Molecular cloning and biochemical characterization of a VCP homolog in African trypanosomes.
Through reverse transcription-polymerase chain reaction using degenerate oligonucleotide primers, a VCP homolog was identified in African trypanosomes. Sequence analysis shows a 72 and 64% deduced amino acid identity, respectively, with mouse VCP and yeast Cdc48p. Southern analysis indicates tbVCP to have a single locus with two alleles. Antibodies generated against recombinant protein recognize a 95 kDa protein in whole cell lysates of both procyclic and bloodstream trypanosomes. There is an approximately four-fold greater expression of TbVCP protein in the procyclic stage of the trypanosome life cycle. Subcellular fractionation and immunofluorescence with anti-TbVCP antibodies indicate the majority of TbVCP to be cytoplasmically localized with a small subset associated with membranes. Sucrose velocity sedimentation and gel filtration size analysis studies suggest that TbVCP is a homohexameric particle as has been demonstrated with other VCP homologs. Also like other VCP homologs, TbVCP contains an NEM-inhibitable ATPase activity. This is the first characterization of an AAA (ATPases Associated with a variety of cellular Activities) family member in African trypanosomes.
Department of Medical Microbiology and Immunology, University of Wisconsin-Madison Medical School, 53706, USA.
J L
JL
Roggy
J D
JD
Bangs
D000251:Adenosine Triphosphatases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D000818:Animals; D002451:Cell Compartmentation; D003001:Cloning, Molecular; D005033:Ethylmaleimide / Q000494:pharmacology; D005455:Fluorescent Antibody Technique; D015870:Gene Expression; D017125:Genes, Protozoan; D008969:Molecular Sequence Data; D011487:Protein Conformation; D015800:Protozoan Proteins / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions; D014346:Trypanosoma brucei brucei / Q000201:enzymology / Q000235:genetics*; D000074405:Valosin Containing Protein
D015800:Protozoan Proteins; D000251:Adenosine Triphosphatases; C118825:VCP protein, Trypanosoma brucei; D000074405:Valosin Containing Protein; D005033:Ethylmaleimide