Proteomic amino-termini profiling reveals targeting information for protein import into complex plastids

Abstract

In organisms with complex plastids acquired by secondary endosymbiosis from a photosynthetic eukaryote, the majority of plastid proteins are nuclear-encoded, translated on cytoplasmic ribosomes, and guided across four membranes by a bipartite targeting sequence. In-depth understanding of this vital import process has been impeded by a lack of information about the transit peptide part of this sequence, which mediates transport across the inner three membranes. We determined the mature N-termini of hundreds of proteins from the model diatom Thalassiosira pseudonana, revealing extensive N-terminal modification by acetylation and proteolytic processing in both cytosol and plastid. We identified 63 mature N-termini of nucleus-encoded plastid proteins, deduced their complete transit peptide sequences, determined a consensus motif for their cleavage by the stromal processing peptidase, and found evidence for subsequent processing by a plastid methionine aminopeptidase. The cleavage motif differs from that of higher plants, but is shared with other eukaryotes with complex plastids.

Figure 1. Protein import into complex plastids.

(a) Schematic diatom plastid surrounded by four membranes, the outermost continuous with the ER. Proteins are synthesized on cytoplasmic ribosomes and co-translationally inserted into the ER lumen where the signal peptide (light blue) is removed by signal peptidase (1). The transit peptide (orange) then targets the proteins across the periplastidal membrane into the periplastidal compartment (2), and then through the translocons of the chloroplast double envelope into the plastid stroma, where the transit peptide is removed by the stromal processing peptidase (3). (b) Schematic structure of a nuclear-encoded plastid-targeted diatom protein precursor.

Figure 2. T. pseudonana protein termini identified by TAILS.

(a) Schematic representation of the TAILS workflow. Proteins with free or naturally modified (black square) N termini are denatured, followed by chemical modification of all primary amines (grey triangle). Specific digestion with trypsin generates peptides amenable to mass spectrometric identification. N-terminal peptides are blocked, whereas internal or C-terminal peptides exhibit a trypsin-generated primary amine at their N terminus that is used to covalenty bind these peptides to an aldehyde-containing polymer which is subsequently removed by filtration. (b) Position of identified N-terminal peptides with respect to curated protein model. N termini matching the protein models at positions 1 and 2 are cytosolic proteins with intact (+M1) or removed (–M1) initiating Met. Black, acetylated N termini; dark grey, protein N termini present in both dimethylated and acetylated forms; light grey, free N termini identified as dimethylated peptides. (c) Sequence logoplot of the first 6 amino acids of 81 N termini of nuclear encoded proteins with intact initiating Met. (d) Sequence logoplot of 231 N termini of nuclear-encoded proteins starting at protein model position 2 because the initiating Met was removed. (e) Combined logoplot of N termini of 22 plastid-encoded proteins starting at position 2 after N-terminal Met excision plus 18 plastid-imported proteins with Met directly preceding the identified peptide.

Figure 3. Approach for determining diatom transit peptide sequences.

Transit peptides (orange) and transit peptide cleavage sites were deduced by mapping identified N termini (green) to the protein model sequence after removal of the ER signal sequence (blue). (a) Cytochrome b₆f complex iron-sulfur protein subunit (PetC). Three peptides identify a single unique N terminus at position 35 of the protein model. (b) Light harvesting antenna complex protein Lhcr2. An acetylated and a dimethylated peptide identify the mature protein N terminus at protein model position 30, and a dimethylated peptide begins at the canonical SP cleavage site (ASA-FAP) at protein model position 16, indicating that this protein was incompletely processed or in transit when isolated. (c) Unknown protein 4820, homologous to a putative higher plant plastid precursor protein. Two peptides identify a mature, partially acetylated N terminus starting at protein model position 49, while a third peptide has an N-terminal Met starting at position 48. Bold, observed peptides; underlined, conserved ASA-FAP motif; arrow, inferred ER signal peptide cleavage site; arrowhead, observed protein termini.

Figure 4. Amino acid occurrences at the transit peptide cleavage site.

IceLogos of sequences surrounding the putative transit peptide cleavage site (dotted line) of 63 plastid-imported proteins (a) based on alignment of the identified N-terminal sequences and the C-terminus of the deduced transit peptides. (b) based on an alignment of the same 63 sequences, with the 18 sequences that showed a Met at -1 shifted by one position to the right on the assumption that N-terminal Met was removed by a plastid Met-aminopeptidase after import and transit peptide cleavage. Note that iceLogos show the difference between the observed amino acid frequency among the 63 identified sequences and the natural amino acid abundance in T. pseudonana proteins, i.e. overrepresented amino acids are indicated above the line and underrepresented amino acids below the line. Only differences with a p-value of 0.05 or smaller are shown.

Figure 5. Conserved processing sites in related proteins from other organisms with complex plastids.

Processing sites for related proteins of other heterokonts were predicted based on alignments of mature protein sequences. (a) Schematic structure of a nuclear encoded plastid-targeted diatom protein. Proteolytic processing steps and proposed cleavage site consensus sequences are indicated. (b) Alignment of glutamate-1-semialdehyde 2,1-aminomutase from four diatom species and a brown alga (c) Alignment of glutamine synthetase from three diatom and two brown algal species. (d) Alignment of glyceraldehyde-3-phosphate dehydrogenase from five diatoms and two brown algae. Bold letters, mature T. pseudonana N terminal sequences identified in this study. Light blue, SP; orange, TP; yellow, a Met that may be removed from the N terminus after SPP processing; green, mature plastid stroma-targeted protein. Thaps, Thalassiosira pseudonana; Phaeo, Phaeodactylum tricornutum ; Fracy, Fragilariopsiscylindrus ; Psemu, Pseudo-nitzschia multiseries; Skeco, Skeletonema costatum ; Odosi, Odontella sinensis ; Ascno, Ascophyllum nodosum ; Ectsi, Ectocarpussiliculosis .

Figure 6. Comparison of transit peptide cleavage sites in higher plants and diatoms.

(a) Sequence logo based on 47 acetylated N-termini of chloroplast imported A. thaliana proteins identified by Zybailov et al. as semi-tryptic peptides within 10 amino acids from the predicted cleavage site [29]. (b) Sequence logo based on 30 acetylated N-termini of plastid-targeted T. pseudonana proteins identified in this study. Only acetylated N-termini were used to exclude a potential bias from comparing acetylated sequences from A. thaliana with non-acetylated ones from T. pseudonana.