Phosphoregulation and depolymerization-driven movement of the Dam1 complex do not require ring formation.
During mitosis, kinetochores form persistent attachments to microtubule tips and undergo corrective detachment in response to phosphorylation by Ipl1 (Aurora B) kinase. The Dam1 complex is required to establish and maintain bi-oriented attachment to microtubule tips in vivo, and it contains multiple sites phosphorylated by Ipl1 (Refs 2, 3, 4, 5, 6, 7, 8, 9, 10). Moreover, a number of kinetochore-like functions can be reconstituted in vitro with pure Dam1 complex. These functions are believed to derive from the ability of the complex to self-assemble into rings. Here we show that rings are not necessary for dynamic microtubule attachment, Ipl1-dependent modulation of microtubule affinity or the ability of Dam1 to move processively with disassembling microtubule tips. Using two fluorescence-based assays, we found that the complex exhibited a high affinity for microtubules (Kd of approximately 6 nM) that was reduced by phosphorylation at Ser 20, a single Ipl1 target residue in Dam1. Moreover, individual complexes underwent one-dimensional diffusion along microtubules and detached 2.5-fold more frequently after phosphorylation by Ipl1. Particles consisting of one to four Dam1 complexes - too few to surround a microtubule - were captured and carried by disassembling tips. Thus, even a small number of binding elements could provide a dynamic, phosphoregulated microtubule attachment and thereby facilitate accurate chromosome segregation.
Gestaut
Daniel R
DR
Department of Biochemistry, University of Washington, Seattle, Washington 98195 USA.
Graczyk
Beth
B
Cooper
Jeremy
J
Widlund
Per O
PO
Zelter
Alex
A
Wordeman
Linda
L
Asbury
Charles L
CL
Davis
Trisha N
TN
D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D003599:Cytoskeleton / Q000378:metabolism / Q000648:ultrastructure; D018386:Kinetochores / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis / Q000502:physiology; D046912:Multiprotein Complexes / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D018797:Cell Cycle Proteins; C116003:DAM1 protein, S cerevisiae; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins