Isolation and analysis of genes encoding HsCen2-interacting proteins.

摘要

Centrins are small, acidic, calcium-binding, phosphoproteins that localize to the centrosome of eukaryotic cells. Genetic evidence from algae and yeast suggests that centrins are essential for proper segregation and duplication of primary microtubule organizing centers. The precise role of centrins in metazoans is poorly understood, although the high degree of sequence conservation strongly suggests conservation of function. Three centrin genes are known in the human. One of the three, HsCen2, appears to be expressed in all cell types (Wolfrum and Salisbury, 1998).;A humanized and fluoresence-enhanded Green Fluorescent Protein fusion to the full length HsCen2 coding sequence was constructed and transfected into HeLa cells. Observation by fluorescence microscopy showed concentrated fluorescence in the centrioles and sometimes in the midbody, as well as generalized staining in the cytoplasm and nucleus. This represents the first determination of the subcellular localization of a specific human centrin.;A molecular genetic screen to identify proteins that interact with HsCen2 protein was performed using the yeast two-hybrid system. Over 200 candidate clones were isolated, and sequence was determined for 70. Bioinformatic analysis of the sequences placed them in a large number of genes, some with known function and some without.;To examine the functions of the candidate genes in human cells, EGFP fusions were constructed to six of the sequences, three known and three unknown. The known sequences came from (1) Chk1, a gene involved in checkpoint control of the cell cycle, (2) FLI1, a gene in involved in organization of the actin cytoskeleton, and (3) FHOS, a gene that may also participate in actin organization. The Chk1 fusion showed nuclear localization. The FLI1 and FHOS fusions localized to the centrosomes and midbody, sites where centrin also is found. Additional evidence for FLI1/centrin colocalization came from indirect immunofluorescence experiments using anti-FLI1 antibodies in cells expressing EGFP-HsCen2. Two of the three unknown sequences also showed centrosome and midbody patterns, consistent with in vivo interaction with centrin.;Finally, a functional in vivo test of FLI1/centrin interaction was performed, made possible by the observation that the cytoplasm becomes solated in HeLa cells expressing high levels of the FLI1-EGFP fusion protein. Suppression of the solation phenotype was observed when the cells were cotransfected with HsCen2, strongly supporting the hypothesis that the FLI1 and Cen2 proteins interact in vivo.