The ATP-dependent helicase RUVBL1/TIP49a associates with tubulin during mitosis.
RUVBL1/TIP49a/Pontin52 is a recently identified multi-functional protein with 2 ATP binding (WALKER) sites, which is essential for cell proliferation. We recovered and identified RUVBL1/TIP49a as a tubulin-binding protein from Triton X-100 lysates of U937 promonocytic cells by protein affinity chromatography and tryptic peptide microsequencing. Performing co-immunoprecipitation using newly generated RUVBL1/TIP49a-specific antibodies (mAb and rabbit polyclonal Ab) and RUVBL1/TIP49a-GST fusion protein-pull down assays we demonstrate co-precipitation of alpha- and gamma tubulin with RUVBL1/TIP49a. Confocal immunoflourescence microscopy reveals that RUVBL1/TIP49a was present not only in the nucleus, as expected, but was also concentrated at the centrosome and at the mitotic spindle in colocalization with tubulin. The topology of RUVBL1/TIP49a at the mitotic spindle varied, depending on the mitotic stage. The protein was localized at the centrosome and at the polar and astral microtubules in metaphase, and was detectable at the zone of polar tubule interdigitation in anaphase B and telophase. During cytokinesis the protein reappeared at the area of decondensing chromosomes. Whereas preincubation of U937 cells with colcemid resulted in inhibition of mitotic spindle formation with subsequent loss of RUVBL1/TIP49a mitotic spindle staining, no relevant influence of colcemid on RUVBL1/TIP49a-tubulin binding was observed. An agonistic effect of RUVBL1/TIP49a on in vitro tubulin assembly is demonstrated. Our results reveal a new functional aspect of RUVBL1/TIP49a.
D000074183:ATPases Associated with Diverse Cellular Activities; D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D002352:Carrier Proteins / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D002455:Cell Division; D004265:DNA Helicases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D003703:Demecolcine / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D006801:Humans; D015151:Immunoblotting; D008938:Mitosis; D008969:Molecular Sequence Data; D014404:Tubulin / Q000378:metabolism*; D050257:Tubulin Modulators; D020298:U937 Cells
D002352:Carrier Proteins; D014404:Tubulin; D050257:Tubulin Modulators; D000255:Adenosine Triphosphate; D000074183:ATPases Associated with Diverse Cellular Activities; D004265:DNA Helicases; C486549:RUVBL1 protein, human; D003703:Demecolcine