Isolation and characterization of subnuclear compartments from Trypanosomabrucei - Identification of a major repetitive nuclear lamina component

Protozoan parasites of the order Kinetoplastida are responsible for a signi ficant proportion of global morbidity and economic hardship. These organism s also represent extremely distal points within the Eukarya, and one such o rganism, 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 proteo mics provide a novel approach to acquiring rapid functional data. However, the vast evolutionary distance between trypanosomes and the higher eukaryot es presents significant problems with functional assignment based on sequen ce similarity, and frequently homologues cannot be identified with sufficie nt confidence to be informative. Direct identification of proteins in isola ted organelles has the potential of providing robust functional insight and is a powerful approach for initial assignment. We have selected the nucleu s of T. brucei as a first target for protozoan organellar proteomics. Our p urification methodology was able to reliably provide both nuclear and subnu clear fractions. Analysis by gel electrophoresis, electron microscopy, and immunoblotting against trypanosome subcellular markers indicated that the p reparations are of high yield and purity, maintain native morphology, and a re well resolved from other organelles. Minor developmental differences wer e observed in the nuclear proteome for the bloodstream and procyclic stages , whereas significant morphological alterations were visible. We demonstrat e by direct sequencing that the NUP-1 nuclear envelope antigen is a coiled coil protein, containing similar to 20 near-perfect copies of a 144-amino a cid sequence. Immunoelectron microscopy localized NUP-1 to the inner face o f the nuclear envelope, suggesting that it is a major filamentous component of the trypanosome nuclear lamina.