Identification and localization of novel basal body proteins

Kerstin Brachhold and Michael Melkonian

University of Cologne, Botanical Institute, Gyrhofstr. 15, 50931 Cologne, Germany

The basal bodies of Chlamydomonas reinhardtii not only nucleate the flagella but they also constitute the ultrastructural and functional equivalent of the centrioles found in animal centrosomes. They have therefore been subjected to intensive studies considering their function and protein composition. To identify this protein composition we established a method to purify basal bodies. Highly purified preparations of basal bodies were then subjected to mass spectrometry using a 1D-gel approach and two independent MudPIT-analyses (multidimensional protein identification technology) with ESI-MS/MS. In addition to well-known basal body proteins a core list of putative basal body proteins was identified. Several novel proteins were further characterized by cloning their partial cDNAs. The cDNAs were expressed in E. coli and the recombinant proteins used to produce polyclonal antibodies in rabbits. The proteins were found to localize to the basal bodies by immunofluorescence. Immunogold localization and functional analyses of these proteins have been initiated.

e-mail address of presenting author: kerstin.brachhold@uni-koeln.de