Abstract
BackgroundMembrane proteins (MPs) play key roles in signal transduction. However, understanding their function at a molecular level is mostly hampered by the lack of protein in suitable amount and quality. Despite impressive developments in the expression of prokaryotic MPs, eukaryotic MP production has lagged behind and there is a need for new expression strategies. In a pilot study, we produced a Drosophila glutamate receptor specifically in the eyes of transgenic flies, exploiting the naturally abundant membrane stacks in the photoreceptor cells (PRCs). Now we address the question whether the PRCs also process different classes of medically relevant target MPs which were so far notoriously difficult to handle with conventional expression strategies.Principal FindingsWe describe the homologous and heterologous expression of 10 different targets from the three major MP classes - G protein-coupled receptors (GPCRs), transporters and channels in Drosophila eyes. PRCs offered an extraordinary capacity to produce, fold and accommodate massive amounts of MPs. The expression of some MPs reached similar levels as the endogenous rhodopsin, indicating that the PRC membranes were almost unsaturable. Expression of endogenous rhodopsin was not affected by the target MPs and both could coexist in the membrane stacks. Heterologous expression levels reached about 270 to 500 pmol/mg total MP, resulting in 0.2–0.4 mg purified target MP from 1 g of fly heads. The metabotropic glutamate receptor and human serotonin transporter - both involved in synaptic transmission - showed native pharmacological characteristics and could be purified to homogeneity as a prerequisite for further studies.SignificanceWe demonstrate expression in Drosophila PRCs as an efficient and inexpensive tool for the large scale production of functional eukaryotic MPs. The fly eye system offers a number of advantages over conventional expression systems and paves the way for in-depth analyses of eukaryotic MPs that have so far not been accessible to biochemical and biophysical studies.
Highlights
Membrane proteins (MPs) represent more than 30% of the cell proteome [1] and play key roles in signal transduction
The successful expression of a functional Drosophila metabotropic glutamate receptor DmGluRA in fly eyes recommended this system for the production of eukaryotic MPs [14] (see Supporting Information: Primer of the fly eye system (Primer S1))
We show that the expression of eukaryotic membrane proteins in the eye of transgenic Drosophila is a powerful tool for the production of functional G protein-coupled receptors (GPCRs), neurotransmitter transporters and channels
Summary
Membrane proteins (MPs) represent more than 30% of the cell proteome [1] and play key roles in signal transduction. Most of them are localized in specialized cells from i.e. the retina for rhodopsin, the lens for aquaporins, the sarcoplasmic reticulum for calcium ATPases and the electric organ of Torpedo for the nicotinic acetylcholine receptor pore. These cells are adapted to the massive production of MPs, which are often densely packed in their respective membrane environment. Membrane proteins (MPs) play key roles in signal transduction.
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