Functional analysis of the trypanosomal AAA protein TbVCP with trans-dominant ATP hydrolysis mutants

TbVCP is a member of the AAA ((A) under bar TPases (A) under bar ssociated with a variety of cellular (A) under bar ctivities) family of proteins cont aining two ATPase domains. Southern analysis indicates TbVCP to have a sing le-locus, two-copy, genomic organization. One copy, but not both, can be di srupted by targeted gene replacement, suggesting that TbVCP is essential fo r trypanosome viability, Site-directed mutagenesis of the ATP hydrolysis mo tifs indicates that the second conserved ATPase domain is essential for TbV CP activity. Constitutive overexpression of TbVCP with a single mutation in the second hydrolysis motif or with mutations in both hydrolysis motifs wa s not possible. Regulated overexpression of these mutants resulted in cell death as a dominant negative phenotype, In each case cell growth arrested a t 24-h post-induction and at all stages of the cell cycle as judged by repl ication of nuclear and kinetoplast genomes. Onset of growth arrest coincide d with the development of severe and characteristic morphological alteratio ns for each mutant. Neither constitutive nor regulated overexpression of wi ld type TbVCP or the single first hydrolysis domain mutant had any overt ef fect on cell viability or morphology, However, the distinct phenotype of th e double mutant indicates that the first hydrolysis domain, although not es sential, does modulate overall TbVCP function. Finally, yeast complementati on studies demonstrated that TbVCP can functionally replace the yeast homol ogue Cdc48p, indicating that protein protein interactions essential to func tion have been maintained over great phylogenetic distances.