Stability of the small gamma-tubulin complex requires HCA66, a protein of the centrosome and the nucleolus.
To investigate changes at the centrosome during the cell cycle, we analyzed the composition of the pericentriolar material from unsynchronized and S-phase-arrested cells by gel electrophoresis and mass spectrometry. We identified HCA66, a protein that localizes to the centrosome from S-phase to mitosis and to the nucleolus throughout interphase. Silencing of HCA66 expression resulted in failure of centrosome duplication and in the formation of monopolar spindles, reminiscent of the phenotype observed after gamma-tubulin silencing. Immunofluorescence microscopy showed that proteins of the gamma-tubulin ring complex were absent from the centrosome in these monopolar spindles. Immunoblotting revealed reduced protein levels of all components of the gamma-tubulin small complex (gamma-tubulin, GCP2, and GCP3) in HCA66-depleted cells. By contrast, the levels of gamma-tubulin ring complex proteins such as GCP4 and GCP-WD/NEDD1 were unaffected. We propose that HCA66 is a novel regulator of gamma-tubulin function that plays a role in stabilizing components of the gamma-tubulin small complex, which is in turn essential for assembling the larger gamma-tubulin ring complex.
Fant
Xavier
X
Wellcome Trust Centre for Cell Biology, University of Edinburgh, Mayfield Road, Edinburgh EH9 3JR, UK.
Gnadt
Nicole
N
Haren
Laurence
L
Merdes
Andreas
A
D000951:Antigens, Neoplasm / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002453:Cell Cycle; D002466:Cell Nucleolus / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D013058:Mass Spectrometry; D008856:Microscopy, Fluorescence; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D046912:Multiprotein Complexes / Q000378:metabolism; D011485:Protein Binding; D055550:Protein Stability; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013997:Time Factors; D014162:Transfection; D014404:Tubulin / Q000378:metabolism*
D000951:Antigens, Neoplasm; D002352:Carrier Proteins; D008869:Microtubule-Associated Proteins; D046912:Multiprotein Complexes; D034741:RNA, Small Interfering; D016601:RNA-Binding Proteins; D011993:Recombinant Fusion Proteins; C112305:TUBGCP2 protein, human; C112306:TUBGCP3 protein, human; D014404:Tubulin; C524211:UTP6 protein, human