphosensitive monoclonal antibody, we mapped the site of p150(Glued) phosphorylation to Ser-19. In vivo and in vitro analysis of phosphorylation site mutants revealed that p150(Glued) phosphorylation mediates dynamic binding to microtubules. To address the function of dynamic binding, we imaged GFP-p150(Glued) during the dynein-dependent transport of Golgi membranes. Live-cell analysis revealed a transient interaction between Golgi membranes and GFP-p150(Glued)-labeled microtubules just prior to transport, implicating microtubules and dynactin in a search-capture mechanism for minus-end-directed organelles.
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN 46556, USA.
Patricia S
PS
Vaughan
Pedro
P
Miura
Matthew
M
Henderson
Belinda
B
Byrne
Kevin T
KT
Vaughan
D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001692:Biological Transport; D019556:COS Cells; D000072159:Dynactin Complex; D004398:Dyneins / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D016297:Mutagenesis, Site-Directed; D010766:Phosphorylation; D011485:Protein Binding
D000072159:Dynactin Complex; D008869:Microtubule-Associated Proteins; D004398:Dyneins