Characterization and reconstitution of Drosophila gamma-tubulin ring complex subunits.
The gamma-tubulin ring complex (gammaTuRC) is important for microtubule nucleation from the centrosome. In addition to gamma-tubulin, the Drosophila gammaTuRC contains at least six subunits, three of which [Drosophila gamma ring proteins (Dgrips) 75/d75p, 84, and 91] have been characterized previously. Dgrips84 and 91 are present in both the small gamma-tubulin complex (gammaTuSC) and the gammaTuRC, while the remaining subunits are found only in the gammaTuRC. To study gammaTuRC assembly and function, we first reconstituted gammaTuSC using the baculovirus expression system. Using the reconstituted gammaTuSC, we showed for the first time that this subcomplex of the gammaTuRC has microtubule binding and capping activities. Next, we characterized two new gammaTuRC subunits, Dgrips128 and 163, and showed that they are centrosomal proteins. Sequence comparisons among all known gammaTuRC subunits revealed two novel sequence motifs, which we named grip motifs 1 and 2. We found that Dgrips128 and 163 can each interact with gammaTuSC. However, this interaction is insufficient for gammaTuRC assembly.
Gunawardane
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RN
Howard Hughes Medical Institute, Carnegie Institution of Washington, Baltimore, Maryland 21210, USA.
Martin
O C
OC
Cao
K
K
Zhang
L
L
Dej
K
K
Iwamatsu
A
A
Zheng
Y
Y
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D000918:Antibody Specificity; D018385:Centrosome / Q000737:chemistry / Q000378:metabolism; D002875:Chromosomes / Q000235:genetics; D003001:Cloning, Molecular; D017124:Conserved Sequence; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000166:cytology / Q000196:embryology / Q000235:genetics / Q000378:metabolism*; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D017403:In Situ Hybridization; D019476:Insect Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008870:Microtubules / Q000737:chemistry* / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D011233:Precipitin Tests; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D016415:Sequence Alignment; D014404:Tubulin / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D029721:Drosophila Proteins; C419558:Grip128 protein, Drosophila; C419559:Grip163 protein, Drosophila; D019476:Insect Proteins; D008869:Microtubule-Associated Proteins; D021122:Protein Subunits; D014404:Tubulin