Nucleotide Dependence and Cytoplasmic Localization

Many different proteins associate with microtubules, influencing their spatial organization and function. These include proteins of a structural nature, which link microtubules to one another or to other cellular organelles and which may stimulate tubulin assembly. The second group, the so-called dynamic microtubuleassociated proteins, move subcellular components along microtubules through nucleotidase action. In this report the effects of nucleotides on a 49-kDa protein which appears to associate with ordered arrays of microtubules within Artemia are described, revealing a protein with novel characteristics. Efficient removal of the 49kDa protein from microtubules assembled in cell-free extracts of Artemia occurred with GTP and some analogues of ATP, nonhydrolyzable or otherwise, but not with ATP itself. The latter nucleotide had a greater impact on cross-linking when microtubules were assembled from purified tubulin. The 49-kDa protein possessed a low level of nucleotidase activity, preferring either ATP or GTP as substrate. Unlike other microtubule-associated proteins, the enzymatic activity of the 49-kDa protein was not stimulated by microtubules, at least under assay conditions in which cross-linking was disrupted by nucleotides. Immunofluorescent staining of Artemia larvae by affinity-purified antibodies indicated that the 49-kDa protein is located in mitotic spindles, midbodies, and setal cells, all regions containing organized microtubules. The 49-kDa microtubule cross-linking protein from Artemia, through its unusual response to nucleotides and its cytoplasmic location, has a unique position within the heterogeneous family of microtubule-associated proteins described to date.