Abstract

Centrins contain four potential Ca2+ binding sites, known as EF-hands, and have essential functions in centrosome duplication and filament contraction. Here we report that centrins from yeast, green algae, and humans bound with high affinity to a peptide of the yeast centrosomal component Kar1p. Interestingly, centrin binding was regulated by physiological relevant changes in [Ca2+], and this Ca2+ dependence was influenced by acidic amino acids within the Kar1p peptide, which also prevented efficient binding of the related yeast calmodulin. However, a hybrid protein with the third and fourth EF-hands from the yeast centrin Cdc31p and the amino-terminal half from yeast calmodulin behaved more like Cdc31p, indicating that the carboxyl-terminal half of Cdc31p influences binding specificity. Besides Kar1p, centrins bound to a yeast calmodulin binding site, explaining the dosage-dependent suppression of a calmodulin mutant by CDC31. Consistent with an essential role of Ca2+ for centrin functions, mutations in the first or the fourth EF-hands of Cdc31p, impairing the Ca2+-induced conformational change of Cdc31p, resulted in nonfunctional proteins in vivo. Our results suggest that centrins are involved in Ca2+ signaling, likely by influencing the properties of target proteins in response to changes in [Ca2+].

Highlights

  • The phenotype of conditional lethal cdc31 mutants being defective in spindle pole body (SPB) duplication under restrictive growth conditions [12]

  • Our results clearly demonstrate that the carboxyl-terminal half of Cdc31p contains features that contribute to the high affinity binding to Kar1p peptide and thereby to the binding specificity of this centrin

  • This conclusion is based on the finding that the four centrins from phylogenetically diverse organisms bind to the Kar1p peptide in a Ca2ϩdependent fashion

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Summary

Construction and properties

MATa ura lys801 ade101 trp1⌬63 his3⌬200 leu2⌬1::pSM41 ⌬cdc31::HIS3 MAT␣ ade ade trp can100 leu leu112 his his ura. ADE3 URA3 CEN4 2 ␮m, LEU2-based yeast-E. coli shuttle vector 2 ␮m, URA3-based yeast-E. coli shuttle vector CDC31 in pT7–7 GST-KAR1 in pGEX-3X CDC31 in pBluescript MET3-CDC31 on the LEU2-based yeast integration vector pRS305 CDC31 in pRS426 GST-⌬⌬kar in pGEX-3X CMD1 in pT7–7 cdc.EF (D142A) in pT7–7 cdc.EF (D33A) in pT7–7 cdc.EF (D69A) in pT7–7 cdc.EF (D106A) in pT7–7 SdCEN in pT7–7 CDC31-CMD1 in pT7–7 GST-kar1-⌬17 in pGEX-3X cdc.EF (D33A) in pRS425 cdc.EF (D69A) in pRS425 cdc.EF (D106A) in pRS425 cdc.EF (D142A) in pRS425 cdc.EF (D33A) in pRS426 cdc.EF (D69A) in pRS426 cdc.EF (D106A) in pRS426 cdc.EF (D142A) in pRS426 CDC31 in pRS425 HsCEN1 in pT7–7 CMD1 in pRS426 CMD1-CDC31 in pT7–7 phage T7 promoter HsCEN2 in pT7–7

Source or reference
EXPERIMENTAL PROCEDURES
RESULTS
NDa NDa NDa
DISCUSSION

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