Dynamic Lipid-dependent Modulation of Protein Topology by Post-translational Phosphorylation

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.

PGC‐1a integrates a metabolism and growth network linked to caloric restriction

“…The PGC‐1a‐associated change in cellular lipid metabolism shown here featured differences in chain length and degree of saturation of phospholipids. These membrane‐resident lipids are likely to impact cellular membrane structure and fluidity both within the cell and at the cell surface (Harayama & Riezman, ), and their influence may even extend to the function of proteins embedded within cellular lipid layers (Vitrac et al, ). The shift in mitochondrial energy metabolism toward increased respiration and enhanced metabolic flexibility was not confined to mitochondrial pathways but extended more broadly to redox metabolism including levels, redox ratios, and chemical properties of NAD(P)H. The detection of discrete nuclear and cytosolic pools of NAD(P)H corroborates recent reports (Cambronne et al, ; Ryu et al, ), but the fact that they appear to be independently responsive to PGC‐1a status raises new questions about how these pools are established, maintained, and independently regulated.…”

How bilayer properties influence membrane protein folding

“…Intriguingly, even charges from posttranslational modifications can affect protein topology. When phosphorylation sites were introduced into LacY, inversion occurred upon addition of kinases 59 . However, because cholesterol and sphingomyelin also affect LacY topology, and because these components affect the electrostatic and mechanical properties of the bilayer, direct correlations between topology and charge were not drawn 59 .…”

Stable membrane orientations of small dual-topology membrane proteins

“…For example, the translocon initially misses 2 of the 6 TMHs of human aquaporin 1, which insert only later (4,5). The transporter LacY from Escherichia coli was shown to flip the topology of 7 of its 12 TMHs upon manipulation of the lipid composition of the membrane or upon phosphorylation, well after the protein has finished translation (6)(7)(8). Finally, the N-terminal TMH in the holin family of phage proteins inserts into the bacterial inner membrane only when the proton-motive force is reduced below a critical threshold (9).…”