Membrane integration

Introducing new proteins to our membrane

Introduction

Membrane Integration

Many reactions rely on optimal conditions like pH and [...]. 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-dependent and an ER-dependent one [1,2].

They rely on a so called mPTS sequence, that is used to mark the proteins for transport to and integration in the peroxisomal membrane [3]. 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 to 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 [4]. 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. [3]

Experimental Work/Design

In order to test our hypothesis we fuse the last 59 amino acids of the C-terminus of human PEX26 (AA 246-305) to an red fluorescent protein, called mRuby2, 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, we are 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 (i) the superfolder-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.

Sources/References

[1] I.A. Sparkes, C. Hawes, A. Baker, AtPEX2 and AtPEX10 are targeted to peroxisomes independently of known endoplasmic reticulum trafficking routes, Plant Physiol. 139 (2005) 690–700.

[2] H.F. Tabak, J.L. Murk, I. Braakman, H.J. Geuze, Peroxisomes start their life in the endoplasmic reticulum, Traffic 4 (2003) 512–518.

[3] 2010, Schueller - The peroxisomal receptor Pex19p forms a helical mPTS recognition domain

[4] 2016, Liu - Assembly of Peroxisomal Membrane Proteins via the Direct Pex19p-Pex3p Pathway