Redox-based topology analysis (ReTA) of membrane proteins
by Christopher Müller & Andreas J. Meyer
Heidelberg Institute for Plant Science, Heidelberg University, 69120 Heidelberg, Germany.
Biological membranes contain a large number of transmembrane (TM) proteins which fulfill important functions in a broad range of biological processes. In Arabidopsis for example approximately 6,500 proteins accounting for ~25% of the entire genome are predicted to contain one or more transmembrane domains (TMD) . Despite the general biological importance of TM proteins the tools for the analysis of the molecular structure and the protein topology are limited. The most frequently applied approach is the use of bioinformatics tools to predict the protein topology based on the known hydrophobicity of amino acids and experimental analysis of the primary protein sequence. Contradictory results of different prediction algorithms, however, highlight the need for experimental analysis of the orientation and the topology within the membrane.
Figure 1. Differences in the glutathione redox potential (EGSH) across the ER membrane can be exploited for redox-based topology analysis of membrane proteins with roGFP2 as topology reporter. The left panel shows a scheme of a single membrane-spanning protein in the ER membrane N- and C-terminally tagged with roGFP2. roGFP2 is depicted in different colors dependent on its redox state. The images on the right show tobacco leaf cells transiently transfected with the C-terminal fusion protein (top row) and the N-terminal fusion protein (bottom row), respectively. Projections were generated from 20 individual serial sections. Arrows point at the nuclei with the clearly visible perinuclear ring typical for ER labeling. Single images collected at 488 nm and 405 nm were color coded in red and blue, respectively, and then merged. The color of the merged image provides immediate information about the orientation of the membrane protein. Scale bars = 20 µm.
Redox-based topology analysis (ReTA) based on the use of redox-sensitive roGFP2 as reporter enables the experimental analysis of TM proteins in the secretory pathway in vivo . The redox potential of the roGFP2 disulfide equilibrates with the local glutathione redox potential (EGSH) and conformational changes associated with oxidation or reduction of the disulfide lead to distinct alterations of the ratiometric fluorescence properties of roGFP2 [3,4] (see also Müller & Meyer, Redox probes). The glutathione pool is highly oxidized within the ER lumen and thus ER-targeted roGFP2 is fully oxidized. RoGFP2 in the cytosol in contrast is almost fully reduced . The ratiometric properties of roGFP2 thus provide a ~5-fold difference in the fluorescence ratio calculated from images excited at 405 nm and 488 nm. Fusion of roGFP2 to the termini of an ER membrane protein results in roGFP2 facing either the cytosol or the ER lumen and thus two completely different redox environments. Equilibration of roGFP2 with the local EGSH thus results in either a highly reduced reporter on the cytosolic side, or a highly oxidized reporter when its facing the ER lumen (Figure 1). This effect can be utilized to decipher the orientation of a membrane protein by fusion to both N- and C-terminal end. To further decipher the complete topology of a membrane protein, predicted membrane-spanning domains can be truncated from the C-terminus and replaced by roGFP2. As a first draft of the topology, consensus models calculated by ARAMEMNON  based on 18 individual prediction algorithms may provide a good starting point to identify suitable truncations for roGFP2 fusions. If a TMD is predicted correctly roGFP2 fused to the respective truncated protein is expected to appear on the opposite site of the ER membrane and thus show a very different fluorescence ratio.
RoGFP2 fusion proteins can be transiently expressed in leaves of Nicotiana tabacum through Agrobacterium infiltration and subsequently observed on a confocal microscope with excitation at 405 nm and 488 nm . A ratio image from the two raw images can be calculated with standard ratio software packages. Alternatively, it is also possible to simply color code the two raw images and then merge the two pseudo-colored images (Figure 1). Because the intensity of the two individual raw images will change dependent on the redox state of roGFP2 the color change of the merge can already provide conclusive results. Correct interpretation of the latter approach, however, depends on collection of appropriate control images for the cytosol and the ER lumen with exactly the same instrument settings, which is only possible when the different constructs are expressed at similar level. Instead of expressing separate control constructs for cytosol and ER it is also possible to treat cells expressing roGFP2 fusion proteins with reducing or oxidizing agents (e.g. DTT or H2O2) to deliberately drive the redox state of the roGFP2 reporter of a given protein into the opposite direction.
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