Direct interaction of Gas11 with microtubules: implications for the dynein regulatory complex.
We previously described the Trypanin family of cytoskeleton-associated proteins that have been implicated in dynein regulation [Hill et al., J Biol Chem2000; 275(50):39369-39378; Hutchings et al., J Cell Biol2002;156(5):867-877; Rupp and Porter, J Cell Biol2003;162(1):47-57]. Trypanin from T. brucei is part of an evolutionarily conserved dynein regulatory system that is required for regulation of flagellar beat. In C. reinhardtii, the trypanin homologue (PF2) is part of an axonemal 'dynein regulatory complex' (DRC) that functions as a reversible inhibitor of axonemal dynein [Piperno et al., J Cell Biol1992;118(6):1455-1463; Gardner et al., J Cell Biol1994;127(5):1311-1325]. The DRC consists of an estimated seven polypeptides that are tightly associated with axonemal microtubules. Association with the axoneme is critical for DRC function, but the mechanism by which it attaches to the microtubule lattice is completely unknown. We demonstrate that Gas11, the mammalian trypanin/PF2 homologue, associates with microtubules in vitro and in vivo. Deletion analyses identified a novel microtubule-binding domain (GMAD) and a distinct region (IMAD) that attenuates Gas11-microtubule interactions. Using single-particle binding assays, we demonstrate that Gas11 directly binds microtubules and that the IMAD attenuates the interaction between GMAD and the microtubule. IMAD is able to function in either a cis- or trans-orientation with GMAD. The discovery that Gas11 provides a direct linkage to microtubules provides new mechanistic insight into the structural features of the dynein-regulatory complex.
Bekker
Janine M
JM
Department of Physiological Sciences, University of California Los Angeles, Los Angeles, CA 90095, USA.
Colantonio
Jessica R
JR
Stephens
Andrew D
AD
Clarke
W Thomas
WT
King
Stephen J
SJ
Hill
Kent L
KL
Crosbie
Rachelle H
RH
D000595:Amino Acid Sequence; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D003598:Cytoskeletal Proteins; D004398:Dyneins / Q000378:metabolism*; D047468:Immunoprecipitation; D051379:Mice; D008870:Microtubules / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011506:Proteins / Q000737:chemistry / Q000378:metabolism*
D003598:Cytoskeletal Proteins; C452182:Gas8 protein, mouse; D011506:Proteins; D004398:Dyneins