tailieunhanh - Báo cáo khoa học: Plastid localization of the PEND protein is mediated by a noncanonical transit peptide

Plastid envelope DNA-binding protein (PEND) is a DNA-binding protein with a chloroplast basic region-zipper domain at its N-terminus and a transmembrane domain at its C-terminus. The localization of PEND to the inner envelope membrane was demonstrated in a targeting experiment using isolated membranes and green fluorescent protein-tagged fusion pro-teins. | ỊFEBS Journal Plastid localization of the PEND protein is mediated by a noncanonical transit peptide Kimihiro Terasawa and Naoki Sato Department of Life Sciences Graduate Schoolof Arts and Sciences University of Tokyo Japan Keywords chloroplast envelope DNA-binding protein green fluorescent protein protein import transit peptide Correspondence N. Sato Department of Life Sciences Graduate School of Arts and Sciences University of Tokyo 3-8-1 Komaba Meguro-ku Tokyo 153-8902 Japan Fax 81 3 5454 6998 Tel 81 3 5454 6631 E-mail naokisat@ Received 21 October 2008 revised 29 December 2008 accepted 13 January 2009 doi Plastid envelope DNA-binding protein PEND is a DNA-binding protein with a chloroplast basic region-zipper domain at its N-terminus and a transmembrane domain at its C-terminus. The localization of PEND to the inner envelope membrane was demonstrated in a targeting experiment using isolated membranes and green fluorescent protein-tagged fusion proteins. An N-terminal sequence analysis showed that the presequence is 15 amino acids long however based on neural network-based prediction tools this short peptide is not predicted to be a chloroplast-targeting sequence. In the present study we confirmed by the digestion of intact chloroplasts that PEND is located in the envelope membrane. We then demonstrated that the N-terminal 88-amino acid sequence is sufficient for plastid import in vitro. The transient expression of green fluorescent protein-tagged fusion proteins revealed that neither the N-terminal 29-amino acid sequence nor the 16-amino acid sequence directed green fluorescent protein to chloroplasts but that the N-terminal 66-amino acid sequence was sufficient for correct targeting. These results suggest that targeting of PEND to the chloroplast requires both the presequence and the basic region whereas postimport processing cleaves only the presequence. Interestingly deletion of the presequence in the green

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