MemPrep, a new technology for isolating organellar membranes provides fingerprints of lipid bilayer stress

Biological membranes have a stunning ability to adapt their composition in response to physiological stress and metabolic challenges. Little is known how such perturbations affect individual organelles in eukaryotic cells. Pioneering work has provided insights into the subcellular distribution of lipids in the yeast Saccharomyces cerevisiae, but the composition of the endoplasmic reticulum (ER) membrane, which also crucially regulates lipid metabolism and the unfolded protein response, remains insufficiently characterized. Here, we describe a method for purifying organelle membranes from yeast, MemPrep. We demonstrate the purity of our ER membrane preparations by proteomics, and document the general utility of MemPrep by isolating vacuolar membranes. Quantitative lipidomics establishes the lipid composition of the ER and the vacuolar membrane. Our findings provide a baseline for studying membrane protein biogenesis and have important implications for understanding the role of lipids in regulating the unfolded protein response (UPR). The combined preparative and analytical MemPrep approach uncovers dynamic remodeling of ER membranes in stressed cells and establishes distinct molecular fingerprints of lipid bilayer stress.This work was funded by the VW foundation (Life?, #93089, #93092, #93090) to RE, MS, and JS, by the Deutsche Forschungsgemeinschaft in the framework of the SFB894 to RE and the SFB1027 to both JH and RE, and by the European Research Council under the European Union’s Horizon 2020 research and innovation program (grant agreement no. 866011) to RE. MS is an incumbent of the Dr. Gilbert Omenn and Martha Darling Professorial Chair in Molecular Genetics

Sialyllactose in Viral Membrane Gangliosides Is a Novel Molecular Recognition Pattern for Mature Dendritic Cell Capture of HIV-1

An accessible sialyllactose moiety on viral membrane gangliosides is shown to be essential for HIV-1 uptake into mature dendritic cells, thereby promoting viral transfer and infection of bystander CD4+ T lymphocytes

Protein-lipid interactions: paparazzi hunting for snap-shots

Photoactivatable groups meeting the criterion of minimal perturbance allow the investigation of interactions in biological samples. Here, we review the application of photoactivatable groups in lipids enabling the study of protein-lipid interactions in (biological) membranes. The chemistry of various photoactivatable groups is summarized and the specificity of the interactions detected is discussed. The recent introduction of ‘click chemistry’ in photocrosslinking of membrane proteins by photoactivatable lipids opens new possibilities for the analysis of crosslinked products and will help to close the gap between proteomics and lipidomics

Sphingolipid Asymmetry and Transmembrane Translocation in Mammalian Cells

Sphingolipids are a typical feature of eukaryotic cells, and indeed, they have been found to fulfi ll a number of intra - and intercellular functions that are specifi c for eukaryotes. Membrane sphingolipids are organized in specialized membrane domains that are involved in the sorting of membrane proteins and lipids along the cellular vesicular transport pathways. In addition, the domains have been invoked in various types of signaling events, like the formation of the T - cell receptor complex and the formation of cell – cell signaling domains. On the other hand, individual sphingolipids act as lipid second messengers, the clearest examples being sphingosine - 1 - phosphate and ceramide. Sphingolipids act at discrete locations, and they are synthesized and degraded at defi ned locations. These are not always on the same side of the membrane, which necessitates transmembrane transport. The sites of transmembrane translocation, the molecular mechanism, and its possible regulation are the topic of the present chapter (Fig. 4.1 )

Sphingolipid Asymmetry and Transmembrane Translocation in Mammalian Cells

Sphingolipids are a typical feature of eukaryotic cells, and indeed, they have been found to fulfi ll a number of intra - and intercellular functions that are specifi c for eukaryotes. Membrane sphingolipids are organized in specialized membrane domains that are involved in the sorting of membrane proteins and lipids along the cellular vesicular transport pathways. In addition, the domains have been invoked in various types of signaling events, like the formation of the T - cell receptor complex and the formation of cell – cell signaling domains. On the other hand, individual sphingolipids act as lipid second messengers, the clearest examples being sphingosine - 1 - phosphate and ceramide. Sphingolipids act at discrete locations, and they are synthesized and degraded at defi ned locations. These are not always on the same side of the membrane, which necessitates transmembrane transport. The sites of transmembrane translocation, the molecular mechanism, and its possible regulation are the topic of the present chapter (Fig. 4.1 )

Trifunctional lipid probes for comprehensive studies of single lipid species in living cells

Lipid-mediated signaling events regulate many cellular processes. Investigations of the complex underlying mechanisms are difficult because several different methods need to be used under varying conditions. Here we introduce multifunctional lipid derivatives to study lipid metabolism, lipid−protein interactions, and intracellular lipid localization with a single tool per target lipid. The probes are equipped with two photoreactive groups to allow photoliberation (uncaging) and photo–cross-linking in a sequential manner, as well as a click-handle for subsequent functionalization. We demonstrate the versatility of the design for the signaling lipids sphingosine and diacylglycerol; uncaging of the probe for these two species triggered calcium signaling and intracellular protein translocation events, respectively. We performed proteomic screens to map the lipid-interacting proteome for both lipids. Finally, we visualized a sphingosine transport deficiency in patient-derived Niemann−Pick disease type C fibroblasts by fluorescence as well as correlative light and electron microscopy, pointing toward the diagnostic potential of such tools. We envision that this type of probe will become important for analyzing and ultimately understanding lipid signaling events in a comprehensive manner.</p

Protein-Sphingolipid Interactions within Cellular Membranes

Each intracellular organelle critically depends on maintaining its specific lipid composition that in turn contributes to the biophysical properties of the membrane. With our knowledge increasing about the organization of membranes with defined microdomains of different lipid compositions, questions arise as to the molecular mechanisms that underly targeting to/segregation from microdomains of a given protein. In addition to specific lipid-transmembrane segment interactions as a basis for partitioning, presence in a given microdomain may alter the conformation of proteins, and, thus, the activity and availability for regulatory modifications. However, for most proteins the specific lipid environment of transmembrane segments as well as its relevance to protein function and overall membrane organization is largely unknown. In order to help filling this gap, we have synthezised a novel photoactive sphingolipid precursor that, together with a precursor for phosphoglycerolipids and with photo-cholesterol, was investigated in vivo with regard to specific protein transmembrane span-lipid interactions. As proof of principle we show specific labeling of the ceramide transporter with the sphingolipid probe, and describe specific in vivo interactions of lipids with caveolin-1, PI-TPß, and the mature form of nicastrin. This novel photolabile sphingolipid probe allows detection of protein-sphingolipid interactions within the membrane bilayer of living cells

Bifunctional Sphingosine for Cell-Based Analysis of Protein-Sphingolipid Interactions

Sphingolipids are essential structural components of cellular membranes and are crucial regulators of cellular processes. While current high-throughput approaches allow for the systematic mapping of interactions of soluble proteins with their lipid-binding partners, photo-crosslinking is the only technique that enables for the proteome-wide mapping of integral membrane proteins with their direct lipid environment. Here we report the synthesis of a photoactivatable and clickable analog of sphingosine. When administered to sphingosine-1-phosphate lyase deficient cells, pacSph allows its metabolic fate and the subcellular flux of de novo synthesized sphingolipids to be followed in a time resolved manner. The chemoproteomic profiling yielded over 180 novel sphingolipid-binding proteins, of which we validated a number, demonstrating the unique value of this technique as a discovery tool. This work provides an important resource for the understanding of the global cellular interplay between sphingolipids and their interacting proteins.status: publishe

The Hsp90 isoforms from S. cerevisiae differ in structure, function and client range.

The molecular chaperone Hsp90 is an important regulator of proteostasis. It has remained unclear why S. cerevisiae possesses two Hsp90 isoforms, the constitutively expressed Hsc82 and the stress-inducible Hsp82. Here, we report distinct differences despite a sequence identity of 97%. Consistent with its function under stress conditions, Hsp82 is more stable and refolds more efficiently than Hsc82. The two isoforms also differ in their ATPases and conformational cycles. Hsc82 is more processive and populates closed states to a greater extent. Variations in the N-terminal ATP-binding domain modulate its dynamics and conformational cycle. Despite these differences, the client interactomes are largely identical, but isoform-specific interactors exist both under physiological and heat shock conditions. Taken together, changes mainly in the N-domain create a stress-specific, more resilient protein with a shifted activity profile. Thus, the precise tuning of the Hsp90 isoforms preserves the basic mechanism but adapts it to specific needs