Biology subjects

Montigny, C.

Publications and source records attributed to Montigny, C..

7 recordsLinked to original sources

bioRxiv2025

Revisiting flippase specificity: Drs2-Cdc50 transports multiple anionic lipid substrates

P4-ATPase lipid flippases maintain transbilayer lipid asymmetry in eukaryotic membranes, which is essential for many cellular processes. In yeast, the Drs2-Cdc50 flippase complex was previously shown to specifically transport phosphatidylserine (PS) from the exoplasmic to the cytosolic leaflet of the trans-Golgi network (TGN), thereby controlling vesicular trafficking in the secretory and endocytic pathways. Using an improved proteoliposome-based lipid flippase assay, we now show that the Drs2-Cdc50 complex transports multiple anionic glycerophospholipids, including PS, phosphatidylinositol, phosphatidylglycerol, and phosphatidic acid. In vivo cell-based lipid uptake assays further support the transport of these lipids. To understand the basis of this substrate promiscuity, we analyzed cryo-EM structures of the complex with occluded lipids. These structures revealed that the water network surrounding the lipid headgroup plays a critical role in enabling Drs2-Cdc50 to recognize different lipids. These data unveil an unexpected broad specificity of the Drs2-Cdc50 complex for anionic lipids, which may significantly impact their transbilayer distribution in the yeast TGN.

biochemistry↗

bioRxiv2025

Ist2 promotes lipid transfer by Osh6 via its membrane tethering and lipid scramblase activities

Lipid transfer proteins (LTPs) are required for the uneven distribution of lipids between cellular membranes, which is essential for many cell functions. In yeast, Osh6 is an LTP that exchanges phosphatidylserine (PS) with phosphatidylinositol 4-phosphate (PI(4)P) between the endoplasmic reticulum (ER) and the plasma membrane (PM), promoting the enrichment of PS in the PM. Here, we address why, to function optimally, Osh6 must bind to Ist2, an ER-resident lipid scramblase able to connect the ER to the PM via an intrinsically disordered region (IDR). We determined in vitro that Osh6 binds to the Ist2 IDR with micromolar affinity, whether empty or bound to its lipid ligands. Moreover, we found that Osh6 efficiently transfers PS at ER-PM contact sites if the Ist2 IDR has a minimal length and its binding site in the IDR is sufficiently removed from the ER surface. Next, we reconstituted the Osh6:Ist2 complex within artificial ER-PM contact sites and demonstrated that the association of Osh6 with Ist2 allows for a fast and directed PS flux between the connected membranes. We identified the Ist2 binding site on the Osh6 surface by validating structural models using our functional assays. Finally, we found that the Osh6-mediated PS transfer can be coupled to the PS scramblase activity of Ist2. These data unveil new functional partnerships between an LTP and a membrane tethering/scramblase protein and point to the general advantage of localizing these processes to membrane contact sites to ensure their efficiency.

biochemistry↗

bioRxiv2025

Ist2, a protein involved in phosphatidylserine transport, is an ER lipid scramblase

Lipid scramblases allow passive flip-flop of phospholipids between bilayer leaflets, thereby promoting membrane symmetry. At the endoplasmic reticulum (ER), where phospholipid synthesis is restricted to one of the two leaflets, scramblase activity should be essential for equilibrated membrane growth. However, phospholipid scramblases at the ER are poorly understood. The yeast protein Ist2 contains an ER domain and a cytosolic tail that binds the plasma membrane (PM) and participates in the transfer of phosphatidylserine (PS). Here, we show both in vitro and in silico that the ER- domain of Ist2, which bears homology to the TMEM16 proteins, possesses a lipid scramblase activity. Ist2 activity is not regulated by Ca2+, in contrast to TMEM16 proteins, but is affected by the lipid composition of the bilayer used in simulations. In cells, we do not find a strong impact of the scramblase domain of Ist2 in on PS distribution; however, its over-expression or deletion affects processes at the ER such as vesicular transport, lipid droplet biogenesis and general phospholipid transport, with a specific contribution of residues important for lipid scrambling. Our study therefore identifies the first dedicated phospholipid scramblase in yeast and demonstrates that membrane asymmetry can impact diverse membrane-remodeling processes at the ER.

cell biology↗

bioRxiv2021

Autoinhibition and regulation by phosphoinositides of ATP8B1, a human lipid flippase associated with intrahepatic cholestatic disorders

P4-ATPases flip lipids from the exoplasmic to the cytosolic leaflet, thus maintaining lipid asymmetry in eukaryotic cell membranes. Mutations in several human P4-ATPase genes are associated with severe diseases, e.g. in ATP8B1 causing progressive familial intrahepatic cholestasis, a rare inherited disorder progressing toward liver failure. ATP8B1 forms a binary complex with CDC50A and displays a broad specificity to glycerophospholipids, but regulatory mechanisms are unknown. Here, we report functional studies and the cryo-EM structure of the human lipid flippase ATP8B1-CDC50A at 3.1 [A] resolution. We find that ATP8B1 is autoinhibited by its N- and C-terminal tails, which form extensive interactions with the catalytic sites and flexible domain interfaces. Consistently, ATP hydrolysis is unleashed by truncation of the C-terminus, but also requires phosphoinositides, most markedly phosphatidylinositol-3,4,5-phosphate (PI(3,4,5)P3), and removal of both N- and C- termini results in full activation. Restored inhibition of ATP8B1 truncation constructs with a synthetic peptide mimicking the C-terminal segment further suggests molecular communication between N- and C-termini in the autoinhibition and demonstrates that the regulatory mechanism can be interfered with by exogenous compounds. A recurring (G/A)(Y/F)AFS motif of the C-terminal segment suggests that this mechanism is employed widely across P4-ATPase lipid flippases in plasma membrane and endomembranes.

biochemistry↗

bioRxiv2020

The SERCA residue Glu340 mediates inter-domain communication that guides Ca2+ transport

The sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) is a P-type ATPase that transports Ca2+ from the cytosol into the SR/ER lumen, driven by ATP. This primary transport activity depends on tight coupling between movements of the transmembrane helices forming the two Ca2+ binding sites and of the cytosolic headpiece mediating ATP hydrolysis. We have addressed the molecular basis for this intramolecular communication by analyzing the structure and functional properties of the SERCA mutant E340A. The mutated Glu340 residue is strictly conserved among the P-type ATPase family of membrane transporters and is located at a seemingly strategic position at the interface between the phosphorylation domain and the cytosolic ends of five out of SERCAs ten transmembrane helices. The mutant displays a marked slowing of the Ca2+-binding kinetics, and its crystal structure in the presence of Ca2+ and ATP analogue reveals a rotated headpiece, altered connectivity between the cytosolic domains and altered hydrogen bonding pattern around residue 340. Supported by molecular dynamics simulations, we conclude that the E340A mutation causes a stabilization of the Ca2+ sites in a more occluded state, hence displaying slowed dynamics. This finding underpins a crucial role of Glu340 in inter-domain communication between the headpiece and the Ca2+-binding transmembrane region.

molecular biology↗

bioRxiv2019

Deciphering the mechanism of inhibition of SERCA1a by sarcolipin using molecular simulations

SERCA1a is an ATPase calcium pump that transports Ca2+ from the cytoplasm to the sarco/endoplasmic reticulum lumen. Sarcolipin (SLN), a transmembrane peptide, regulates the activity of SERCA1a by decreasing its Ca2+ transport rate, but its mechanism of action is still not well understood. To decipher this mechanism, we have performed normal modes analysis in the all-atom model, with the SERCA1a-SLN complex or the isolated SERCA1a embedded in an explicit membrane. The comparison of the results allowed us to provide an explanation for the action of SLN that is in good agreement with experimental observations. In our analyses, the presence of SLN locally perturbs the TM6 transmembrane helix and as a consequence modifies the position of D800, one of the key metal-chelating residues. Additionally, it reduces the flexibility of the gating residues, V304 and E309 in TM4, at the entrance of the Ca2+ binding sites, which would decrease the affinity for Ca2+. Unexpectedly, SLN has also an effect on the ATP binding site more than 35 [A] away, due to the straightening of TM5, a long helix considered as the spine of the protein. The straightening of TM5 modifies the structure of the P-N linker that sits above it, and which comprises the 351DKTG354 conserved motif, resulting in an increase of the distance between ATP and the phosphorylation site. As a consequence, the turn-over rate could be affected. All this gives SERCA1a the propensity to go toward a Ca2+-deprived E2-like state in the presence of SLN and toward a Ca2+ high-affinity E1-like state in the absence of SLN, although the SERCA1a-SLN complex was crystallized in an E1-like state. In addition to a general mechanism of inhibition of SERCA1a regulatory peptides, this study also provides an insight in the conformational transition between the E2 and E1 states. Statement of SignificanceThe role of sarco/endoplasmic reticulum calcium ATPase in muscle relaxation is essential. Impairment of its function may result in either cardiac diseases, or myopathies, and also thermogenesis defects. Inhibition of the ATPase by regulatory peptide such as sarcolipin remains unclear. The structure of the ATPase in complex with this peptide was studied by all-atom normal modes analysis, an in silico technique which allows us to decipher the mechanism of inhibition of calcium transport by sarcolipin at a molecular level. Our results open the way to understanding the impact of in vivo misregulation of the ATPase activity by sarcolipin. Development of tools enhancing or preventing interaction between the ATPase and its regulatory peptide could be considered as new therapeutic approaches.

biophysics↗

bioRxiv2019

Structure and autoregulation of a P4-ATPase lipid flippase

P4-ATPases are lipid flippases that drive active transport of phospholipids from the exoplasmic or lumenal to the cytosolic leaflets of eukaryotic membranes to maintain their asymmetric lipid composition. The molecular architecture of P4-ATPases and how they work in lipid recognition and transport has remained elusive. Using cryo-electron microscopy we have determined the structures of a P4-ATPase, specifically of the Saccharomyces cerevisiae Drs2p-Cdc50p, which is a phosphatidylserine and phosphatidylethanolamine specific lipid flippase. Drs2p-Cdc50p is autoinhibited by the Drs2p C-terminal tail and activated by phosphatidylinositol-4 phosphate (PI4P). We present three structures representing an autoinhibited, an intermediate, and a fully activated state. The analysis highlights specific features of P4-ATPases and reveals sites of auto-inhibition and PI4P-dependent activation. We observe the opening of a putative flippase pathway engaging conserved residues Ile508 of transmembrane segment 4 and Lys1018 and polar residues of transmembrane segment 5 in the centre of the lipid bilayer.

molecular biology↗