Isolation and Characterization of Subnuclear Compartments from Trypanosoma brucei

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Abstract
Protozoan parasites of the order Kinetoplastida are responsible for a significant proportion of global morbidity and economic hardship. These organisms also represent extremely distal points within the Eukarya, and one such organism,Trypanosoma brucei, has emerged as a major system for the study of evolutionary cell biology. Significant technical challenges have hampered the full exploitation of this organism, but advances in genomics and proteomics provide a novel approach to acquiring rapid functional data. However, the vast evolutionary distance between trypanosomes and the higher eukaryotes presents significant problems with functional assignment based on sequence similarity, and frequently homologues cannot be identified with sufficient confidence to be informative. Direct identification of proteins in isolated organelles has the potential of providing robust functional insight and is a powerful approach for initial assignment. We have selected the nucleus of T. brucei as a first target for protozoan organellar proteomics. Our purification methodology was able to reliably provide both nuclear and subnuclear fractions. Analysis by gel electrophoresis, electron microscopy, and immunoblotting against trypanosome subcellular markers indicated that the preparations are of high yield and purity, maintain native morphology, and are well resolved from other organelles. Minor developmental differences were observed in the nuclear proteome for the bloodstream and procyclic stages, whereas significant morphological alterations were visible. We demonstrate by direct sequencing that the NUP-1 nuclear envelope antigen is a coiled coil protein, containing ∼20 near-perfect copies of a 144-amino acid sequence. Immunoelectron microscopy localized NUP-1 to the inner face of the nuclear envelope, suggesting that it is a major filamentous component of the trypanosome nuclear lamina.