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 (ASA-FAP), 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.

Proteomic Analysis of a Sea-Ice Diatom: Salinity Acclimation Provides New Insight into the Dimethylsulfoniopropionate Production Pathway

“…5). Although there is weak evidence for a chloroplast presequence on the REDOX gene and no signal/transit peptide predicted for the DECARB, diatom studies have established that while this motif predicts chloroplast localization, it is not always present in chloroplast proteins (Gruber et al, 2007). Previous work by Grö ne and found DMSP synthesis to be inhibited by addition of protease inhibitors, indicating that protein turnover was a source for DMSP precursors.…”

“…Scores from these online tools are presented in Supplemental Table S6. Signal peptides were also manually screened for the most essential amino acids of the well-characterized diatom chloroplast localization motif, ASAFAP; the underlined amino acids have been experimentally determined to be the most integral for chloroplast import, although certain substitutions (G/S for A and W/Y/L for F) are also prevalent (Gruber et al, 2007). Chloroplast bipartite presequences are predicted for the AT, SAMmt, and DiDECARB ( Fig.…”

“…Asterisk (*) indicates significant probability of chloroplast-targeting presequence based on Target P and Signal P predictions. Bold letters, predicted signal peptide; gray highlight, predicted cleavage site motif; yellow highlight, manually predicted cleavage site based on motif characterization by Gruber et al (2007); underlined, predicted transit peptide; red letters, conserved domain of mature protein.…”

“…html) and Phaeodactylum tricornutum (http://genome.jgi-psf.org/Phatr2/ Phatr2.home.html) were also screened for the presence of chloroplast bipartite targeting signals. Signal peptides and cleavage site predictions were performed by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) and manually evaluated for the conserved diatom chloroplast motif ASAFAP at the signal peptide cleavage site (Gruber et al, 2007). Plastid transit peptide domains were identified by TargetP (http://www.cbs.dtu.dk/services/ TargetP/).…”

Diatom plastids depend on nucleotide import from the cytosol

“…Moreover, a conserved phenylalanine (or in rare cases tryptophan, tyrosine or leucine) in the sequence motif (ASAFAP) at the cleavage site of the signal peptide was shown to be essential for successful stroma targeting (15). The latter characteristics help to identify plastidial precursor proteins (15).…”