Peptide deformylase (DEF; EC 126.96.36.199) catalyzes the removal of the N-formyl group from the methionine residue of organellar- and prokaryotic-translated nascent polypeptides as the first step in cotranslational protein processing and is an essential enzyme for eubacterial survival. Analyses of mitochondria and chloroplasts targeting of two nuclear-encoded DEF proteins has resulted in published discrepancies. Transient expression studies in onion epidermal cells of N-terminal sequences of AtDEF1 and 2 fused with green fluorescent protein (GFP) localized AtDEF2 to mitochondria and chloroplasts and restricted AtDEF1 to mitochondria (EMBO J. 19 (2000) 5916). However, according to immunological analysis of chloroplast lysates and in vitro uptake and processing using precursor forms of AtDEF1, chloroplasts also import AtDEF1 (Plant Physiol. 127 (2001) 97). To further evaluate the subcellular localization of AtDEF1, we fused full length AtDEF1 and 2 proteins with GFP for transient expression studies using onion epidermal cells (as a direct comparison with previous work) and photosynthetically active tobacco leaves. Because two inframe AUGs exist at the 5′ UTR of the AtDEF1 mRNA and the translation start in vivo is unknown, AtDEF1 fusion proteins that begin with each of the putative AUG start codons were tested. Our results indicate that the upstream AUG codon specifically targets AtDEF1: GFP to the mitochondria, whereas the AtDEF1: GFP with the second AUG codon localized the fusion protein to both mitochondria and chloroplasts. A single N-terminal sequence that targets a protein to both organelles is termed an ambiguous presequence and both AtDEF1 and AtDEF2 have such sequences for dual organellar localization.
|Number of pages||8|
|State||Published - Oct 1 2003|
Bibliographical noteFunding Information:
The first two authors (R.D.D. and H.M.C.) contributed equally to this research. We thank Carl Redmond for help on the BioRad PDS 1000, Kay MacAlester and Marisa Belcastro for technical support, Nemo Peeters (Cornell University) for providing the CoxIV::GFP plasmid. This research was supported in part by a Kentucky Tobacco Research and Development Center grant (awarded to MAW, LMAD, and RLH) and by the DOE, Division of Energy Bioscience (grant DE-FG02-92ER 20075 to RLH). Part of this research was conducted by H.M.C. as part of the Agricultural Biotechnology ABT395 undergraduate laboratory project requirement. This paper (No. 03-06-047) is published with the approval of the Director of the Kentucky Agricultural Experiment Station.
- Chloroplast and mitochondrial targeting
- GFP fusion protein
- N-terminal processing
- Peptide deformylase
- Transit peptide
ASJC Scopus subject areas
- Agronomy and Crop Science
- Plant Science