bioRxiv Science⌕ Search

Biology subjects

Loewith, R.

Publications and source records attributed to Loewith, R..

11 recordsLinked to original sources

Atonosomes, compartments involved in membrane tension decrease

To ensure survival, cells need to buffer the effects of environmental stress on their plasma membrane, yet the structural mechanisms by which this is acutely achieved remain largely unknown. Here, we propose atonosomes as a unifying identity for a class of previously observed but enigmatic, tension-responsive, plasma membrane-derived compartments that arise across contexts of acute and chronic membrane tension loss. Leveraging unprecedented high resolution cryo-FIB-ET imaging in yeast, we show that atonosomes are complex, organelle-containing structures bounded by membranes and cell wall material, spanning hundreds of nanometers, and displaying a remarkable morphological diversity. Atonosomes form within seconds in response to reduced plasma membrane tension, and their emergence appears to require no dedicated molecular machinery, arising instead as a direct consequence of membrane biophysics. Upon formation, they recruit key membrane-associated proteins, including TORC2, Slm1, and septins. Under conditions of chronic disruption of PM homeostasis, atonosomes become constitutively present. Their stability and reversibility are further modulated by the cell wall, whose polymerization state influences atonosome dynamics. Structural conservation in fungi and ichthyosporea, demonstrates that atonosomes are a conserved stress-triggered response of cell-wall enclosed organisms. Together, these findings establish atonosomes as a novel compartment that mediates cellular responses to plasma membrane tension variation, coupling membrane remodeling and lipid homeostasis to preserve cellular integrity under stress.

cell biology↗

Structural basis for TORC2 activation

The Target of Rapamycin Complex 2 (TORC2) is a central node in signaling feedback loops serving to maintain biophysical homeostasis of the plasma membrane (PM). How TORC2 is regulated by mechanical perturbation of the PM is not well understood. To address this, we determined the cryo-electron microscopy structure of endogenous yeast TORC2 to up to 2.2 [A] resolution. Our model refines the position and interactions of TORC2-specific subunits, providing a structural basis for the differential assembly of Tor2 into TORC2. Furthermore, we observe the insertion of the pleckstrin-homology domain of the Avo1 subunit into the Tor2 active site, providing a regulatory mechanism by phosphoinositides. Structure-guided functional experiments reveal a potential TORC2 membrane binding surface and a positively charged pocket in the Avo3 subunit that is necessary for TORC2 activation. Collectively, our data suggest that signaling phosphoinositides activate TORC2 by membrane-induced structural rearrangements via concerted action of conserved regulatory subunits.

biochemistry↗

TORC2-regulated sterol redistribution mediates recovery from membrane perturbation by small amphipathic molecules.

To maintain plasma membrane (PM) integrity, cells need to acutely regulate PM lipid composition. The Target Of Rapamycin (TOR) complex 2 is a protein kinase that acts as a central regulator of PM homeostasis, but the mechanisms by which it monitors and reacts to membrane stresses are poorly understood. To address this knowledge gap, we characterized a family of amphiphilic molecules that physically perturb PM organization and in doing so inhibit TORC2 in yeast and mammalian cells. Using fluorescent lipid associated reporters in budding yeast, we show that these small molecules first cause a transient increase in the amount of biochemically accessible ergosterol at the PM. Contemporaneous TORC2 inhibition stimulates a rapid removal of accessible ergosterol from the PM by the PM-ER sterol transporters Lam2 and Lam4, necessary for TORC2 reactivation. Thus, we show that TORC2 acts in a feedback loop to control active sterol levels at the PM and introduce sterols as possible TORC2 signalling modulators.

cell biology↗

Structure and function of the yeast amino acid-sensing SEAC-EGOC supercomplex

The Seh1-associated complex (SEAC) transduces amino acid signals to the Target of Rapamycin Complex 1 (TORC1), a master regulator of cell growth located on the vacuole membrane. The SEAC acts as a GTPase activating protein (GAP) for Gtr1, a small GTPase that forms a heterodimer with Gtr2, and as part of the EGO complex (EGOC), relays nutrient signals to TORC1. The SEAC is composed of two subcomplexes, SEACIT, an inhibitor of TORC1 that contains the GAP activity, and SEACAT, that has been proposed to regulate the activity of SEACIT. However, molecular details of its regulation are unclear. Here, we determined the cryo-electron microscopy structure of the SEAC-EGOC supercomplex and studied its function in TORC1 amino acid signalling. A single SEAC can interact with two EGOC molecules via SEACIT, binding exclusively to the "active" version of the EGOC as their interaction depends on Gtr1 being loaded with GTP. SEACAT does not modulate nor interact with the EGOC. The GAP activity of the SEAC is essential for the regulation of TORC1 by amino acids, and its loss phenocopies the lack of Gtr1-Gtr2, establishing the SEAC-EGOC complex as an amino acid-sensing hub. Despite being located far from the active site, deletion of Sea2, or its N-terminal {beta}-propeller domain, also results in defects in amino acid signalling to TORC1. We propose that the SEAC-EGOC supercomplex integrates coatomer-like mechanisms of regulation via bidirectional feedback between GAP-GTPase (SEACIT-EGOC) and coat (SEACAT) modules that explain the functional interaction between Sea2 and the GAP activity. Given the conservation between the SEAC and its mammalian ortholog GATOR, we envision that this mechanism is similar in different organisms.

biochemistry↗

CryoEM architecture of a native stretch-sensitive membrane microdomain

Biological membranes are partitioned into functional zones containing specific lipids and proteins, termed membrane microdomains. Their composition and organization remain controversial owing to a paucity of techniques that can visualize lipids in situ without disrupting their native behavior1,2. The yeast eisosome, a membrane compartment scaffolded by the BAR-domain proteins Pil1 and Lsp1, senses and responds to mechanical stress by flattening and releasing sequestered factors3-7. Here, we isolated native eisosomes as helical filaments of Pil1/Lsp1 lattice bound to plasma membrane lipids and solved their structures by helical reconstruction. We observe remarkable organization within the lipid bilayer density from which we could assign headgroups of PI(4,5)P2 and phosphatidylserine bound to Pil1/Lsp1 and a pattern of membrane voids, signatures of sterols, beneath an amphipathic helix. We verified these assignments using in vitro reconstitutions and molecular dynamics simulations. 3D variability analysis of the native eisosomes revealed a dynamic stretching of the Pil1/Lsp1 lattice that affects functionally important lipid sequestration, supporting a mechanism in which membrane stretching liberates lipids otherwise anchored by the Pil1/Lsp1 coat. Our results provide mechanistic insight into how eisosome BAR-domain proteins create a mechanosensitive membrane microdomain and, more globally, resolve long-standing controversies about the architecture and nature of lipid microdomains.

biophysics↗

Dynamic metabolome profiling uncovers potential TOR signaling genes

Although the genetic code of the yeast Saccharomyces cerevisiae was sequenced 25 years ago, the characterization of the roles of genes within it is far from complete. The lack of a complete mapping of functions to genes hampers systematic understanding of the biology of the cell. The advent of high-throughput metabolomics offers a unique approach to uncovering gene function with an attractive combination of cost, robustness, and breadth of applicability. Here we used flow-injection time-of-flight mass spectrometry (FIA-MS) to dynamically profile the metabolome of 164 loss-of-function mutants in TOR and receptor or receptor-like genes under a time-course of rapamycin treatment, generating a dataset with over 7,000 metabolomics measurements. In order to provide a resource to the broader community, those data are made available for browsing through an interactive data visualization app hosted at https://rapamycin-yeast-metabolome.herokuapp.com/. We demonstrate that dynamic metabolite responses to rapamycin are more informative than steady state responses when recovering known regulators of TOR signaling, as well as identifying new ones. Deletion of a subset of the novel genes causes phenotypes and proteome responses to rapamycin that further implicate them in TOR signaling. We found that one of these genes, CFF1, was connected to the regulation of pyrimidine biosynthesis through URA10. These results demonstrate the efficacy of the approach for flagging novel potential TOR signaling-related genes and highlights the utility of dynamic perturbations when using functional metabolomics to deliver biological insight.

systems biology↗

Ultrastructure Expansion Microscopy reveals the nanoscale cellular architecture of budding and fission yeast

The budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe have served as invaluable model organisms to study various fundamental and highly conserved cellular processes. While super-resolution (SR) microscopy has in recent years paved the way to a better understanding of the spatial organization of molecules in cells, its wide use in yeast models has remained limited due to the specific know-how and specialized instrumentation required, contrasted with the relative ease of endogenous tagging and live cell fluorescence microscopy in these systems. To facilitate SR microscopy in yeasts, we have extended the ultrastructure expansion microscopy (U-ExM) method to both S. cerevisiae and S. pombe, enabling 4-fold isotropic expansion in both systems. We demonstrate here that U-ExM allows the nanoscale imaging of the microtubule cytoskeleton and its associated spindle pole body (SPB), notably unveiling a conserved Sfi1p/Cdc31p spatial organization on the appendage bridge structure. In S. pombe, we validate the method by quantifying the homeostatic regulation of nuclear pore complex (NPC) number through the cell cycle. Combined with pan-labelling (NHS ester), which provides a global cellular context, U-ExM unveils the subcellular organization of the eukaryote yeast models S. cerevisiae and S. pombe. This easy-to-implement imaging with conventional microscopes provides nanoscale resolution and adds a powerful new method to the already extensive yeast toolbox.

cell biology↗

Multiple roles for the ESCRT machinery in maintaining plasma membrane homeostasis

The endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

cell biology↗

Chemical genetics of AGC-kinases reveals shared targets of Ypk1, Protein Kinase A and Sch9

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take centre stage in regulating this process in S. cerevisiae. To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signalling through Ypk1 is therefore more closely related to PKA- and Sch9-signalling than previously appreciated and may perform functions previously only attributed to the latter kinases.

cell biology↗

The Aspartic Protease Ddi1 Contributes to DNA-Protein Crosslink Repair in Yeast

Naturally occurring or drug-induced DNA-protein crosslinks (DPCs) interfere with key DNA transactions if not timely repaired. The unique family of DPC-specific proteases Wss1/SPRTN targets DPC protein moieties for degradation, including topoisomerase-1 trapped in covalent crosslinks (Top1ccs). Here we describe that the efficient DPC disassembly requires Ddi1, another conserved predicted protease in Saccharomyces cerevisiae. We found Ddi1 in a genetic screen of the tdp1wss1 mutant defective in Top1cc processing. Ddi1 is recruited to a persistent Top1cc-like DPC lesion in an S-phase dependent manner to assist eviction of crosslinked protein from DNA. Loss of Ddi1 or its putative protease activity hypersensitize cells to DPC trapping agents independently from Wss1 and 26S proteasome, implying its broader role in DPC repair. Among potential Ddi1 targets we found the core component of RNAP II and show that its genotoxin-induced degradation is impaired in ddi1. Together, we propose that the Ddi1 protease contributes to DPC proteolysis.

molecular biology↗

TORC2 Affects Endocytosis through Plasma Membrane Tension

Target Of Rapamycin complex 2 (TORC2) is a conserved protein kinase that regulates multiple plasma membrane (PM)-related processes including endocytosis. Direct, chemical inhibition of TORC2 arrests endocytosis but with kinetics that are relatively slow and therefore inconsistent with signaling being mediated solely through simple phosphorylation cascades. Here, we show that, in addition to regulation of the phosphorylation of endocytic proteins, TORC2 also controls endocytosis by modulating PM tension. Elevated PM tension, upon TORC2 inhibition, impinges on endocytosis at two different levels: first, by severing the bonds between the PM adaptor proteins Sla2 and Ent1 and the actin cytoskeleton; and, second, by hindering recruitment of Rvs167, an N-BAR-containing protein important for vesicle fission, to endocytosis sites. These results underline the importance of biophysical cues in the regulation of cellular and molecular processes.

cell biology↗