Figure 2.
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.