title: Protein targeting into complex diatom plastids: functional characterisation of a specific targeting motif.
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.
authors.affiliation: Plant Ecophysiology, University of Konstanz, Universitätsstrasse 10, 78464, Konstanz, Germany.
authors.forename: Ansgar
authors.initials: A
authors.lastname: Gruber
authors.forename: Sascha
authors.initials: S
authors.lastname: Vugrinec
authors.forename: Franziska
authors.initials: F
authors.lastname: Hempel
authors.forename: Sven B
authors.initials: SB
authors.lastname: Gould
authors.forename: Uwe-G
authors.initials: UG
authors.lastname: Maier
authors.forename: Peter G
authors.initials: PG
authors.lastname: Kroth
mesh_terms: D000465:Algorithms; D000595:Amino Acid Sequence; D017377:Diatoms / Q000235:genetics* / Q000254:growth & development; D017930:Genes, Reporter; D016678:Genome; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018087:Plastids; D011506:Proteins / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
chemical_list: D010455:Peptides; D011506:Proteins; D011993:Recombinant Fusion Proteins