Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif |
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Authors: | Ansgar Gruber Sascha Vugrinec Franziska Hempel Sven B Gould Uwe-G Maier Peter G Kroth |
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Institution: | 1. Plant Ecophysiology, University of Konstanz, Universit?tsstra?e 10, 78464, Konstanz, Germany 2. Cell Biology, Philipps-University Marburg, Karl-von-Frisch Stra?e 8, 35042, Marburg, Germany 3. School of Botany, University of Melbourne, Melbourne, VIC, 3010, Australia
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Abstract: | Plastids of diatoms and related algae evolved by secondary endocytobiosis, the uptake of a eukaryotic alga into a eukaryotic
host cell and its subsequent reduction into an organelle. As a result diatom plastids are surrounded by four membranes. Protein
targeting of nucleus encoded plastid proteins across these membranes depends on N-terminal bipartite presequences consisting
of a signal and a transit peptide-like domain. Diatoms and cryptophytes share a conserved amino acid motif of unknown function
at the cleavage site of the signal peptides (ASAFAP), which is particularly important for successful plastid targeting. Screening
genomic databases we found that in rare cases the very conserved phenylalanine within the motif may be replaced by tryptophan,
tyrosine or leucine. To test such unusual presequences for functionality and to better understand the role of the motif and
putative receptor proteins involved in targeting, we constructed presequence:GFP fusion proteins with or without modifications
of the “ASAFAP”-motif and expressed them in the diatom Phaeodactylum tricornutum. In this comprehensive mutational analysis we found that only the aromatic amino acids phenylalanine, tryptophan, tyrosine
and the bulky amino acid leucine at the +1 position of the predicted signal peptidase cleavage site allow plastid import,
as expected from the sequence comparison of native plastid targeting presequences of P. tricornutum and the cryptophyte Guillardia theta. Deletions within the signal peptide domains also impaired plastid import, showing that the presence of F at the N-terminus
of the transit peptide together with a cleavable signal peptide is crucial for plastid import.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
A. Gruber and S. Vugrinec contributed equally to this work. |
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Keywords: | Chloroplast Diatom Evolution Import Presequence |
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