Over-expression of just these residues disrupts recruitment of c-tubulin to the centrosome because this region of NEDD1 binds to c-tubulin and keeps it away from the centrosome, L-Ornithine thereby acting as a dominant negative. Importantly, we identify specific residues that are required for the NEDD1/c-tubulin interaction, but do not compromise the tetrameric conformation of the C-terminus of NEDD1. Mutation of these residues reverses the dominant negative effect of this C-terminal fragment of NEDD1, suggesting that they are critical for its interaction with c-tubulin. Previous studies have shown that NEDD1 interacts with ctubulin. We first confirmed this interaction with endogenous proteins by immunoprecipitating NEDD1 from Selenomethionine mammalian cells, and detecting bound c-tubulin. The reciprocal immunoprecipitation of c-tubulin did not result in any interacting NEDD1 visible on immunoblot, but this could be due to the low levels of endogenous NEDD1. As others have reported, the Cterminal residues of NEDD1 are required for this interaction and endogenous c-tubulin is only immunoprecipitated by Myc-tagged NEDD1 constructs that contain residues 572�C660. We next assessed whether the interaction between NEDD1 and ctubulin occurs directly or is mediated by other proteins. The GSTtagged carboxyl-terminal domain of NEDD1,and His-tagged c-tubulin were expressed in E. coli. Both purified proteins were added together to glutathione sepharose beads either with, or without a mammalian cell lysate that would contain any additional binding partners. We found that c-tubulin was bound to NEDD1 in the presence, or absence, of HEK293T lysate. This indicates that NEDD1 CTD can bind to c-tubulin directly. Previously it has been shown that the C-terminal half of NEDD1 does not localize to the centrosome, but over-expression of this truncated protein causes a loss of c-tubulin from the centrosome by keeping it in the cytoplasm. To investigate if residues 599�C660 of NEDD1 were sufficient for binding c-tubulin in vivo, we tested whether this region could also interfere with c-tubulin localization to the centrosome.