Identification of ribonucleotide reductase protein R1 as an activator of microtubule nucleation in Xenopus egg mitotic extracts.
Microtubule nucleation on the centrosome and the fungal equivalent, the spindle pole body (SPB), is activated at the onset of mitosis. We previously reported that mitotic extracts prepared from Xenopus unfertilized eggs convert the interphase SPB of fission yeast into a competent state for microtubule nucleation. In this study, we have purified an 85-kDa SPB activator from the extracts and identified it as the ribonucleotide reductase large subunit R1. We further confirmed that recombinant mouse R1 protein was also effective for SPB activation. On the other hand, another essential subunit of ribonucleotide reductase, R2 protein, was not required for SPB activation. SPB activation by R1 protein was suppressed in the presence of anti-R1 antibodies or a partial oligopeptide of R1; the oligopeptide also inhibited aster formation on Xenopus sperm centrosomes. In accordance, R1 was detected in animal centrosomes by immunofluorescence and immunoblotting with anti-R1 antibodies. In addition, recombinant mouse R1 protein bound to gamma- and alpha/beta-tubulin in vitro. These results suggest that R1 is a bifunctional protein that acts on both ribonucleotide reduction and centrosome/SPB activation.
Takada
S
S
Cellular and Molecular Biology Laboratory, RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan.
Shibata
T
T
Hiraoka
Y
Y
Masuda
H
H
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002457:Cell Extracts / Q000737:chemistry; D018385:Centrosome / Q000378:metabolism; D007399:Interphase; D008297:Male; D008870:Microtubules / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009842:Oligopeptides / Q000378:metabolism; D010063:Ovum / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012264:Ribonucleotide Reductases / Q000235:genetics* / Q000302:isolation & purification / Q000502:physiology*; D017385:Sequence Homology; D013094:Spermatozoa / Q000378:metabolism; D008941:Spindle Apparatus / Q000378:metabolism*; D014404:Tubulin / Q000378:metabolism; D014981:Xenopus; D029867:Xenopus Proteins
D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D009842:Oligopeptides; D011994:Recombinant Proteins; D014404:Tubulin; D029867:Xenopus Proteins; C498691:RRR1 protein, Xenopus; D012264:Ribonucleotide Reductases