Most of the proteins which are transported across the eukaryotic ER are translocated through a protein conducting channel (for reviews see (Rapoport 2007, Rapoport et al. 2004, Rapoport et al. 1996)). In prokaryotes there is a similar process, except that the proteins are transported across and are integrated into the plasma membrane (Rapoport 2007, Rapoport et al. 1996). The translocation happens in both cases by a conserved membrane-protein complex, called Sec61 in eukaryotes or SecY in bacteria (Rapoport 2007, Rapoport et al.
2004). The polypeptide chains can be transferred co-translationally to the channel by ribosomes, which is initiated by the recognition of a signal or transmembrane sequence and a signal-recognition particle (SRP). The SRP is then interacting with its membrane receptor and the polypeptide is released into the associated channel (Figure 2A, (Rapoport 2007)).
Figure 2: Different translocation processes across the ER membrane via Sec protein complexes (Rapoport 2007).
A) Co-translational transport with interaction of a signal sequence, a signal recognition particle (SRP) and its receptor. B) Post-translational transport in eukaryotes (ratcheting mechanism). The ER luminal chaperone prevents the protein from moving back. C) Post-translational transport in prokaryotes (pushing mechanism). See Text for details.
Some proteins are transported after translation, post-translationally, probably due to a second post-targeting signal recognition event in the ER (Jungnickel & Rapoport 1995). It was postulated that this process is more common in bacteria and yeast, possibly due to fast-growing cells, where translocation would not keep pace with translation (Rapoport 2007).
However, this mechanism is used mostly by soluble proteins, which possess only moderate hydrophobic domains and remain loosely folded after release from the ribosome (Huber et al.
2005a, Huber et al. 2005b, Ng et al. 1996). In yeast, and probably in all eukaryotes, post-translational transport requires the interaction of the protein complexes, Sec61 and Sec62/Sec63, and the luminal chaperone BiP (Deshaies et al. 1991, Panzner et al. 1995). The translocation starts with the binding of a signal sequence to the channel (Figure 2B, (Rapoport 2007)). The cytosolic chaperones release the substrate and the polypeptide is
2007)). The translocation is starting with the binding of a cytosolic chaperone (SecB) (Randall et al. 1997), which is accepted by SecA. After insertion of the polypeptide chain into the channel, the substrate is translocated by a ‘pushing’ mechanism, which is not clear in detail, but possibly caused by the peptide-binding groove of SecA pushing the substrate towards the channel (Economou & Wickner 1994).
Due to gene transfers from the genome of the endosymbiont to the host nucleus, the majority of plastid proteins in diatoms is nucleus-encoded and depends on a targeting- and transport-mechanisms strongly correlated to their bipartite presequences (Apt et al. 2002, Lang et al.
1998). The characteristic bipartite presequence consists of a signal peptide and a transit peptide domain (Figure 3). The N-terminal region is necessary for transport to the ER, while the C-terminal region facilitates the protein to traverse the other envelope membranes (Apt et al. 2002). This import system includes the transport of plastidial proteins via the ER and therefore requires the presence of a signal peptide domain to allow the import into the ER (Apt et al. 2002, Bhaya & Grossman 1991). Once entered the ER lumen, the transition of the pre-protein could occur either by further transport through other transporter proteins present in the remaining membranes (‘translocator model’) or by vesicle trafficking across the periplastidial space (‘vesicle shuttle model’) (Cavalier-Smith 1999, Gibbs 1979, McFadden 1999, van Dooren 2001, Vugrinec et al. 2011). In both cases, the process is initiated by the co-translational import of the polypeptide chain into the ER lumen via the Sec protein translocator after recognition of a signal peptide (Kilian & Kroth 2005). The signal peptide is cleaved off after entering the ER lumen and further transport is possibly mediated by the
‘translocator model’ (Figure 3): the symbiont-derived ER-like machinery (SELMA) in the second outermost membrane (Hempel et al. 2009) would be the next transporter, followed by proteins similar to the Tic/Toc complex of land plant plastids and being situated in the two innermost membranes (Gruber et al. 2007). Here, a protein derived from the cyanobacterial Omp85 is thought to take over the translocation of proteins across the second innermost membrane in P. tricornutum (PtOmp85) and to be the equivalent to the Toc75 protein in the outer envelope of chloroplasts (Bullmann et al. 2010). Transport across the innermost membrane is the same in both models and would be mediated by a Tic related translocon. The ‘vesicle shuttle model’ is based on electron microscopy (Gibbs 1979), where vesicles were found in the periplastidial space and would be responsible for transporting the proteins from one compartment or membrane to the next one (Figure 3).
For integral membrane proteins with multiple transmembrane domains, the translocation is thought to happen in a multistep process and co-translationally by releasing the polypeptide chain into the ER translocon Sec61 to subsequently insert the transmembrane domains into the phospholipid layer (for review see (Martinez-Gil et al. 2011, Shao & Hegde 2011)). The targeting process is also supposed to be dependent on the composition of the presequences and transport across the membranes would probably also occur either via the ‘translocator model’ or the ‘vesicle shuttle model’.
Furthermore, since the protein transport into complex plastids requires the transition of the ER lumen, protein modifications would be feasible. Protein glycosylation during the transport could be possible, since N-glycosylation for example has been shown to occur in microalgae (Baiet et al. 2011, Mathieu-Rivet et al. 2014, Mathieu-Rivet et al. 2013).
Accordingly, any plastidial protein could be involved in glycosylation, which might play a crucial role for protein import into plastids or integration processes of membrane proteins of complex plastids. Besides, it was already shown, that proteins can still be transported across translocation machineries in P. tricornutum despite bulky glycosylation sites (Peschke et al.
2013).
Figure 3: Schematic illustration of a characteristic bipartite presequence of nucleus-encoded plastid proteins in diatoms. The presequence consists of a signal (red) and a transit (green) peptide proceeding the mature protein (yellow). A so-called ‘ASAFAP’-motif is crucial for import into the stroma. Depending on the composition of the presequence, the proteins are directed and transported across the membranes via different translocators into the subcompartments and inserted into the membranes of the plastid. (Gruber et al. 2007, Kilian & Kroth 2005, Lang et al. 1998)
1.3 Nucleotide metabolism in prokaryotes and eukaryotic organisms