Experimental determination of organelle targeting-peptide cleavage sites using transient expression of green fluorescent protein translational fusions
Section snippets
Plant material and growth conditions
Arabidopsis plants (Arabidopsis thaliana (L.) Heynh, Col-0 ecotype) were grown in Jiffy7 pots (Jiffy Products International, Oslo, Norway) in a growth chamber (23 °C, 75% relative humidity, 16 h light with an intensity of 80–100 μmol m−2 s−1).
DNA constructs
Full-length cDNA clones were ordered from OriGene (NM-000284, human PDHA1) and from the Arabidopsis Biological Resource Center (At3g10920.1, MSD1; At5g38430.1, RBCS; At4g05180.1, PSBQ2; At2g42540.2, COR15A; At2g42530.1, COR15B). Plasmids purified from clones
Results and discussion
The method we describe here to determine the targeting-peptide cleavage sites for proteins imported into mitochondria or plastids is shown schematically in Fig. 1. It takes advantage of the high level of accumulation of GFP translational fusions that typically occurs during transient expression in transfected animal cells or plant protoplasts. Proteins of interest are genetically fused upstream of GFP, in order not to mask the N-terminal targeting peptide. During the expression period the
Acknowledgments
We are very grateful to Jennifer Alban for technical assistance with human cell transfection, to Mathilde Joint for expert help with mass spectrometry experiments, and to Marjorie Juchaux (IMAC) for assistance with confocal microscopy. A.C. and P.P. acknowledge the support of Ph.D. fellowships from the Région-Pays-de-la-Loire/INRA and Angers Loire Metropole, respectively. We are thankful to Professor David Logan for careful proofreading and helpful suggestions.
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These authors have equally contributed to this work.