Processing of preproteins

To study whether there are differences in the processing of aBLSs and nBLSs prepro-teins by proteases, we performed western blot analysis with cell lysates of P. tricor-nutum transformants expressing modified and unmodified presequence:GFP proteins which however, lead to the same phenotype and are both probably located in the periplastidic space. We used protein extracts from a cell line expressing GFP without any presequence (leading to accumulations of GFP in the cytosol) and from a cell line expressing GFP fused to the incomplete presequence of the P. tricornutum oxygen evolving enhancer 1 (PtOEE1; leading to accumulation of GFP in the stroma due to the missing thylakoid import signal) as controls. The calculated molecular size of the mature GFP is 26.9 kDa (according to EditSeq, Lasergene). Using antibodies against GFP, we detected for the GFP construct without any presequence one band presumably of about 27kDain size (Figure 4.4AandB). For the GFP fusion protein encoding the incomplete PtOEE1 we could observe two bands. A smaller one, in size of the mature GFP and a bigger one corresponding to GFP fusion protein with the tran-sit peptide of PtOEE1 (calculated size of 30 kDa; according to EditSeq, Lasergene) (Figure 4.4A andB). In the case of the modified fusion proteins PtOEE1pre∆F:GFP and PtOEE1preFR:GFP, we detected one band of about 27 kDa, corresponding to mature GFP protein without a presequence as this band runs on the same level as the GFP control. (Figure 4.4A and B). For TpNTT3pre:GFP fusion proteins, we observed two bands at the same height of the bands of PtOEE1, corresponding to GFP fusion proteins with and without a transit peptide, respectively, (calculated size 30,5 kDaand 26,9 kDa) indicating that the processing efficiency is not as high as in PtOEE1pre∆F:GFP and PtOEE1preFR:GFP, where only one band can be observed (Figure 4.4A and B).

In view of these findings it is possible that a transit peptidase is located in the pps, which cleaves off the transit peptide-like domain from periplastidic space targeted proteins, but it seems to be unimportant whether those proteins are nBLSs or aBLSs.

This result would fit with the prediction of Cavalier-Smith [27], that a transit peptidase must act within the pps, where it cleaves the transit peptide-like domains of bipartite presequences of pps targeted proteins. In western blot analyses of cells expressing a GFP fusion protein encoding the presequence of a heat shock protein 70 (Hsp70), Gould et al. [51] could also show that the fusion protein is processed to about 27 kDa, approximately the size of GFP. In another study made by Kilian and Kroth [81], it is reported that a modified ATPC:GFP fusion protein (ATP-synthase subunit C;

kDa

Figure 4.4.: Western blot analysis from nBLS and aBLS samples using a GFP antibody. (A)GFP is present in two different sizes (composed of the transit peptide from the gene of interest together with the mature GFP protein and the mature GFP alone). TpNTT3pre1/TpNTT3pre2: two independent protein extractions ofP. tricornutumcells expressing TpNTT3pre:GFP;(B)Schematic representation of the TpNTT3pre:GFP, PtOEE1∆Fpre:GFP and PtOEE1FRpre:GFP fusion proteins. The positions of the signal peptide (sp), the transit peptide (tp) and the GFP are indicated. Scissors mark predicted processing points. Underlined: Calculated fragment size. TpNTT3: T. pseudonananucleotide translo-cator; PtOEE1∆F:P. tricornutum oxygen evolving enhancer 1 (lacking the phenylalanine at the +1 position of the transit peptide-like domain); PtOEE1FR: P. tricornutumoxygen evolving enhancer 1 (substitution of the phenylalanine at the +1 position of the transit peptide-like domain with arginine).

substitution of F to T at signal peptide cleavage site), which also leads to accumulation of GFP in a BLS is not processed, resulting in one band, which corresponds to a GFP fusion protein with a transit peptide. Compared to our findings it is unclear where this processing differences come from. It can be speculated, that during the cloning procedures the recognition site for the transit peptidase is getting damaged or modified in a way, that the peptidase is not able to recognise the cleavage site of the transit peptide-like domain. For further investigations it could be advantageous, when full-length constructs would be used. There might be more recognition and targeting informations, within the protein, which could have an influence on processing. How this additional information might look like is unclear. Even though Felsner et al.

(2010) [43] could show that charges of the transit peptide-like domain play a role in

protein import, it is unlikely that this charge dependent effect occurs here, because the transit peptide-like domains do not differ in their charge.

Conclusion

The results observed in our co-localisation and processing analyses indicate that there is no difference in the localisation and processing between native and artificial prese-quences that lead GFP into the previously described “blob”-like structures. Based on the processing experiments we conclude that a transit peptidase acts within the periplastidic space, where it apparently cleaves off the transit peptide-like domains of accumulating preproteins, regardless whether these substrates represent native quences leading to accumulation of GFP in BLS or modified stromal targeting prese-quence that lead to the same phenotype.

Acknowledgements

This work was supported by the Universität Konstanz and the DFG. We thank D.

Ballert for technical assistance as well as N. Poulsen and N. Kröger (Georgia Tech, Atlanta, GA, USA) for kindly providing theThalassiosira pseudonana transformation vectors.