Subcellular localization of HGPRT-I, HGPRT-II and HGXPRT in the bloodstream form of <i>T</i>. <i>brucei</i>.

<p>(A) Immunoblot analysis of BF cells over-expressing v5-tagged HGPRT-I, HGPRT-II and HGXPRT was performed to reveal the subcellullar localization of these proteins. Cytosolic (CYT) and organellar (ORG) fractions were obtained by digitonin fractionation. Purified fractions were analyzed by immunoblot with the following antibodies: anti-V5, anti-enolase (cytosol), anti-hexokinase (organellar fraction, glycosomes), anti-mt hsp70 (organellar fraction, mitochondrion). The relevant sizes of the proteins are indicated on the left. (B) Immunofluorescence microscopy of the same cell lines as in (A) was used to determine subcellular localization of the v5-tagged HGPRT-I, HGPRT-II and HGXPRT proteins within the cell. HGPRT-I, HGPRT-II and HGXPRT were visualized by immunostaining using a monoclonal anti-v5 antibody and anti-mouse secondary antibody conjugated with fluorescein isothiocyanate (FITC). Antibodies against enolase and hexokinase served to mark cytosolic and glycosomal localization, respectively. The DNA content (nucleus and kinetoplast) was visualized using DAPI (4,6-diamidino-2-phenylindole).