Identification of a complex between centrin and heat shock proteins in CSF-arrested Xenopus oocytes and dissociation of the complex following oocyte activation.
Coimmunoprecipitation experiments using a monoclonal anti-centrin antibody (20H5) and cytostatic factor (CSF)-arrested Xenopus oocyte extracts specifically precipitates oocyte centrin (20-kDa) and two associated proteins of 70- and 90-kDa. Microsequence analysis of a tryptic peptide fragment of the 70-kDa protein reveals 100% identity with a 13-amino-acid peptide sequence from Xenopus heat shock protein hsp-70. Western blot analysis of immunoprecipitates using anti-hsp monoclonal antibodies (N27 and AC-88) confirms the identity of the 70-kDa protein as hsp-70 and identifies the 90-kDa protein as hsp-90. The centrin/hsp complex is also immunoprecipitated when anti-hsp-70 or anti-hsp-90 monoclonal antibodies (BB70 and 4F3, respectively) are used as primary antibodies during immunoprecipitation. The centrin/hsp complex is sensitive to pH and Ca2+ concentration. The complex shows differential dissociation of hsp-70 and hsp-90 under a variety of conditions, suggesting that each hsp can bind to centrin independently of the other. When oocytes are first activated by electric shock or ionophore treatment, followed by immunoprecipitation using anti-centrin monoclonal antibody 20H5, centrin precipitates with significantly reduced levels of hsp-70 in the complex, and these complexes contain no apparent hsp-90. We conclude that, in CSF-arrested oocytes, the centrosomal protein, centrin, is associated as a complex with the heat shock proteins, hsp-70 and hsp-90, and that this complex dissociates upon activation of the oocyte. The functional consequences of the formation of complexes between centrin and these hsps are unknown. However, based on the roles that have been defined for heat shock proteins in other systems, several possibilities are suggested.
Uzawa
M
M
Mayo Clinic Foundation, Rochester, Minnesota 55905, USA.
Grams
J
J
Madden
B
B
Toft
D
D
Salisbury
J L
JL
D000595:Amino Acid Sequence; D000818:Animals; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism*; D005260:Female; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism*; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D009865:Oocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D011233:Precipitin Tests; D017231:Proto-Oncogene Proteins c-mos / Q000494:pharmacology*; D014982:Xenopus laevis
D002135:Calcium-Binding Proteins; D002868:Chromosomal Proteins, Non-Histone; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; C056608:caltractin; D017231:Proto-Oncogene Proteins c-mos