Periodic Reporting for period 3 - MemDense (Cellular control of membrane protein density in the endoplasmic reticulum via the unfolded protein response)
Période du rapport: 2023-04-01 au 2024-09-30
The MemDense project aims to identify how eukaryotic cells control the ratio of proteins and lipid in their membranes. Despite the enormous relevance of these biomolecules for virtually every cellular function, it remains unknown how cells balance the production of proteins and lipids. It has been known for decades that the different organelles (e.g. the endoplasmic reticulum (ER) or mitochondria) feature remarkably distinct protein-to-lipid ratios. Barely anything is known about, how different organelles control the exchange and flux of lipids for maintaining these largely distinct packing densities, and if an overcrowding of proteins in the membrane induces cellular stress.
We pursue the hypothesis that the protein-to-lipid ratio is sensed via the unfolded protein response (UPR), a conserved pathway, which controls the relative rate of lipid and protein production and which serves as a diagnostic marker for ER stress. We study this mechanistic connection in two model systems: The baker's yeast Saccharomyces cerevisiae and cultured, mammalian cells. We aim to understand, how communication and exchange of lipid metabolites between organelles support cell functions during acute, prolonged, and chronic ER stresses.
The MemDense project is important for society, because a large number of chronic inflammatory and complex metabolic diseases, such as type II diabetes and non-alcoholic fatty liver disease have been firmly associated with chronic ER stress, while the molecular basis of ER stress remains unresolved. The MemDense project establishes new experimental pipelines to test the contribution of the ER membrane and its composition to ER stress and UPR activation in health and disease.
Our goals are
1) Isolating organelles from cells with unprecedented purity
2) Establishing lipid fingerprints of ER stress, in other words: What are the characteristic lipid compositions of the stressed ER membrane?
3) Studying the cooperation of cellular organelles, e.g. the ER, mitochondria, and the vacuole, in dealing with ER stress
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER stress
We pursue the hypothesis that the protein-to-lipid ratio is sensed via the unfolded protein response (UPR), a conserved pathway, which controls the relative rate of lipid and protein production and which serves as a diagnostic marker for ER stress. We study this mechanistic connection in two model systems: The baker's yeast Saccharomyces cerevisiae and cultured, mammalian cells. We aim to understand, how communication and exchange of lipid metabolites between organelles support cell functions during acute, prolonged, and chronic ER stresses.
The MemDense project is important for society, because a large number of chronic inflammatory and complex metabolic diseases, such as type II diabetes and non-alcoholic fatty liver disease have been firmly associated with chronic ER stress, while the molecular basis of ER stress remains unresolved. The MemDense project establishes new experimental pipelines to test the contribution of the ER membrane and its composition to ER stress and UPR activation in health and disease.
Our goals are
1) Isolating organelles from cells with unprecedented purity
2) Establishing lipid fingerprints of ER stress, in other words: What are the characteristic lipid compositions of the stressed ER membrane?
3) Studying the cooperation of cellular organelles, e.g. the ER, mitochondria, and the vacuole, in dealing with ER stress
4) Figuring out how the protein-to-lipid ratio is balanced in healthy cells and how membrane protein overcrowding causes chronic ER stress
The MemDense project emphasizes the impact of macromolecular crowding in biological membranes, creating resources for membrane biochemistry, biophysics, and cell biology through innovative preparative and analytical methods.
Our team has made significant strides towards the project's objectives. We've developed techniques to characterize ER stress signatures in organellar membranes and track subtle changes in protein-to-lipid ratios under stress.
PUBLICATION 1: Our study, "Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1" (doi.org/10.1083/jcb.202011078) shows that different ER stress signals converge in a single, signaling-active configuration of IRE1, supporting a hydrophobic mismatch-based oligomerization in the stressed ER.
PUBLICATION 2: We analyzed the relationships between protein folding, growth, and lipid metabolism in "A quantitative analysis of cellular lipid compositions during acute proteotoxic ER stress reveals specificity in the production of asymmetric lipids" (doi.org/10.3389/fcell.2020.00756) detailing how two proteotoxic agents affect lipid metabolism. We plan to publish similar comprehensive lipidome data for two mammalian cell lines by 2025 or early 2026.
PUBLICATION 3: In "The Unfolded Protein Response as a Guardian of the Secretory Pathway" (doi.org/10.3390/cells10112965) we review UPR activation mechanisms proposing a framework for how UPR transducers monitor protein crowding and membrane stiffness.
PUBLICATION 4: This preprint (now Publication 6) contributes to the understanding of how stress affects organelle compositions and membrane properties.
PUBLICATION 6: Our work on identifying new membrane contact sites appears in "Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution" (eLife, doi.org/10.7554/eLife.74602) using lipidomic analyses to validate membrane preparation purity.
PUBLICATION 8: We established methods for isolating vacuole and ER membranes, revealing insights into lipidome changes during different growth phases in "Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)" (doi.org/10.1016/j.bpj.2023.01.009) and characterizing lipid bilayer stress in "MemPrep, a new technology for isolating organellar membranes" (doi.org/10.1038/s44318-024-00063-y). These studies suggest that anionic lipids may negatively regulate the UPR, connecting membrane protein folding with lipid metabolism.
PUBLICATION 9: Our perspective on membrane biogenesis, linking protein production and lipid biosynthesis, is presented in "Membrane homeostasis beyond fluidity: control of membrane compressibility" (doi.org/10.1016/j.tibs.2023.08.004). We argue that membrane compressibility affects transmembrane protein throughout their entire lifecycle and clarify the concept of "membrane fluidity."
PUBLICATION 10: "Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response" (doi.org/10.1101/cshperspect.a041400) discusses lipid roles during ER stress and highlights differences between yeast and mammalian ER lipid compositions.
PUBLICATION 11: Addressing technical challenges with UPR transducers, we introduced "Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting" (doi.org/10.1101/2024.09.28.615506). This method allows us to modulate lipid composition and study membrane proteins in complex, native-like environments.
PUBLICATION 12, 13: We’ve contributed commentaries for The EMBO Journal and Nature Reviews in Molecular and Cell Biology.
Our team has made significant strides towards the project's objectives. We've developed techniques to characterize ER stress signatures in organellar membranes and track subtle changes in protein-to-lipid ratios under stress.
PUBLICATION 1: Our study, "Cysteine cross-linking in native membranes establishes the transmembrane architecture of Ire1" (doi.org/10.1083/jcb.202011078) shows that different ER stress signals converge in a single, signaling-active configuration of IRE1, supporting a hydrophobic mismatch-based oligomerization in the stressed ER.
PUBLICATION 2: We analyzed the relationships between protein folding, growth, and lipid metabolism in "A quantitative analysis of cellular lipid compositions during acute proteotoxic ER stress reveals specificity in the production of asymmetric lipids" (doi.org/10.3389/fcell.2020.00756) detailing how two proteotoxic agents affect lipid metabolism. We plan to publish similar comprehensive lipidome data for two mammalian cell lines by 2025 or early 2026.
PUBLICATION 3: In "The Unfolded Protein Response as a Guardian of the Secretory Pathway" (doi.org/10.3390/cells10112965) we review UPR activation mechanisms proposing a framework for how UPR transducers monitor protein crowding and membrane stiffness.
PUBLICATION 4: This preprint (now Publication 6) contributes to the understanding of how stress affects organelle compositions and membrane properties.
PUBLICATION 6: Our work on identifying new membrane contact sites appears in "Systematic analysis of membrane contact sites in Saccharomyces cerevisiae uncovers modulators of cellular lipid distribution" (eLife, doi.org/10.7554/eLife.74602) using lipidomic analyses to validate membrane preparation purity.
PUBLICATION 8: We established methods for isolating vacuole and ER membranes, revealing insights into lipidome changes during different growth phases in "Remodeling of yeast vacuole membrane lipidomes from the log (one phase) to stationary stage (two phases)" (doi.org/10.1016/j.bpj.2023.01.009) and characterizing lipid bilayer stress in "MemPrep, a new technology for isolating organellar membranes" (doi.org/10.1038/s44318-024-00063-y). These studies suggest that anionic lipids may negatively regulate the UPR, connecting membrane protein folding with lipid metabolism.
PUBLICATION 9: Our perspective on membrane biogenesis, linking protein production and lipid biosynthesis, is presented in "Membrane homeostasis beyond fluidity: control of membrane compressibility" (doi.org/10.1016/j.tibs.2023.08.004). We argue that membrane compressibility affects transmembrane protein throughout their entire lifecycle and clarify the concept of "membrane fluidity."
PUBLICATION 10: "Endoplasmic Reticulum Membrane Homeostasis and the Unfolded Protein Response" (doi.org/10.1101/cshperspect.a041400) discusses lipid roles during ER stress and highlights differences between yeast and mammalian ER lipid compositions.
PUBLICATION 11: Addressing technical challenges with UPR transducers, we introduced "Reversible tuning of membrane sterol levels by cyclodextrin in a dialysis setting" (doi.org/10.1101/2024.09.28.615506). This method allows us to modulate lipid composition and study membrane proteins in complex, native-like environments.
PUBLICATION 12, 13: We’ve contributed commentaries for The EMBO Journal and Nature Reviews in Molecular and Cell Biology.
The MemPrep technology (PUBLICATION 8) opens new horizons in cell biology, by providing the possibility for the first time, to 1) quantitatively study the composition of organelle subdomains, 2) study comprehensively the lipid composition of organelles with functional and dysfunctional lipid exchange mechanisms, and 3) identify the spectrum of clients for inter-organelle protein transport (e.g. from the ER to mitochondria or from the ER to the Golgi) by quantitative proteomics.
We found that the MemPrep technology for organelle isolations from baker's yeast can be modified and optimized for isolating ER membrane subdomains from cultured mammalian cells. This success goes well beyond the expected results and opens new horizons for understanding the role of organelle homeostasis in mammalian cells. In fact, the MemPrep procedure is one of the key aspects of the Excellence Cluster Application SCALE by the Goethe University Frankfurt to the Deutsche Forschungsgemeinschaft DFG (among the 25 most important publications for the Cluster of Excellence).
We have established new reconstitution schemes (PUBLICATION 11) allowing us to adjust the lipid environment of a reconstutited transmembrane protein in liposomes after successful reconstitution. This technology represents a key step toward studying transmembrane proteins in naive-like, protein-rich, complex, asymmetric, sterol-rich membrane environments. Furthermore, this technology allows us to study the impact of the lipid environement on membrane protein insertion and lipid flipflop in isolated organelle membranes.
We found that the MemPrep technology for organelle isolations from baker's yeast can be modified and optimized for isolating ER membrane subdomains from cultured mammalian cells. This success goes well beyond the expected results and opens new horizons for understanding the role of organelle homeostasis in mammalian cells. In fact, the MemPrep procedure is one of the key aspects of the Excellence Cluster Application SCALE by the Goethe University Frankfurt to the Deutsche Forschungsgemeinschaft DFG (among the 25 most important publications for the Cluster of Excellence).
We have established new reconstitution schemes (PUBLICATION 11) allowing us to adjust the lipid environment of a reconstutited transmembrane protein in liposomes after successful reconstitution. This technology represents a key step toward studying transmembrane proteins in naive-like, protein-rich, complex, asymmetric, sterol-rich membrane environments. Furthermore, this technology allows us to study the impact of the lipid environement on membrane protein insertion and lipid flipflop in isolated organelle membranes.