Abstract
The Escherichia coli Min system plays an important role in the proper placement of the septum ring at mid-cell during cell division. MinE forms a pole-to-pole spatial oscillator with the membrane-bound ATPase MinD, resulting in MinD concentration being the lowest at mid-cell. MinC, the direct inhibitor of the septum initiator protein FtsZ, forms a complex with MinD at the membrane, mirroring its polar gradients. Therefore, MinC-mediated FtsZ inhibition occurs away from mid-cell. Min oscillations are often studied in living cells by time-lapse microscopy using fluorescently labelled Min proteins. Here, we show that, despite permitting oscillations to occur in a range of protein concentrations, the enhanced yellow fluorescent protein (eYFP) C-terminally fused to MinE impairs its function. Combining in vivo, in vitro and in silico approaches, we demonstrate that eYFP compromises the ability of MinE to displace MinC from MinD, to stimulate MinD ATPase activity and to directly bind to the membrane. Moreover, we reveal that MinE-eYFP is prone to aggregation. In silico analyses predict that other fluorescent proteins are also likely to compromise several functionalities of MinE, suggesting that the results presented here are not specific to eYFP.
Highlights
The discovery of the green fluorescent protein (GFP) and its deployment as a fluorescent tag to be fused to proteins of interest has brought a real revolution to molecular biology
We introduced the DNA sequence coding for a short flexible linker and the eyfp gene downstream of the minE gene into pBAD33MinCDE giving rise to pBAD33MinCDE-enhanced yellow fluorescent protein (eYFP)
We selected this form because it represents the cytosolic state prior to MinD binding, and we considered the monomer because we asked the question whether dimerization would occur when eYFP is C-terminally fused to MinE
Summary
The discovery of the green fluorescent protein (GFP) and its deployment as a fluorescent tag to be fused to proteins of interest has brought a real revolution to molecular biology. 30 kDa), they do not, in most cases, perturb the localization or function of the protein they are fused to [5,6]. Often the problem is caused by the position of GFP within the fusion protein: where an N-terminal fusion may be perturbed, for instance, a C-terminal fusion may be fully functional [7,8,9]. GFP is too large for the protein of interest, impairing the function/localization of the latter even when placed in a location where a smaller tag would be tolerated [10]
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