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Membrane Curvature and the ABC Transporter BmrA: A Yin & Yang Story
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Edité par CCSD ; Biophysical Society -
International audience. Matching protein shape to membrane curvature provides a possible targeting mechanism for membrane proteins. However, how much membrane curvature can affect membrane protein function and reciprocally, how protein conformational change modulates its localization has not been investigated. We have studied BmrA, a bacterial homologue of the human ABC (ATP Binding Cassette) transporter P-glycoprotein, to understand the feedback between protein function, conformation and the physicochemical properties of the surrounding membrane including lipid composition and membrane curvature. Upon ATP binding and hydrolysis, BmrA switches conformation between an apo- and a post-hydrolytic state. The protein was reconstituted in small and giant liposomes (GUVs). We could first demonstrate with small liposomes that different negatively charged and PE lipids stimulate the protein ATPase activity. Furthermore, we showed that the activity is reduced by at least two-fold when decreasing the diameter of the liposomes from 150 nm down to 30 nm. Next, from the analysis of fluorescent proteins' enrichment in nanotubes pulled from GUVs containing BmrA at surface fraction ≈ 1%, we could demonstrate that the trans-membrane domain of BmrA exhibit 2 contrasted spontaneous curvature Cp: a high positive Cp in the apo-state, and a negative Cp (−1/7.5 nm−1) in the post-hydrolytic state. In the apo-state, BmrA sorting kinetics is very slow but leads to a gradual constriction of the tube at constant membrane tension, down to tube radius ≈ 30 nm and an enrichment factor of the order of 30. Eventually, analysis of protein sorting in the presence of ATP suggests that BmrA remains a large fraction of its cycle in the post-hydrolytic state. Globally, this shows that localization of this type of proteins in cell membranes should strongly depend on their activity, which itself is modulated by membrane shape.
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