Mutation of the fourth EF-hand impairs centriole localization. A, HeLa cells were transfected either with VSV-tagged HsCEN1 or HsCEN3, or P99A mutant form of HsCEN3, or with His-tagged D147,149,151A mutant form of HsCEN3, fixed, and analyzed by double immunofluorescence with antitag and anticentriole antibodies. The percentage of cells with centrosomal localization of the tagged protein was estimated 3, 24, or 48 h after transfection. A′, HeLa cells transfected with HsCEN3 were processed for antitag immunofluorescence 48 h after transfection. Three cells in this field present centrosome labeling (arrows). Bar, 2 μm. B, Soluble (S) and insoluble (I) Triton X-100 proteins from 10⁵ cells were migrated on 12% SDS-PAGE, transferred to nitrocellulose, and revealed either with anti-HsCen1p antibodies or with anti-HsCen3p antibodies. Note that overexpression of HsCen1p (detection in alkaline phosphatase) is more efficient than overexpression of wild-type or mutant HsCen3p (detection in ECL). In all cases, the major part of the overexpressed protein is extracted by Triton X-100 treatment. Note also the existence of an insoluble pool of D147,149,151A mutant protein, despite the lack of centriole localization.

HsCen3p specifically blocks cell growth. Yeast strains transformed with a 2μ (pYES) or a CEN (centromeric) empty vector, or a 2μ vector encoding HsCen1p, HsCen2p, HsCen3p, yeast Cdc31p, or a CEN vector encoding HsCen3p were grown on glucose plates (repressive condition) or on raffinose + galactose plates (inducing condition) for 3 d at 30°C. For each condition, two dilutions were spotted. Cells expressing HsCen3p on a 2μ or on a CEN plasmid did not grow in inducing condition.

Yeast cells expressing HsCen3p contain an unduplicated SPB. A and B, 12 h after induction, cells expressing HsCen3p were fixed and double labeled with an anti–α-tubulin (A1) or an anti-Cen3p (A2) antibody, or an anti-Spc110p (B1) or an anti-Cen3p (B2) antibody. DNA was labeled with DAPI (A3 and B3). Large-budded cells did not contain any spindle, but diverging microtubules radiating from the nucleus trapped in the bud neck (A1). Anti-Spc110p antibody detected only one dot on the nucleus trapped in the bud neck (B1). No specific localization of HsCen3p was detected (A2 and B2). Bar, 5 μm. C and C′, 12 h after induction, GFP-Spc42p bearing strain overexpressing HsCen3p was observed for GFP in the fluorescein channel (1), for DNA staining (DAPI; 2), and in Nomarski optics (3). In both cases, a single dot per nucleus was detected. Bar, 5 μm. D 1, Electron micrograph of a cell 12 h after induction of HsCen3p expression. A single SPB is observed at the bud neck. The contour of the nucleus can be easily identified by the nuclear envelope bearing nuclear pores (black dots). Microtubules diverging from the SPB can be seen in the nucleus and in the cytoplasm. D 2 and 3, SPBs of two individual cells. Note the bent profile of the unduplicated SPB. Bars, 0.3 μm. E, Five serial sections of a cell expressing HsCen3p 12 h after induction. A single unduplicated SPB is observed in sections 2, 3, and 4 (arrows) and is enlarged tenfold in the corresponding insets. Bars, 2 μm and 0.2 μm, respectively.

HsCen3p interacts with Kar1p extracts of yeast cells overexpressing HsCen3p, Cdc31p. HsCen3p in a Δkar1/cdc31-16 background, or HsCen2p were immunoprecipitated with anti-HsCen3p affinity-purified antibodies or with anti-HsCen2p antibodies. Immunoprecipitated proteins were separated on SDS-PAGE, transferred onto nitrocellulose, and revealed with anti-HsCen3p, anti-Cdc31p, or anti-Kar1p antibodies, or with anti-HsCen2p and anti-Kar1p antibodies. In cells overexpressing HsCen3p, HsCen3p and Kar1p are coimmunoprecipitated. It is noteworthy that in the conditions we have used, Cdc31p and Kar1p are not coimmunoprecipitated, whereas HsCen2p and Kar1p are.

CDC31 and centrin from Chlamydomonas reinhardtii define two divergent subfamilies. Note that human and murine CEN3 and CDC31 belong to the same subfamily, whereas HsCEN1, HsCEN2, and centrin from Chlamydomonas belong to the other subfamily. Accession numbers are indicated for the S. pombe centrin gene sequence, which has been found in the genome sequencing project and for the two divergent Giardia genes. Bar, mutation frequency.

6His-HsCen3p induces blastomeres undercleavage in Xenopus laevis embryos. A, Two-cell stage embryos microinjected either with 6 mg/ml His-HsCen3p (1), 12 mg/ml His-HsCen3p (2), 12 mg/ml heat-treated His-HsCen3p (3), or 12 mg/ml protease-treated His-HsCen3p (4) were observed 7 h after fertilization. Undercleaved blastomeres are present in the first three conditions (arrows). Bar, 1 mm. B, An embryo, injected with His-HsCen3p, methanol-fixed 8 h after microinjection, and labeled with anti–α-tubulin antibodies, contains large blastomeres with one or two microtubule asters in the injected half, whereas the uninjected cells gave rise to small blastomeres with one or two asters (inset). Bar, 50 μm. C, Large blastomere of a His-HsCen3p–injected embryo exhibiting one aster in a confocal section (1) and two additional asters in another section (2), whereas the uninjected cells gave rise to small blastomeres with one or two asters (bottom left). Bar, 50 μm. D, An embryo, injected with 12 mg/ml HsCen2p, methanol-fixed 8 h after microinjection, and labeled with anti–α-tubulin antibodies, contains large blastomeres with numerous asters (left) in the injected half, whereas the uninjected cells gave rise to small blastomeres with one or two asters (right). Bar, 50 μm.

Yeast cells expressing HsCen3p present a large bud. A, Yeast strain expressing HsCen3p or Cdc31p were grown in inducing condition (raffinose + galactose), repressing condition (glucose), or in raffinose medium (the Gal promotor is neither repressed or induced). The number of cells were determined at hourly intervals. Cells expressing HsCen3p stopped growing in raffinose + galactose and in raffinose medium at 6 and 10 h, respectively. B, Proteins from yeast strain expressing HsCen3p growing in glucose, raffinose, or raffinose + galactose medium were prepared and analyzed by Western blot with anti-HsCen3p antibodies. HsCen3p is rapidly and efficiently induced in raffinose + galactose and a very low signal is detected in raffinose after a 12-h culture. C, Yeast strain expressing HsCen3p was grown in inducing condition (raffinose + galactose). The number of unbudded, small-budded, or large-budded cells were determined at hourly intervals. Cells presenting a large bud start accumulating at 6 h. D, Phase-contrast microscopy of yeast strain expressing HsCen3p grown in inducing conditions. Most of the cells present a large bud. Bar, 10 μm. E, FACS analysis of yeast strain expressing HsCen3p or Cdc31p grown in inducing conditions (raffinose + galactose). At 12 h, cells expressing HsCen3p have mainly a G2 DNA content, whereas control cells have a G1 DNA content.

Cdc31p can overcome HsCen3p-induced growth arrest in a dose-dependent manner. A, Cells expressing: a fusion between either amino acids 1–23 of HsCen3p and amino acids 18–161 of Cdc31p, or amino acids 1–17 of Cdc31p and amino acids 24–167 of HsCen3p; or overexpressing 2μ plasmids coding for HsCen3p and Cdc31p; or overexpressing both HsCen3p cloned on a centromeric (CEN) plasmid and Cdc31p cloned on a 2μ plasmid; or expressing HsCen3p and Cdc31-16p in a Δkar1 background were grown on glucose plates (repressive condition) or on raffinose + galactose plates (inducing condition) for 3 d at 30°C. For each condition, two dilutions were spotted. Cells expressing a fusion between amino acids 1–17 of Cdc31p and amino acids 24–167 of HsCen3p, both HsCen3p and Cdc31p, or expressing HsCen3p and Cdc31-16p in a Δkar1 background did not grow in inducing condition. B, 12 h after induction, GFP-Spc42p bearing strain overexpressing HsCen3p in a Δkar1 background was observed for GFP in the fluorescein channel, for DNA staining (DAPI), and in Nomarski optics. In both cases, a single dot per nucleus was detected. Bar, 5 μm.