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Posttranslational modifications of proteins, such as phosphorylation and dephosphorylation, play critical roles in cellular functions through diverse cell signaling pathways. Protein kinases and phosphatases have been described early on as key regulatory elements of the phosphorylated state of proteins. Tight spatial and temporal regulation of protein kinase and phosphatase activities has to be achieved in the cell to ensure accurate signal transduction. We demonstrated previously that phosphorylation of a membrane protein can lead to its topological rearrangement. Additionally, we found that both the rate and extent of topological rearrangement upon phosphorylation are lipid charge– and lipid environment–dependent. Here, using a model membrane protein (the bacterial lactose permease LacY reconstituted in proteoliposomes) and a combination of real-time measurements and steady-state assessments of protein topology, we established a set of experimental conditions to dissect the effects of phosphorylation and dephosphorylation of a membrane protein on its topological orientation. We also demonstrate that the phosphorylation-induced topological switch of a membrane protein can be reversed upon protein dephosphorylation, revealing a new regulatory role for phosphorylation/dephosphorylation cycles. Furthermore, we determined that the rate of topological rearrangement reversal is correlated with phosphatase activity and is influenced by the membrane's lipid composition, presenting new insights into the spatiotemporal control of the protein phosphorylation state. Together, our results highlight the importance of the compartmentalization of phosphorylation/dephosphorylation cycles in controlling membrane protein topology and, therefore, function, which are influenced by the local lipid environment of the membrane protein.

The N-terminal six-transmembrane domain (TM) bundle of lactose permease of Escherichia coli is uniformly inverted when assembled in membranes lacking phosphatidylethanolamine (PE). Inversion is dependent on the net charge of cytoplasmically exposed protein domains containing positive and negative residues, net charge of the membrane surface, and low hydrophobicity of TM VII acting as a molecular hinge between the two halves of lactose permease (Bogdanov, M., Xie, J., Heacock, P., and Dowhan, W. (2008) J. Cell Biol. 182, 925–935). Net neutral lipids suppress the membrane translocation potential of negatively charged amino acids, thus increasing the cytoplasmic retention potential of positively charged amino acids. Herein, TM organization of sucrose permease (CscB) and phenylalanine permease (PheP) as a function of membrane lipid composition was investigated to extend these principles to other proteins. For CscB, topological dependence on PE only becomes evident after a significant increase in the net negative charge of the cytoplasmic surface of the N-terminal TM bundle. High negative charge is required to overcome the thermodynamic block to inversion due to the high hydrophobicity of TM VII. Increasing the positive charge of the cytoplasmic surface of the N-terminal TM hairpin of PheP, which is misoriented in PE-lacking cells, favors native orientation in the absence of PE. PheP and CscB also display co-existing dual topologies dependent on changes in the charge balance between protein domains and the membrane lipids. Therefore, the topology of both permeases is dependent on PE. However, CscB topology is governed by thermodynamic balance between opposing lipid-dependent electrostatic and hydrophobic interactions.

International audience; Gap junctions are intercellular channels that allow the passage of ions, small molecules, and second messengers that are essential for the coordination of cellular function. They are formed by two hemichannels, each constituted by the oligomerization of six connexins (Cx). Among the 21 different human Cx isoforms, studies have suggested that in the heart, Cx40 and Cx43 can oligomerize to form heteromeric hemichannels. The mechanism of heteromeric channel regulation has not been clearly defined. Tissue ischemia leads to intracellular acidification and closure of Cx43 and Cx40 homomeric channels. However, coexpression of Cx40 and Cx43 in Xenopus oocytes enhances the pH sensitivity of the channel. This phenomenon requires the carboxyl-terminal (CT) part of both connexins. In this study we used different biophysical methods to determine the structure of the Cx40CT and characterize the Cx40CT/Cx43CT interaction. Our results revealed that the Cx40CT is an intrinsically disordered protein similar to the Cx43CT and that the Cx40CT and Cx43CT can interact. Additionally, we have identified an interaction between the Cx40CT and the cytoplasmic loop of Cx40 as well as between the Cx40CT and the cytoplasmic loop of Cx43 (and vice versa). Our studies support the “particle-receptor” model for pH gating of Cx40 and Cx43 gap junction channels and suggest that interactions between cytoplasmic regulatory domains (both homo- and hetero-connexin) could be important for the regulation of heteromeric channels.

The translocation of the catalytic domain through the membrane of the endosome to the cell cytoplasm is a key step of intoxication by botulinum neurotoxin (BoNT). This step is mediated by the translocation (T) domain upon endosome acidification, although the mechanism of interaction of the T domain with the membrane is still poorly understood. Using physicochemical approaches and spectroscopic methods, we studied the interaction of the BoNT/A T domain with the membrane as a function of pH. We found that the interaction with membranes does not involve major secondary or tertiary structural changes, as reported for other toxins like diphtheria toxin. The T domain becomes insoluble around its pI value and then penetrates into the membrane. At that stage, the T domain becomes able to permeabilize lipid vesicles. This occurs for pH values lower than 5.5, in agreement with the pH encountered by the toxin within endosomes. Electrostatic interactions are also important for the process. The role of the so-called belt region was investigated with four variant proteins presenting different lengths of the N-extremity of the T domain. We observed that this part of the T domain, which contains numerous negatively charged residues, limits the protein-membrane interaction. Indeed, interaction with the membrane of the protein deleted of this extremity takes place for higher pH values than for the entire T domain. Overall, the data suggest that acidification eliminates repulsive electrostatic interactions between the T domain and the membrane, allowing its penetration into the membrane without triggering detectable structural changes.