Interactome analysis of the structure of the trypanosome nuclear lamina and other complexes. (360G-Wellcome-093723_Z_10_Z)

Defining function and the role of proteins in biological processes requires information at many levels. Genome sequences facilitate a parts list. A level of functional definition is possible by gene knockdown or knockout strategies. In the African trypanosome these approaches are powerful, but a complete definition of function requires determining the physiological context in which specific proteins operate. Protein interactions cannot at present be reliably predicted by in silico approaches, an d direct evidence is essential. It is proposed to use a new set of techniques to examine protein-protein interactions in trypanosomes. Using a GFP-tagged version of the protein (integrated at the endogenous gene locus), coupled with a supercooled ball mill procedure at -186˚C to generate a fine grindate, protein complexes have been isolated in high yield from total trypanosome cells. It is proposed to use this technology to explore how generally applicable the technique will be by focusin g on several complexes involved in intracellular transport. Proteins will be identified by MS/MS. If, as anticipated, the method is general, this will provide a powerful addition to the analytic methodology available for study of trypanosomes and potentially other (pathogenic) protists.