Abstract
Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding. We previously demonstrated that the lipid environment is also a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition. However, the effect on protein topology of post-assembly phosphorylation of amino acids localized within initially cytoplasmically oriented extramembrane domains has never been investigated. Here, we show in a controlled in vitro system that phosphorylation of a membrane protein can trigger a change in topological arrangement. The rate of change occurred on a scale of seconds, comparable with the rates observed upon changes in the protein lipid environment. The rate and extent of topological rearrangement were dependent on the charges of extramembrane domains and the lipid bilayer surface. Using model membranes mimicking the lipid compositions of eukaryotic organelles, we determined that anionic lipids, cholesterol, sphingomyelin, and membrane fluidity play critical roles in these processes. Our results demonstrate how post-translational modifications may influence membrane protein topology in a lipid-dependent manner, both along the organelle trafficking pathway and at their final destination. The results provide further evidence that membrane protein topology is dynamic, integrating for the first time the effect of changes in lipid composition and regulators of cellular processes. The discovery of a new topology regulatory mechanism opens additional avenues for understanding unexplored structure-function relationships and the development of optimized topology prediction tools.
Highlights
Membrane protein topology and folding are governed by structural principles and topogenic signals that are recognized and decoded by the protein insertion and translocation machineries at the time of initial membrane insertion and folding
We previously demonstrated that the lipid environment is a determinant of initial protein topology, which is dynamically responsive to post-assembly changes in membrane lipid composition
We demonstrated that topogenic signals can be decoded by the membrane lipid profile during initial membrane protein insertion (14 –17) and that membrane proteins can undergo transmembrane domains (TMDs) flipping after initial assembly in response to changes in the lipid environment, both in vivo and in vitro (18 – 21)
Summary
Phosphorylation of a Membrane Protein Can Lead to a Change in Its Topological Arrangement—We sought to determine the effects of one of the most common post-translational modifications of EMDs on membrane protein dynamic organization, as a function of the lipid environment. We previously demonstrated that changing the net charge of cytoplasmic EMDs C2/C4/C6 of LacY (Fig. 1A) from ϩ2/ϩ2/ϩ2 (WT LacY) to Ϫ2/Ϫ2/Ϫ2 results in topological inversion of the N-terminal six-TMD bundle of LacY in wild type E. coli cells, whereas a change to Ϫ2/0/Ϫ2 did not result in inversion [19]. Native LacY does not contain any known phosphorylation site; we engineered phosphoinositide-dependent protein kinase 1 (PDK1) and liver kinase B1 (LKB1) EMD sites based on the lack of charged amino acids in their kinase consensus sequences. We sought to determine the effect of phosphorylation on steady state LacY topology when reconstituted in proteoliposomes made of E. coli total lipids.
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