[Mitochondrial signal peptide guides EGFP-GRP75 fusion proteins into mitochondria]

Objective To investigate the role of mitoch-ondrial signal peptide in guiding enhanced green fluorescent protein-glucose-regulated protein 75 fusion proteins (EGFP-GRP75) into mitochondria. Methods At first, the signal peptide gene and GRP75 domain genes were spliced by overlap extension PCR. Unphosphorylatable mutant T62A/S65A, phospho-mimic mutant T62D/S65D and substrate binding-defective mutant V482F were further created through site-directed mutagenesis PCR. The fusion gene fragments were ligated into pEGFPC1 expression plasmid, respectively. The expressions of EGFP-GRP75 fusion constructs in HeLa cells were examined directly with Western blotting and laser scanning confocal microscopy. Results All the fusion proteins were highly expressed. Signal peptide remarkably reduced the expression of EGFP-GRP75 fusions compared with recombinant plasmids without signal peptide. Fluorescence was seen exclusively located in mitochondria of the cells transfected by signal peptide-contained plasmids, whereas signal peptide-absent EGFP-GRP75 fusion proteins were homogeneously distributed in the whole cell body. In addition, no changes were observed in the subcellular localization of EGFP-GRP75 fusion proteins that contained double or triple mutations at Thr 62 residues, Ser 65 residues and Val 482 residues. Conclusion Signal peptide is essential for targeting EGFP-GRP75 fusion proteins into mitochondria, and 62 threonine/65 serine/482 valine residues contribute to phosphorylation or substrate-binding characteristics are dispensable in the mitochondrial targeting function.