Abstract
Microtubules are highly dynamic structures, composed of alpha/beta-tubulin heterodimers. Biosynthesis of the functional dimer involves the participation of several chaperones, termed cofactors A-E, that act on folding intermediates downstream of the cytosolic chaperonin CCT (1, 2). We show that cofactor D is also a centrosomal protein and that overexpression of either the full-length protein or either of two centrosome localization domains leads to the loss of anchoring of the gamma-tubulin ring complex and of nucleation of microtubule growth at centrosomes. In contrast, depletion of cofactor D by short interfering RNA results in mitotic spindle defects. Because none of these changes in cofactor D activity produced a change in the levels of alpha-or beta-tubulin, we conclude that these newly discovered functions for cofactor D are distinct from its previously described role in tubulin folding. Thus, we describe a new role for cofactor D at centrosomes that is important to its function in polymerization of tubulin and organization of the mitotic spindle.
Highlights
Formation of ␣/ tubulin heterodimers is promoted by five tubulin-specific co-chaperones, termed cofactors A–E, that act
Demonstration of the roles for these five cofactors in folding tubulin heterodimers comes from in vitro folding assays, which allowed purification of the five co-chaperones [1, 10]. This function is consistent with genetic studies in Saccharomyces cerevisiae [11, 12], Schizosaccharomyces pombe [13,14,15], Arabidopsis thaliana [16, 17], and Caenorhabditis elegans [18] in which mutations in cofactor D (CoD) yielded defects in microtubule-dependent processes, including maintenance of chromosome number
CoD Is a Component of Centrosomes—Rabbit polyclonal antibodies were raised against a 15-mer peptide, located near the N terminus of human CoD and conserved in several mammalian CoD orthologs
Summary
Cloning and Plasmids—The plasmid directing expression in mammalian cells of bovine CoD fused to GFP was obtained from Nicholas Cowan (New York University) [19] and was used as a template to generate our HA-tagged bovine CoD expression constructs. A plasmid containing the entire open reading frame of human CoD was obtained from ATCC (clone MGC1583; Manassas, VA) and used to generate both full-length and truncation mutant expression plasmids by PCR amplification using custom oligonucleotide primers. Quantification of centrosomal staining intensities for CoD, ␥-tubulin, centrin-2, pericentrin, and GCP-WD were performed by comparison with the staining observed in control cells and were scored by eye. Standard epifluorescence images were obtained in each case, to facilitate viewing of structures or staining in different focal planes. This results in lower resolution in several of the images shown in the figures, as compared with the use of confocal data.
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