Nucleotide dependence and cytoplasmic localization of a 49-kDa microtubule cross-linking protein from the brine shrimp, Artemia.
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 microtubule-associated 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 49-kDa 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.
Zhang
J
J
Department of Biology, Dalhousie University, Halifax, Nova Scotia, Canada.
MacRae
T H
TH
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001156:Artemia / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D007814:Larva; D008854:Microscopy, Electron; D008869:Microtubule-Associated Proteins / Q000009:adverse effects / Q000302:isolation & purification / Q000378:metabolism* / Q000648:ultrastructure; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D008970:Molecular Weight; D012265:Ribonucleotides / Q000494:pharmacology*; D008941:Spindle Apparatus / Q000378:metabolism / Q000648:ultrastructure; D014404:Tubulin / Q000378:metabolism / Q000648:ultrastructure
D008869:Microtubule-Associated Proteins; D012265:Ribonucleotides; D014404:Tubulin; C085638:microtubule cross-linking protein, Artemia; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate