The protein translocation systems in plants - composition and variability on the example of Solanum lycopersicum

Abstract

Background: Protein translocation across membranes is a central process in all cells. In the past decades the molecular composition of the translocation systems in the membranes of the endoplasmic reticulum, peroxisomes, mitochondria and chloroplasts have been established based on the analysis of model organisms. Today, these results have to be transferred to other plant species. We bioinformatically determined the inventory of putative translocation factors in tomato (Solanum lycopersicum) by orthologue search and domain architecture analyses. In addition, we investigated the diversity of such systems by comparing our findings to the model organisms Saccharomyces cerevisiae, Arabidopsis thaliana and 12 other plant species.

Results: The literature search end up in a total of 130 translocation components in yeast and A. thaliana, which are either experimentally confirmed or homologous to experimentally confirmed factors. From our bioinformatic analysis (PGAP and OrthoMCL), we identified (co-)orthologues in plants, which in combination yielded 148 and 143 orthologues in A. thaliana and S. lycopersicum, respectively. Interestingly, we traced 82% overlap in findings from both approaches though we did not find any orthologues for 27% of the factors by either procedure. In turn, 29% of the factors displayed the presence of more than one (co-)orthologue in tomato. Moreover, our analysis revealed that the genomic composition of the translocation machineries in the bryophyte Physcomitrella patens resemble more to higher plants than to single celled green algae. The monocots (Z. mays and O. sativa) follow more or less a similar conservation pattern for encoding the translocon components. In contrast, a diverse pattern was observed in different eudicots.

Conclusions: The orthologue search shows in most cases a clear conservation of components of the translocation pathways/machineries. Only the Get-dependent integration of tail-anchored proteins seems to be distinct. Further, the complexity of the translocation pathway in terms of existing orthologues seems to vary among plant species. This might be the consequence of palaeoploidisation during evolution in plants; lineage specific whole genome duplications in Arabidopsis thaliana and triplications in Solanum lycopersicum.

Figure 1

The analysis of the orthologous species. (a) The phylogenetic relation of the plant species analysed via OrthoMCL (Additional files 2 and 4) is given. (b) Correlation of the number of protein sequences to the number of orthologues for all 14 plant species discussed (c) The according orthologues in S. lycopersicum, S. cerevisiae and A. thaliana (left scatterplot) and S. lycopersicum to A. thaliana (right scatterplot) have been analysed with respect to amino acid number (protein length in amino acids). The line in the right scatterplot represent the least square fit analysis to y=a*x with a=0.992. (d) Domain architecture of cpSecA2 orthologues from A.thaliana and S. lycopersicum. AT1G21650 corresponds to Solyc11g005020 whereas AT1G21651 corresponds to the combination of Solyc11g005040 and Solyc11g005030. (e) RT-PCR results confirming that Solyc11g005040 and Solyc11g005030 is ‘one gene’; lanes 1 to 4: cDNA, genomic DNA, no-RT control (without reverse transcriptase), negative control (water), respectively.

Figure 2

The ER & ERAD translocation system according to yeast. (a) In the co-translational pathway, SRP binds to the emerging polypeptide to form a RNC. Then, SRP is recognized by the SR composed of SRα and SRβ. The RNC is transferred to Sec61, which translocates the emerging protein into or across the ER membrane. (b) In the post-translational pathway, the precursor is guided by chaperones to the Sec62/63 and Sec61 complex. The final insertion or translocation of polypeptides is assisted by BiP, the ER-resident Hsp70 isoform [21]. (c) TA proteins are transferred to Get3 in a Sgt2/Get4/Get5 mediated manner. The Get3-TA protein complex interacts with Get1-Get2 complex, the latter facilitating the release of TA protein into the membrane. (d) In the ERAD-L pathway, misfolded substrates are recognized by ER-resident chaperones. This complex interacts with the luminal domain of Hrd3, the latter being complexed with the Hrd1/Der3 [22]. Their substrates are translocated via Sec61, Der1 or the E3 ligases. A complex of cytosolic E3-ligases (anchored to the membrane; Doa10, Hrd1/Der3) and E2-conjugating enzymes (Ubc6, Ubc7 bound to Cue1) ubiquitinate the substrates in the cytosol. Hrd1/Der3 interacts with Der1 via Usa1, while Dfm1 forms complexes with Doa10, Hrd1/Der3 and Cdc48. After ubiquitination, the substrates are pulled out by the AAA ATPase machinery (Cdc48, in complex with Npl4, Ufd1 and Ubx2) and degraded by the 26S proteasome. Note: The colour indicates the number of genes encoding the component. Green: equal number of genes found in Arabidopsis and tomato; blue: more genes in tomato than in A. thaliana; pink: less genes in tomato than in A. thaliana; yellow: no orthologues found in plants; red: only found in plants; purple: multiple subunits unified for better presentation; brown: not found in tomato; white: not included in our discussion.

Figure 3

Peroxisomal protein import. Pex5 and Pex7 recognize proteins with PTS1 and PTS2, respectively. Pex18 and Pex21 act as co-receptors for Pex7. The receptor-cargo complex is recognized by Pex13, Pex14 and Pex17. Pex5 shuttles between cytosol and membrane (and may be lumen, which at state is controversially discussed), and mediates the transfer of the cargo protein and becomes subsequently monoubiquitinated by Pex22-anchored Pex12 and Pex4. Pex5 is exported by Pex1 and Pex6 (anchored by Pex15). Malfunction of Pex5 recycling induces the RADAR mechanism, by which Pex5 becomes polyubiqunated via the E2-conjugating enzyme Ubc4 or Ubc5 and the E3-ubiquitin ligase Pex2 and Pex10. The polyubiquitinylated Pex5 is exported and degraded in the cytosol. (b) Type-II PMPs interact with Pex19, which docks to Pex3 and releases the PMP into the membrane. For color code see legend of Figure  2.

Figure 4

Mitochondrial and chloroplast protein import machineries. (a,b) Import of mitochondrial proteins generally unifies at TOM complex. Tom20 acts as initial receptor for preproteins and transfers them to Tom22 and Tom40 channel. (a) Presequence pathway: in IMS preproteins interact with Tim50 / Tim21 and Tim17 / Tim23 translocates preproteins across IM assisted by mtHsp70, Tim44, Pam18, 16 and 17. SAM pathway: in IMS Tim9-Tim10 and Tim8-Tim13 guide β-barrel proteins to SAM complex (Sam50, Sam35, Sam37). In plants, Sam35 and Sam37 are likely replaced by metaxin. (b) Carrier pathway: IMPs with cleavable sequence are recognized by OM receptor Tom70 (replaced by OM64 in plants). In IMS Tim9-Tim10 / Tim8-Tim13 guide protein to TIM22 complex, laterally releasing the protein into the membrane. Mia pathway: Mia40 recognizes cysteine residue in IMS preproteins and leads to the formation of a disulfide bond in an Erv1/Hot13-dependent manner. The Mim pathway promotes the insertion of α-helical proteins into the outer membrane. (c) Preproteins bind to Toc64, Toc159 and Toc34, which transfer them to Toc75. Toc64 forms ‘IMS complex’ with Toc12, imsHsp70 and Tic22. Tic110, Tic20 and Tic21 have a discussed translocation-channel function, while Tic62, Tic55 and Tic32 regulate the translocon activity. Tic40, stHsp70 and stHsp93 provide energy for final translocation. (d) cpSRP pathway: cpSRP is composed of cpSRP54 and cpSRP43, which form a transit complex with substrates and targets them to thylakoid membrane via cpFtsY and Alb3. Spontaneous pathway does not require proteinaceous factors. Twin-arginine translocon: TAT signal containing preproteins bind to Hcf6 and cpTatC, leading to the assembly with oligomeric subunit, Tha4 and transient translocon formation. Sec pathway: cpSecA binds to signal sequence and guides it to cpSecY, cpSecE and Alb3. For color code see legend of Figure 2. The question mark on Toc12 indicates that localization of this factor is under debate.