In a recent study published in Nature, researchers used cryo-electron microscopy (cryo-EM) to visualize the structure and dynamics of a membrane microdomain in yeast cells. They aimed to understand how proteins and lipids work together to create a functional zone within the plasma membrane that responds to mechanical stress.
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Background
The plasma membrane is the outer layer of a cell, separating it from its environment and regulating molecular exchange. It consists of a lipid bilayer with hydrophilic heads and hydrophobic tails, along with various proteins. The plasma membrane has distinct regions called membrane microdomains, which have different lipid and protein compositions and are involved in processes like signal transduction and membrane trafficking.
Studying these microdomains is challenging because it requires observing lipids and proteins in their natural state. Many techniques involve chemical modifications or fluorescent labels, which can change their properties. Cryo-EM is an emerging technique that overcomes these limitations by allowing the observation of biological samples in their near-native state, at high resolution, and in three dimensions.
About the Research
In this paper, the authors studied a membrane microdomain called the membrane compartment containing Can1 (MCC) in the yeast Saccharomyces cerevisiae. The MCC is a furrow-like structure supported by a protein coat made of two related proteins, Pil1 and Lsp1, which are part of the Bin-amphiphysin-Rvs (BAR) domain protein family.
BAR-domain proteins are known to sense and create membrane curvature by interacting with negatively charged lipids on the membrane surface. The MCC is also mechanosensitive, meaning it can detect and respond to membrane tension or pressure changes by flattening and releasing sequestered factors that affect signaling or transport functions.
The researchers isolated MCC microdomains from yeast cells using a gentle purification process that preserved the Pil1 and Lsp1 protein lattice bound to the plasma membrane bilayer. They then used cryo-EM to reconstruct the three-dimensional (3D) structure of the MCC microdomains, which appeared as helical filaments of varying diameters. They resolved the structure of the Pil1 and Lsp1 proteins, as well as the lipid density within the membrane bilayer, at a resolution of around 3.2 Å.
Research Findings
The study revealed a remarkable organization of lipids within the MCC microdomain and identified specific interactions between the Pil1 and Lsp1 proteins and various lipids. They found that the N-terminal region of Pil1 and Lsp1 contains an amphipathic helix, a feature common in BAR-domain proteins that helps them bind to membranes.
This helix is embedded in the membrane's cytoplasmic leaflet and stabilized by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), a negatively charged lipid crucial for membrane dynamics and signaling.
PI(4,5)P2 also creates a pocket on the membrane-facing surface of Pil1 and Lsp1, where another negatively charged lipid, phosphatidylserine (PS), is bound. The researchers observed voids in the membrane density beneath the amphipathic helix, likely due to sterols like ergosterol, the main sterol in yeast cells. Sterols modulate membrane fluidity and permeability and are often associated with membrane microdomains.
Lipid density assignments were confirmed by reconstituting MCC microdomains in vitro using recombinant Pil1 protein and lipid mixtures of known composition. Molecular dynamics simulations verified the positioning and interactions of individual lipid molecules within the MCC microdomain.
PI(4,5)P2 and sterols were found to be crucial for the insertion and stabilization of the amphipathic helix, with PS binding enhanced by the presence of sterols. Additionally, the binding of these lipids reduced the mobility of other lipids within the MCC microdomain, creating a distinct lipid environment.
Furthermore, 3D variability analysis (3DVA) was performed to investigate the dynamics of the MCC microdomain. It showed that the Pil1 and Lsp1 lattice exhibited spring-like stretching and compression, correlated with changes in lipid density. Stretching the lattice disrupted the binding of PI(4,5)P2 and PS and blurred the pattern of sterols, suggesting these lipids became more mobile in response to mechanical stress.
Applications
The paper demonstrates how proteins and lipids create a functional membrane microdomain that senses and responds to mechanical stress. The stretching of the Pil1 and Lsp1 lattice releases lipids that affect other proteins in the MCC microdomain. Mutations in the lipid-binding sites of Pil1 and Lsp1 alter the microdomain's shape and function, impacting yeast cell growth and stress response.
The authors also demonstrate the effectiveness of cryo-EM in visualizing lipids and proteins in membrane microdomains without chemical modifications. This technique can be applied to study other membrane microdomains across different organisms, revealing their structure and function.
Conclusion
Cryo-EM effectively revealed the structure and dynamics of a membrane microdomain in yeast cells, showing how protein-lipid interactions create a lipid environment sensitive to mechanical stress. The outcomes offered insight into the formation and regulation of membrane microdomains and their role in cellular processes.
Cryo-EM also holds the potential for studying membrane lipids and proteins in their near-native state, resolving long-standing questions about the architecture and nature of lipid microdomains.
Journal Reference
Kefauver, JM., et al. (2024). Cryo-EM architecture of a near-native stretch-sensitive membrane microdomain. Nature. DOI: 10.1038/s41586-024-07720-6
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