Membrane integration
Adding new proteins to our membrane
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 [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 mehanism, (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 [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]
