We previously found that the peroxisomal citrate synthase of Saccharomyces cerevisiae, Cit2p, contains a cryptic targeting signal for both peroxisomes (PTS) and mitochondria (MTS) within its 20–amino acid N-terminal segment [Lee et al. (2000) J. Biochem. 128, 1059–1072]. In the present study, the fine structure of the cryptic signal was scrutinized using green fluorescent protein fusions led by variants of the N-terminal segment. The minimum ranges of the cryptic signals for mitochondrial and peroxisomal targeting were shown to consist of the first 15– and 10–amino acid N-terminal segments, respectively. Substitution of the 3rd Val, 6th Leu, 7th Asn, or 8th Ser with Ala abolished the cryptic MTS function, however, no single substitution causing an obvious defect in PTS function was found. Neither the 15–amino acid N-terminal segment nor the C-terminal SKL sequence (PTS1) was necessary for Cit2p to restore the glutamate auxotrophy caused by the double Δcit1 Δcit2 mutation. The Cit2p variant lacking PTS1 [Cit2(ΔSKL)p] partially restored the growth of both the Δcit1 Δcit2 and Δcit1 mutants on acetate, while that carrying intact PTS1 or lacking the N-terminal segment [Cit2p, Cit2(ΔNΔSKL)p, and Cit2(ΔN)p] did not. It is thus suggested that the potential of the N-terminal segment as an ambidextrous targeting signal can be unmasked by deletion of PTS1.

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