In vivo topology and function of Tic110

The in vivo topology of Tic110 was analyzed using the genetically encoded tag miniSOG.

MiniSOG is capable of producing singlet oxygen, that in presence of diaminobenzidine leads to the production of an osmiophilic precipitate and can be visualized during electron microscopy.

Three distinct miniSOG-tagged Tic110 proteins were generated. MiniSOG was placed in both external loops that are protruding into the intermembrane space as well as at the very C-terminus as a control for stromal localization. It was possible to successfully target and express all miniSOG-tagged variants transiently in tobacco chloroplasts, which was confirmed by either fluorescence microscopy or immunoblot analyses by using antisera against atTic110 and miniSOG. Immunoblot analyses could confirm successful expression under the control of both promoters (35S and endogenous Tic110), although expression was higher using the 35S promoter. Photooxidation was carried out using transiently infected tobacco leave tissues and in some cases, a dot-like structure in the intermembrane space could be observed that might indicate a specific reaction of Tic110-miniSOG (residues 226-311) generated singlet oxygen with DAB.

Due to our technical setup it was not possible to visually control the photooxidation process.

However, as the ultra-structure of chloroplasts was still preserved, it could be argued that blue light illumination had no detrimental effect on the tissue. To get a stronger signal a possibility would be to create tandem-miniSOG fusion proteins. However, this would increase the risk of interference of the tag with the structure and/or function of the tagged protein. So far, the miniSOG-tagging method has been established in a variety of organisms

5 Discussion or tissues (cell cultures, C. elegans, brain tissue of mouse). None of these organisms contain chlorophyll which absorbs predominantly blue (400 nm - 500 nm) as well as the red (600 nm - 700 nm) light. These wavelengths partially overlap with the absorption maximum of miniSOG. Thus, it cannot be excluded that the presence of chlorophyll interferes with the blue light mediated singlet oxygen production by miniSOG. One possibility to avoid that drawback is to use etioplasts. Etioplasts derive from plants that have not been exposed to light. However, as Tic110 is present in all kinds of plastids it should remain possible to perform a photooxidation experiment with a functionally expressed Tic110. Even though the detection of an unambiguous precipitate in the intermembrane space will remain problematic, it should be possible to discriminate between stroma and intermembrane space. By using the C-terminal control it should be possible do clearly identify parts that are located in the stroma, which are not visible by using intermembrane space-tagged Tic110.

Improvements could be achieved by analyzing how much DAB reagent is required for image detection. This has to be considered very carefully, avoiding under- and over-labeling of the tissue, and multiple parameters, such as protein expression level, labeling density, fixation conditions, heavy metal staining and microscope settings play a role in the EM visualization of the labeled product. All these parameters must be carefully addressed before further experiments can lead to promising results.

The correct function of a protein is affiliated with its native topology. Although it could be shown that targeting to the chloroplast is not impaired by inserting miniSOG in various parts of Tic110 it cannot be excluded that its native topology is altered. To confirm the correct assembly of the channel-like structure, complementation lines of heterozygous TIC110/

tic110 Arabidopsis plants with the various miniSOG-tagged Tic110 constructs under the control of its native promoter were generated. Only if the variants can complement the phenotype of heterozygous mutant plants, it can be deduced that topology and thus function is not impaired in transgenic plants. To this end, TIC110/tic110 plants were transformed with all three constructs, however so far no homozygous progeny plants could be isolated.

Furthermore, in case of transformation with miniSOG at residue 257, the plants look even more chlorotic than their heterozygous parents. The plants look obviously chlorotic and yellowish, which implies that the import machinery is not functioning properly, and required photosynthetic compounds are not assembled correctly. This effect became even more prominent when plants were grown on soil and not on sugar-containing plates. At this stage, miniSOG-tagged Tic110_ims1 transformed plants are hampered in assembling a fully working photosynthetic machinery. So far it is not clear whether this chlorotic phenotype is due to the absence (non-expression of protein) of miniSOG-tagged Tic110_ims1 or dominant negative effects. Especially regarding the fact that close to this region the proposed transit peptide of

5 Discussion preproteins binding domain is localized (Inaba et al., 2005), one can speculate that due to competition with intrinsic Tic110 interaction with incoming preproteins is negatively affected.

It has been shown by others with different truncated constructs that they have a dominant negative effect on intrinsic Tic110 (Inaba et al., 2005). In this case, immunoblot analysis showed that intrinsic Tic110 is not reduced in transgenic plants, although plants look more chlorotic. However, the dominant negative effect is still more likely as the absence of miniSOG-tagged proteins simply should result in a heterozygous Tic110-like phenotype because no interference is expected, although it cannot be excluded that regulations on the RNA level occurred, leading to feedback mechanisms. So far, detection of miniSOG-tagged Tic110 with antiserum against miniSOG in stable Arabidopsis lines was not successful, thus no conclusion about protein levels of the recombinant protein can be drawn yet.

It would be interesting to see if the actual amount of some photosynthetic precursors such as pSSU in the chloroplast is reduced in transgenic plants compared to untransformed heterozygous plants.

As these complementation studies do not allow to draw reliable conclusion concerning correct topology of the Tic110 fusion proteins, another idea would be to perform limited proteolysis with intact chloroplasts from transgenic plants or transiently expressing tobacco leaves. Trypsin can penetrate the outer envelope but not the inner envelope. The degradation pattern can now be tracked with the miniSOG specific antibody, at least for transiently expressing tobacco leaves. The signal of domains of the intermembrane space loops carrying miniSOG should be disappear very early during protease treatment, whereas the signal using the construct with miniSOG at the C-terminus should remain stable even with increasing trypsin concentrations, as miniSOG at that position is protected by the inner membrane.

Beside genetic tagging, the in vivo topology of Tic110 could also be addressed by spatially restricted enzymatic tagging. Ascorbate peroxidase (Apex) can be used, which normally catalyses the reduction of H₂O₂ and the simultaneous oxidation of ascorbate. However, upon mutation of the active centre the enzyme also accepts other substrates, e.g. biotin-phenol (Rhee et al., 2013). In the presence of H₂O₂ the mutated Apex (mApex) will covalently transfer biotin-phenol to electron rich amino acids like tyrosine or histidine to every peptide in close proximity. Modified amino acids can be identified by mass spectrometry. This approach was performed by Rhee et al. who targeted mApex to different mitochondrial sub-compartments and solved the topology of many outer and inner mitochondrial membrane proteins (Rhee et al., 2013). Recently, this approach was even more improved by applying desthiobiotin as a substrate for mAPEX (Lee et al., 2017). By targeting mApex to different sub-compartments of the chloroplast – equivalent to the mitochondria – namely stroma,

5 Discussion inner envelope membrane or intermembrane space, it will be possible to tag Tic110 from different orientations which should result in differentially labeled pattern identified with mass spectrometry. This experimental approach would not only allow to determine the topology of a single membrane-spanning protein like Tic110, but offers also great applicability to map the architecture of chloroplast membrane proteins in living cells in a high throughput manner.

Tic110 contains six fully conserved cysteines which might be involved in disulfide bridge formation. In vitro data showed that mutation of at least two of them influenced the behavior of the protein in solution. To analyze the role of the cysteines in vivo, heterozygous TIC110/tic110 plants were transformed with various mutated cysteine variants. To this end, the presence of a construct having a mutated cysteine at position 526 in a heterozygous background led to a chlorotic phenotype, although the total amount of Tic110 was unaffected compared to wild type plants. It has to be mentioned that this cysteine is not among the six fully conserved ones, nevertheless, it is present in many plant species. Interestingly, the protein amount of Tic110 in plants transformed with the construct carrying C526S was higher than in the heterozygous parent plants, suggesting that the mutated protein is indeed expressed at normal levels. It remains to be established if this mutation has an impact on e.g. protein import or interaction with other proteins. Furthermore, the in vivo role of this cysteine under stress conditions could also be addressed.