SUBCELLULAR LOCALIZATION OF NADPH DEPENDENT CYTOCHROME P450 REDUCTASES FROM TRYPANOSOMA CRUZI

DE VAS MG; SCHLESINGER M; PORTAL P; FLAWIA M; TORRES H; F VILLAMIL S; ALONSO GD; PAVETO C

San Miguel de Tucumán. Tucumán, Argentina.

Congreso; XLV Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular (SAIB).; 2009

The presence of TcCPR-A and TcCPR-B in cytosolic fractions and TcCPR-C in microsomal fractions from T. cruziepimastigote cells was previously demonstrated by Western blot using polyclonal mouse antisera raised against the recombinant proteins. To study specific subcellular localization of TcCPRs by indirect immunofluorescence we designed different constructs using the vectors pTREX and pTEX-GFP. Therefore we analyzed their co-localization with cytosolic GFP in GFP-expressing parasites and the endoplasmic reticulum and glycosome markers calreticulin/BiP and GAPDH, respectively. TcCPR-A was detected in the cytosol coincident with GFP localization. TcCPR-C showed a strong co-localization with the ER markers around the nucleus and kinetoplast. A different approach consisting in GFP fusions at the C-terminal end of each expressed CPR supported the subcellular localization observed for TcCPR-A and TcCPR-C using the respective antisera. We could also analyze TcCPR-B concluding a cytosolic pattern of localization similar to the one observed for TcCPR-A. Subcellular localization of TcCPR-A and TcCPR-C was assessed using immunogold electron microscopy. TcCPR-A signal was not homogeneous in the cytosol, confirming previous results obtained by immunofluorescence. TcCPR-C was located in some areas of the endoplasmic reticulum, closely associated with the mitochondrial and nuclear membrane.