On the problem of establishing the subcellular localization of Dictyostelium retrotransposon TRE5-A proteins by biochemical analysis of nuclear extracts

At first sight a protein that is enriched in extracts prepared from nuclei by means of biochemical methods can be considered to be a nuclear protein i n vivo. Although this assumption will hold true for most of the analyzed pr oteins, it could also lead to false interpretations. We analyzed the subcel lular distribution of endogenous and plasmid-borne proteins derived from th e retrotransposon TRE5-A of Dictyostelium discoideum. In biochemical fracti onation experiments the proteins encoded by TRE5-A open reading frame 1 (OR F1p) and the putative endonuclease encoded in ORF2 (ENp) were found in the detergent-insoluble material containing the nuclei. However, salt extractio n of isolated nuclei did not considerably release the TRE5-A proteins. Inst ead, the TRE5-A proteins were strongly enriched in a fraction that containe d the chromosomal DNA after removal of most cytoskeletal and histone protei ns. These observations implied that ORF1p and ENp were both attached to chr omatin in vivo, but this conclusion was disproved by the expression of gene tic fusions of green fluorescent protein with either ORF1p or ENp. We show conclusive evidence that both fusion proteins were located as large aggrega tes of native protein in the cytoplasm of D. discoideum cells. (C) 2001 Aca demic Press.