<figcaption><b>Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:</b>.<br>
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<span style="color: rgb(0, 0, 0);">Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for membrane marker mRuby-PEX26 is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.</span></figcaption>
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<p>In order to have full control over the amount of expressed protein, we designed our plasmids with the inducible galactose promoter "pGAL1". Not only were we able to see that our fluorescent marked protein anchors from <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> would localize at specific points inside our cells but also to show that it was in deed the peroxisome they were accumulating at. For that we coexpressed each of our fluorescent membrane anchors together with a GFP protein that was fused to a PTS1 sequence and thus imported into the peroxisome.Fluorescent microscopy was used to colocalize both, the green fluorescing GFP and the red fluorescing mRuby and it is clearly visible, that our anchors integrated into the peroxisomal membrane.</p>
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<h4>Figure 1: <span style="color: rgb(0, 0, 0);">Bacteriorhodopsin expression and integration</span></h4>
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Finally we used the same approach to direct a mRuby-tagged <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> as well as <a href="http://www.uniprot.org/uniprot/P28795">Pex3</a> and <a href="http://www.uniprot.org/uniprot/Q7Z412">PEX26</a> were successfully integrated into the membrane of our compartment. Since <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a> is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.
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<h4>Outlook</h4>
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<p>The ultimate goal of this subproject is, to have a complete set of ready to transform membrane proteins that could be combined with any promoter to create the optimal conditions for each desired situation. Besides <a href="http://www.uniprot.org/uniprot/P02945">bacteriorhodopsin</a>, we also started to work with sugar translocators, since yeast does not posses the ability to import it into or export it from the peroxisome. This would open up a whole new chapter of peroxisomal usage, from example as a temporary storage compartment.
<figcaption><b>Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:</b>.<br>
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<span style="color: rgb(0, 0, 0);">Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for membrane marker mRuby-PEX26 is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.</span></figcaption>
Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:. Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for membrane marker mRuby-PEX26 is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.
In order to have full control over the amount of expressed protein, we designed our plasmids with the inducible galactose promoter "pGAL1". Not only were we able to see that our fluorescent marked protein anchors from Pex3 and PEX26 would localize at specific points inside our cells but also to show that it was in deed the peroxisome they were accumulating at. For that we coexpressed each of our fluorescent membrane anchors together with a GFP protein that was fused to a PTS1 sequence and thus imported into the peroxisome.Fluorescent microscopy was used to colocalize both, the green fluorescing GFP and the red fluorescing mRuby and it is clearly visible, that our anchors integrated into the peroxisomal membrane.
Figure 1: Bacteriorhodopsin expression and integration
Fluorescent microscopy of bacteriorhodopsin coexpressed with sf-GFP:. Microscopy pictures were taken with a Zeiss Elyra PS. Peroxisomes were labeled with GFP-PTS1 (green). The green fluorescent spots (on the right) shows a typical peroxisomal shape. The signal for the membrane marked bacteriorhodopsin is shown in red (on the left). Both signals show colocalization when merged (middle), which indicates that the protein gets integrated into the membrane.
Finally we used the same approach to direct a mRuby-tagged bacteriorhodopsin to our compartment. In coexpressing it with the same GFP as in the previous steps, we could show that the bacteriorhodopsin as well as Pex3 and PEX26 were successfully integrated into the membrane of our compartment. Since bacteriorhodopsin is a rather complex protein, we're very optimistic about integrating other proteins into the membrane using the same approach.
Outlook
The ultimate goal of this subproject is, to have a complete set of ready to transform membrane proteins that could be combined with any promoter to create the optimal conditions for each desired situation. Besides bacteriorhodopsin, we also started to work with sugar translocators, since yeast does not posses the ability to import it into or export it from the peroxisome. This would open up a whole new chapter of peroxisomal usage, from example as a temporary storage compartment.
