Identification of a cryptic N-terminal signal in Saccharomyces cerevisiae peroxisomal citrate synthase that functions in both peroxisomal and mitochondrial targeting

Saccharomyces cerevisiae has three distinct citrate synthases, two located in mitochondria (mature Cit1p and Cit3p) and one in peroxisomes (mature Cit 2p). While the precursor of the major mitochondrial enzyme, Cit1p, has a si gnal for mitochondrial targeting at its N-terminus (MTS), Cit2p has one for peroxisomal targeting (PTS1) at its C-terminus, We have previously shown t hat the N-terminal segment of Cit2p is removed during import into peroxisom es [Lee, H.S. et al, (1994) Kor. J. Microbiol. 32, 558-564], which implied the presence of an additional N-terminal sorting signal. To analyze the fun ction of the N-terminal region of Cit2p in protein trafficking, we construc ted the N-terminal domain-swapped versions of Cit1p and Cit2p, Both fusions , Cit1::Cit2 and Cit2::Cit1, complemented the glutamate auxotrophy caused b y the double-disruption of the CIT1 and CIT2 genes. In addition, part of th e Cit2::Cit1 fusion protein, as well as Cit1::Cit2, was shown to be transpo rted into both mitochondria and peroxisomes, The subcellular localization o f the recombinant fusion proteins containing various N-terminal segments of Cit2p fused to a mutant version of green fluorescent protein (GFP2) was al so examined. As a result, we found that the 20-amino acid N-terminal segmen t of Cit2p contains a cryptic cleavable targeting signal for both peroxisom es and mitochondria, In addition, we show that the peroxisomal import proce ss mediated by the N-terminal segment of Cit2p was not affected by the disr uption of either PEX5 (encoding PTS1 receptor) or PEX7 (encoding PTS2 recep tor).