Functional analysis of the trypanosomal AAA protein TbVCP with trans-dominant ATP hydrolysis mutants.
TbVCP is a member of the AAA (ATPases Associated with a variety of cellular Activities) family of proteins containing two ATPase domains. Southern analysis indicates TbVCP to have a single-locus, two-copy, genomic organization. One copy, but not both, can be disrupted by targeted gene replacement, suggesting that TbVCP is essential for trypanosome viability. Site-directed mutagenesis of the ATP hydrolysis motifs indicates that the second conserved ATPase domain is essential for TbVCP activity. Constitutive overexpression of TbVCP with a single mutation in the second hydrolysis motif or with mutations in both hydrolysis motifs was not possible. Regulated overexpression of these mutants resulted in cell death as a dominant negative phenotype. In each case cell growth arrested at 24-h post-induction and at all stages of the cell cycle as judged by replication of nuclear and kinetoplast genomes. Onset of growth arrest coincided with the development of severe and characteristic morphological alterations for each mutant. Neither constitutive nor regulated overexpression of wild type TbVCP or the single first hydrolysis domain mutant had any overt effect on cell viability or morphology. However, the distinct phenotype of the double mutant indicates that the first hydrolysis domain, although not essential, does modulate overall TbVCP function. Finally, yeast complementation studies demonstrated that TbVCP can functionally replace the yeast homologue Cdc48p, indicating that protein.protein interactions essential to function have been maintained over great phylogenetic distances.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, Madison, Wisconsin 53706, USA.
J R
JR
Lamb
V
V
Fu
E
E
Wirtz
J D
JD
Bangs
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D019943:Amino Acid Substitution; D000818:Animals; D016054:DNA, Protozoan / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D005838:Genotype; D016297:Mutagenesis, Site-Directed; D015388:Organelles / Q000201:enzymology / Q000235:genetics; D015800:Protozoan Proteins / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D014346:Trypanosoma brucei brucei / Q000166:cytology / Q000201:enzymology* / Q000235:genetics*; D000074405:Valosin Containing Protein
D016054:DNA, Protozoan; D015800:Protozoan Proteins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; C118825:VCP protein, Trypanosoma brucei; D000074405:Valosin Containing Protein