Dual Organellar Targeting of Aminoacyl-tRNA Synthetases in Diatoms and Cryptophytes

Abstract

The internal compartmentation of eukaryotic cells not only allows separation of biochemical processes but it also creates the requirement for systems that can selectively transport proteins across the membrane boundaries. Although most proteins function in a single subcellular compartment, many are able to enter two or more compartments, a phenomenon known as dual or multiple targeting. The aminoacyl-tRNA synthetases (aaRSs), which catalyze the ligation of tRNAs to their cognate amino acids, are particularly prone to functioning in multiple subcellular compartments. They are essential for translation, so they are required in every compartment where translation takes place. In diatoms, there are three such compartments, the plastid, the mitochondrion, and the cytosol. In cryptophytes, translation also takes place in the periplastid compartment (PPC), which is the reduced cytoplasm of the plastid's red algal ancestor and which retains a reduced red algal nucleus. We searched the organelle and nuclear genomes of the cryptophyte Guillardia theta and the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana for aaRS genes and found an insufficient number of genes to provide each compartment with a complete set of aaRSs. We therefore inferred, with support from localization predictions, that many aaRSs are dual targeted. We tested four of the predicted dual targeted aaRSs with green fluorescent protein fusion localizations in P. tricornutum and found evidence for dual targeting to the mitochondrion and plastid in P. tricornutum and G. theta, and indications for dual targeting to the PPC and cytosol in G. theta. This is the first report of dual targeting in diatoms or cryptophytes.

Keywords: Guillardia; PPC; Phaeodactylum; pheRS; protein targeting; syfB.

Fig. 1.—

Maximum-likelihood (ML) phylogenies of pheRSs. Left: pheRS alpha subunit, including monomeric type typically found in mitochondria. Right: pheRS beta subunit. Eukaryotes typically encode multiple pheRSs: An alpha and a beta subunit for the cytosol (salmon-colored box, left and right tree, respectively), a monomer-type for the mitochondrion (yellow box), and, in algae, an additional pheRS for the plastid. In the diatoms T. pseudonana and F. cylindrus, the plastid is served by mitochondrial-type monomeric pheRSs, whereas in P. tricornutum, the beta subunit (green box, right tree) is encoded in the plastid genome while the alpha subunit (indicated by white text on black background, left tree) is related to cyanobacterial homologs and therefore appears to be a product of endosymbiotic gene transfer. Numbers at nodes indicate ML bootstrap support out of 1,000 replicates/Bayesian posterior probabilities.

Fig. 2.—

Schematic representation of N-terminal extensions of aaRSs from G. theta and P. tricornutum chosen for GFP localization experiments, see text for details. Predicted signal peptides are represented by yellow fill, predicted mitochondrial presequences are represented by blue boxes, and the locations of all methionine residues are indicated by M. Bottom axis measures amino acid residues. Green ellipses labeled “GFP” indicate the location of GFP, chosen to correspond to the predicted start of the mature aaRS domain, and are not to scale.

Fig. 3.—

Fluorescence localization of aaRSs. Constructs of N-terminal targeting sequences from aaRSs fused to eGFP were expressed in the diatom P. tricornutum. (A) Localization of aaRS proteins from P. tricornutum, arginyl-tRNA synthetase 2 (Pt_argRS2) and asparaginyl-tRNA synthetase 2 (Pt_asnRS2). GFP fluorescence can be seen in a region adjacent to the plastid as well as colocalizing with PAF, indicative of dual targeting to the mitochondrion and the plastid. (B) Localization of tyrosyl-tRNA synthetase proteins from the cryptophyte G. theta (Gt_tyrRS1 and Gt_tyrRS2). The Gt_tyrRS1 construct shows diffuse fluorescence throughout the cytosolic area as well as in a small concentrated region characteristic of PPC localization (the “blob-like structure”), seen here as a small, hourglass-shaped, bright green spot. The Gt_tyrRS2 construct shows GFP fluorescence adjacent to and colocalizing with PAF, indicative of dual targeting to the mitochondrion and plastid. TL, transmitted light; Scale bars: 10 μm.