Identification of proteins binding the native tubulin dimer.
Microtubules play an essential role in eukaryotic cells, where they perform a wide variety of functions. In this paper, we describe the characterization of proteins associated to tubulin dimer in its native form, using affinity chromatography and mass spectrometry. We used an immunoaffinity column with coupled-monoclonal antibody directed against the alpha-tubulin C-terminus. Tubulin was first loaded onto the column, then interphase and mitotic cell lysates were chromatographed. Tubulin-binding proteins were eluted using a peptide mimicking the alpha-tubulin C-terminus. Elution fractions were analyzed by SDS-PAGE, and a total of 14 proteins were identified with high confidence by mass spectrometry. These proteins could be grouped in four classes: known tubulin-binding proteins, one microtubule-associated protein, heat shock proteins, and proteins that were not shown previously to bind tubulin dimer or microtubules.
Gache
Vincent
V
INSERM Unité 366, DRDC/CS, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble Cedex 9, France.
Louwagie
Mathilde
M
Garin
Jérôme
J
Caudron
Nicolas
N
Lafanechere
Laurence
L
Valiron
Odile
O
D000818:Animals; D002417:Cattle; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D019281:Dimerization; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D014404:Tubulin / Q000737:chemistry* / Q000378:metabolism*; D014443:Tyrosine / Q000378:metabolism
C405804:EB1 microtubule binding proteins; D050883:HSC70 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; C494371:HSPA8 protein, human; D008869:Microtubule-Associated Proteins; D014404:Tubulin; D014443:Tyrosine