NTT1 and NTT2. The lack of a characteristic plastid presequence, low sequence similarities to any NTT investigated so far and an accumulation of positively charged amino acids emphasised the peculiarity of PtNTT5. The latter indicated ER localisation, since it is known that ER proteins contain several positively charged amino acids at the N-terminus, flanked by hydrophobic residues (for review see (Zimmermann et al. 2011)). Interestingly, this accumulation was present at the C-terminus of PtNTT5. Moreover, C-terminal GFP fusion apparently perturbs the targeting and/or folding processes, since all truncated versions of PtNTT5 ended in patchy accumulations inside the cells, maybe associated to the plastid, but not very likely for an insertion into a membrane surrounding the plastid. Anyhow, PtNTT5 harbours hydrophobic transmembrane (TM) domains, thus it is conceivable that it has to integrate into a membranous environment. It has been reported that a vesicle-like network exists in the periplastidial space of P. tricornutum, but its function remains to be unknown (Flori et al. 2016). One possible function could be the temporary storage of defective proteins prior to degradation, which can be kept either in a soluble state inside these vesicles or inserted into the vesicle membrane, if the proteins are hydrophobic like in the case of PtNTT5. In contrast, N-terminal GFP fusion in combination with C-terminal truncations of the protein changed the localisation, indicating that these features are crucial for a proper targeting function. Additionally, the targeting process of PtNTT5 was suggested to take place post-translationally (Chu et al. 2016b), and thus would differ from other nucleus-encoded plastid proteins, like the fucoxanthin chlorophyll proteins (FCPs), which were suggested to be targeted co-translationally depending on their precursor polypeptides (Bhaya & Grossman 1991). Surprisingly, despite the lack of a signal peptide domain and even with an N-terminal attachment of GFP, PtNTT5 was directed to the ER membrane. This implies that the transporter protein was first translated prior to the insertion into the ER membrane (see Figure 26A).
It would be conceivable that PtNTT5 harbours a so-called C-tail anchor (TA) domain. Such domains contain hydrophobic amino acids, which help in holding transmembrane polypeptides within the phospholipidlayer, being reported to be found in mitochondrial, ER and chloroplast membranes (Borgese et al. 2007, Borgese et al. 2003, Borgese & Fasana
functions in both targeting to organelles and insertion into the membrane in a post-translational manner (Borgese et al. 2003). Several in vitro studies revealed that insertion pathways of TA proteins can range from spontaneous unassisted insertion into the membranes to energy requiring mechanisms, mediated by chaperones or protein complexes (Abell et al. 2007, Borgese & Fasana 2011, Shao & Hegde 2011). Furthermore, it was shown that insertion pathways of TA proteins and multi-span proteins might overlap (Dukanovic &
Rapaport 2011, Otera et al. 2007). For PtNTT5, a similar scenario is possible. After translation, the protein would be kept in an unfolded state, with the help of cytosolic chaperones. A C-terminal post-targeting signal could be present in PtNTT5, facilitating transport to the ER. This mechanism could proceed without the interaction of a signal recognition particle (SRP), the protein is then drawn towards the ER membrane by the hydrophobic core of a signal peptide-like domain at the C-terminus. A similar phenomenon without the presence of a SRP was also described by (Jungnickel & Rapoport 1995), where the translocon Sec61 complex was demonstrated to be sufficient for binding the ribosome to the ER membrane, due to the interaction of the hydrophobic part of the signal sequence with the Sec61 complex and phospholipids (Martoglio et al. 1995). Since Sec61 is known to mediate co- and post translational translocation across the ER membrane for soluble proteins (Rapoport 2007), there might be similar mechanism for hydrophobic proteins, either involving the same or a translocation system homologous to the Sec system.
After translocation across a Sec-like channel, PtNTT5 could then be folded from the inside of the cER lumen into the cER membrane, possibly stepwise and assisted by chaperones, starting from the C-terminus due to the hydrophobic domains.
The truncations of the C-terminal part of PtNTT5 could define the location of a potential targeting domain. Possibly the ninth TM domain contains a crucial targeting domain, since the shorter versions which lack this area were not even entering the secretory pathway and the subcellular GFP fluorescence was observed in the cytosol. This is a further indication for the post-translational insertion of PtNTT5.
In contrast, the C-terminal GFP attachment to PtNTT5 showed a clearly different GFP-pattern: GFP seemed to be accumulated in or associated to the plastid. Most likely, the recognition of the targeting domain was still taking place in that case and the protein was also translocated across a Sec-like translocon (Figure 26B). However, the insertion of the membrane protein would be impaired, possibly by the GFP-molecule impeding the integration process of the membrane proteins or resulting in an unsteady insertion into the membrane due to restricted folding properties. A consequence would be the degradation of the protein. The symbiont-derived ERAD (ER-associated degradation) - like machinery (SELMA) is suggested to be situated in the second outermost membrane of diatoms and being part of a preprotein transport system across this membrane (Hempel et al. 2009).
Furthermore, components of the SELMA are possibly involved in ubiquitination and proteasomal subunits are present in the periplastidial space (Sommer et al. 2007, Stork et al.
2012). Accordingly, it is very likely that the PPS is a place in which proteins that are misled to plastidial compartments are degraded. Soluble proteins destined for degradation could be released from the cER lumen to the cytosolic proteasome via the ERAD translocation machinery, which is most likely retained in diatoms from the host ER. Instead highly hydrophobic membrane proteins could be guided through the SELMA towards the PPS and might be ubiquitinated in parallel, followed by a direct insertion into the PPS-membrane in order to keep the hydrophobic proteins in a membranous environment prior to degradation.
could be guided to this location and why GFP accumulates inside the cells in patchy structures, probably near to the plastid or associated to plastidial membranes.
Figure 26: Schematic illustration of a putative post-translational targeting and insertion process for PtNTT5 GFP fusion proteins. A) N-terminal GFP fusion: the protein is kept in an unfolded state with the help of chaperones and is translocated through a Sec-like translocon into the cER lumen. The C-terminal targeting and tail-anchor domain help to insert the protein into the ER membrane and to keep it within the membrane. B) C-terminal GFP fusion: the protein is also translocated into the cER lumen across a Sec-like translocon. After unsuccessful integration of the protein into the ER membrane, it is determined for degradation. Therefore, it could be guided across the SELMA into the PPS. For defective soluble proteins it is feasible to release them directly into the cytosol for proteasomal degradation. The hydrophobic proteins in the PPS might be ubiquitinated prior to integration into vesicle-like structures. The formation of vesicles would not require the ATP-dependent chaperone interaction and thus could keep the proteins temporarily in a membranous environment before they will be introduced into the degradation process. Alternatively, the proteins could form aggregates, which are finally also degraded. TM = transmembrane domain; Sec-L = Sec-like translocation channel; ERAD = ER-associated degradation system;
SELMA = symbiont-derived ERAD-like machinery; ER = chloroplast ER; PPS = periplastidial space
7.5 What tools could help to locate proteins in plastidial