Team:Cologne-Duesseldorf/Membrane-integration

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MEMBRANE INTEGRATION

Description

To optimize the luminal conditions of our compartment we focused on integrating new proteins to its membrane. This way we can alter specific properties or supply reactions inside with necessary co-factors. To test the import and integration mechanism, we fused our designed membrane anchors to fluorescent marker proteins and finally integrated the proton pump bacteriorhodopsin into the membrane to acidify our compartment

Introduction

Many reactions rely on optimal conditions like pH and co-factors. Thus, this subproject aims at the optimization of those circumstances through the integration of new membrane proteins, which alter specific properties of the peroxisomal lumen. Such an approach promises to be very useful for metabolic engineering projects as it can help to adjust the pH, provide cofactors to enzymes or increase/decrease the concentrations of metabolites inside to peroxisome. In nature two distinct mechanisms exist, which are used for the integration of membrane proteins into the peroxisomal membrane – a Pex19-Pex3 dependent and an ER-dependent one (2005, Sparkes et al.) .

They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration in the peroxisomal membrane (2003, H.F. Tabak et al.) . We will try to utilize the capability of both mechanisms to incorporate new proteins into the peroxisomal membrane. However, to test whether yeast can integrate and use the foreign proteins in its peroxisomal membrane, we will design three different constructs, which will hopefully give us insights into the mechanisms and its efficiency to incorporate new proteins into the peroxisomal membrane.

As a proof of concept, we will incorporate three proteins through three different approaches into the peroxisomal membrane: (i) mRuby2-PEX26 as a proof for the Pex19-dependent mechanism, (ii) Pex3-mRuby2 itself to showcase the ER-dependent mechanism and (iii) bacteriorhodopsin, a unidirectional proton pump, fused to the N-terminal anchor of Pex3.

Pex19-dependent Mechanism

The exact mechanisms of mPTS binding, Pex3/Pex19 disassembly, mPTS-PMP binding, and release from the Pex3/Pex19 mediated mPTS-PMP docking to the full integration into the membrane are yet unknown (2010, Schueller et al.) . However, general principles of the integration of a new peroxisomal membrane protein (PMP) through Pex19 and Pex3 are studied. Most PMPs feature a membrane targeting signal (mPTS), multiple binding sites for Pex19p, and at least one transmembrane domain (TMD). The mPTS can appear in two different ways, either located in the middle of the primary amino acid sequence, which is the rather complex form, or it can be found at the N-terminal part of the PMP as in Pex25. Pex19p is a cytosolic protein, which recognizes the mPTS of the PMP to be incorporated. In the first step Pex19p attaches to the PMP by binding to the mPTS and acts like a chaperone, guiding it to the peroxisome. Next, Pex19p binds N-terminally to the peroxisomal membrane protein Pex3p, which is attached to the peroxisomal membrane through an N-terminal membrane anchor. This will bring the PMP in close proximity to the peroxisomal membrane. Last, Pex19p initiates the membrane integration of the PMP. .

Additional Sources/References

2001, Jones - Multiple Distinct Targeting Signals in Integral Peroxisomal Membrane Proteins

2004, Jones - PEX19 is a predominantly cytosolic chaperone and import receptor for class 1 peroxisomal membrane proteins

2004, Rottensteiner - Peroxisomal Membrane Proteins Contain Common Pex19p-binding Sites that Are an Integral Part of Their Targeting Signals

2016, Mayerhofer - Targeting and insertion of peroxisomal membrane proteins ER trafficking versus direct delivery to peroxisomes

2016, Hua - Multiple paths to peroxisomes Mechanism of peroxisome maintenance in mammals

2016, Giannopoulou - Towards the molecular mechanism of the integration of peroxisomal membrane proteins

Design

In our design we worked with both, the cytosolic import way and the the ER-dependant one. We first tested our hypothesis and then created membrane anchors that can be fused to you protein of interest C-terminally as well as N-terminal. This allows you to add proteins to the compartment that never been introduced to the peroxisomal membrane before.

Scientific background

Peroxisomal membrane proteins are synthesized on free polysomes in the cytosol and afterwards integrated into the membrane via two major pathways: one dependent on the endoplasmatic reticulum and one dependent on PEX19 and PEX3.

PEX19-dependent

ER-dependent

Experimental Work/Design

In order to test our hypothesis we fused the last 59 amino acids of the C-terminus of human PEX26 (AA 246-305) to a red fluorescent protein, to further elucidate the Pex3/Pex19-dependent import. mRuby is generally used as a marker in combination with a fluorescent microscope to visualize the localization of the fusion protein. The C-terminus of PEX26 contains a helical signal-anchor, which serves as both, a mPTS and transmembrane domain. We designed our construct with mRuby2 fused to the N-terminal side of the PEX26-C-terminus, this way the mRuby should face the cytosolic side of the peroxisomal membrane. Quite similar to our mRuby-PEX26 approach, we designed a construct for the ER-dependent import. Therefore, we fused the mRuby2 fluorescent protein to the N-terminus of Pex3 (AA 1-39). This construct should be N-terminally anchored in the peroxisomal membrane, with mRuby2 again facing the cellular lumen.

Our main goal is to introduce a rather complex membrane protein to the peroxisome that can alter specific traits. For that we fused the Pex3 N-terminus (AA 1-39) to a Halobacterium salinarum bacteriorhodopsin protein (AA 16-262), replacing the first 16 amino acids (Pex3-BacR). The original archaeal bacteriorhodopsin acts as a proton pump by capturing light energy to move protons across the membrane out of the cell. The resulting proton gradient is subsequently converted into chemical energy. Our assumption is that the first transmembrane segment determines the orientation of the following protein and that therefore due to the N-terminal anchoring signal the bacteriorhodopsin will be inserted in reverse orientation, pumping the protons into the peroxisome. This way the pH of the peroxisomal lumen could actively be controlled and adjusted.

Finally, our project involved combining the work of other subteams to verify the localization of our constructs in the peroxisome and analyze the effects they have on the import. Therefore, we are using the superfolded-GFP protein, another fluorescent marker, which is in our case fused to the peroxisomal import sequence PTS1, and a version of Pex11 that is fused to the fluorescent marker Venus. Both markers emit light in the green light spectrum, were as mRuby2 emits light in the red part of the spectrum, giving us a strong contrast and an easy way of differentiating between the two under the fluorescent microscope.

To physically create our constructs, we researched the DNA sequences of bacteriorhodopsin, Pex3 and PEX26 via UniProt and pre-designed our fusion constructs with the software tool „Geneious“. We ordered the synthesis of three separate parts (Pex3, PEX26 and Pex3-BacR) from IDT. To ease out the process of assembling our plasmids, we used the „Dueber Toolbox", containing various parts such as promoters and terminators, to tailor the plasmids specific to your needs. Finally, to combine all the selected parts, we used the „Golden Gate” assembly method.

Results

Not only can we completely control the import into our compartment we also managed to integrate two marker proteins, namely Pex3 from yeast and PEX26 from human cells into our membrane. Furthermore did we manage to integrate the proton pump bacteriorhodopsin that containing seven membrane domains and was a much bigger challenge.

Figure 1: PEX26 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 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.