Biology subjects

Menon, A. K.

Publications and source records attributed to Menon, A. K..

15 recordsLinked to original sources

bioRxiv2025

Proposed mechanism for Rft1-mediated scrambling of a dolichol-linked oligosaccharide

The membrane protein Rft1 is proposed to play an essential role in yeast and human cells by scrambling the glycolipid Man5GlcNAc2-PP-dolichol (M5-DLO) across the endoplasmic reticulum (ER) for protein N-glycosylation. While this activity has been demonstrated in liposomes reconstituted with purified Rft1, biochemical evidence of additional M5-DLO scramblases and the viability of Rft1-null Trypanosoma brucei suggest that scrambling may be a moonlighting function of Rft1 rather than its essential cellular role. To investigate this paradox, we used AlphaFold3 and Chai-1 to model the conformational dynamics of yeast Rft1-M5-DLO complexes. The models suggest an alternating access mechanism, typical of Multidrug/Oligosaccharidyl-lipid/Polysaccharide (MOP) superfamily transporters, in which a cationic central cavity coordinates the anionic headgroup of M5-DLO, while the dolichol tail of the lipid is accommodated through a lateral portal formed by two transmembrane helices. We used the models to design mutations to disrupt the interaction between Rft1 and the M5-DLO headgroup, and to engineer a salt bridge to block the portal and stall transport. Using a Tet-off yeast reporter strain, we tested 26 central cavity mutants and identified two that supported cell growth poorly despite being well-expressed. Strikingly, the portal-blocking mutant which lacks scramblase activity supported robust growth. These data suggest that while M5-DLO binding is important for Rft1s essential function, scrambling activity is dispensable. We propose that Rft1s essential role may be as an M5-DLO chaperone, capturing and routing M5-DLO propitiously on the cytoplasmic side of the ER to coordinate DLO biosynthesis. ImportanceCell surface and secreted proteins are decorated with sugar chains. These chains are first assembled on a lipid carrier. Initial stages of assembly occur on the cytoplasmic side of a subcellular structure called the endoplasmic reticulum (ER). To complete assembly, the partially assembled lipid-linked sugar chain must be flipped across the ER. Here we use computationally guided cell-based assays to examine the role of the Rft1 protein in this process.

biochemistry↗

bioRxiv2025

Opsins are Phospholipid Scramblases in All Domains of Life

Opsins are highly abundant retinal proteins in the membranes of photoheterotrophic bacteria. However, some microbial genomes encode an opsin but lack the gene for the final enzyme in retinal synthesis. To account for this paradox, we hypothesized that bacterial opsins play a role in membrane structure and/or biogenesis independent from their potential for light-driven signaling or proton pumping. After purifying actinorhodopsin from a cell-free expression system and from E. coli membranes upon overexpression, we demonstrated both in vitro and in silico that actinorhodopsin from Nanopelagicus ca. is a phospholipid scramblase, serving in its pentameric state as a retinal-independent phospholipid diffusion channel. Phospholipid headgroups move along a transbilayer path between actinorhodopsin protomers, to equilibrate lipid content in the inner and outer leaflets. Two profound activities, membrane biosynthesis and capture of light energy, are thus facilitated by one ancient bacterial polypeptide. Light-dependent activity and light-independent phospholipid scrambling are shared functions of eukaryotic, archaeal, and bacterial rhodopsins. ImportanceCells are surrounded by membranes which concentrate metabolites and protect cellular contents. Most biomembranes are phospholipid bilayers, in which the phospholipids of each leaflet orient their greasy tails inward and polar groups outward. Bilayer biogenesis depends on phospholipids synthesized on the cytofacial side of the membrane reorienting to the extracellular membrane leaflet. This reorientation requires proteins, termed scramblases, and it was shown that rhodopsins -- 7-helix photoactive membrane proteins bound to the cofactor retinal -- from organisms as widely divergent as mammals and archaea possess scramblase activity. Now we conclusively demonstrate using purified proteins in laboratory membranes as well as computational approaches, that bacterial rhodopsins are also phospholipid scramblases. This work is important because it highlights a surprising commonality among bacteria, archaea and eukaryotes and because it shows that rhodopsins - ancient proteins found in the last universal common ancestor - manifest two seemingly unrelated biochemical functions in one protein.

biophysics↗

bioRxiv2025

A single vesicle fluorescence microscopy platform to quantify phospholipid scrambling

Scramblases play important roles in physiology by translocating phospholipids bidirectionally across cell membranes. For example, scrambling facilitated by dimers of the Voltage-Dependent Anion Channel 1 (VDAC1) enables endoplasmic reticulum-derived phospholipids to cross the outer membrane to enter mitochondria. Precise quantification of lipid scrambling, while critical for mechanistic understanding, cannot be obtained from ensemble averaged measurements of reconstituted scramblases. Here, we describe a microscopy platform for high-throughput imaging of single vesicles reconstituted with fluorescently labeled phospholipids and heterogeneously crosslinked VDAC1 dimers. For each vesicle, we quantify size, protein occupancy and scrambling rate. Notably, we find that individual VDAC1 dimers have different activities, ranging from <100 to >10,000 lipids per second. This kinetic heterogeneity, masked in ensemble measurements, reveals that only some dimer interfaces are capable of promoting rapid scrambling, as suggested by molecular dynamics simulations. We extend our analyses to opsin, a monomeric G protein-coupled receptor scramblase, thereby demonstrating the versatility of our platform for quantifying transbilayer lipid transport and exploring its regulation.

biophysics↗

bioRxiv2024

Molecular characterization of Rft1, a membrane protein associated with congenital disorder of glycosylation type 1N

The oligosaccharide needed for protein N-glycosylation is assembled on a lipid carrier via a multi-step pathway. Synthesis is initiated on the cytoplasmic face of the endoplasmic reticulum (ER) and completed on the luminal side after transbilayer translocation of a heptasaccharide lipid intermediate. More than 30 Congenital Disorders of Glycosylation (CDGs) are associated with this pathway, including RFT1-CDG which results from defects in the membrane protein Rft1. Rft1 is essential for the viability of yeast and mammalian cells and was proposed as the transporter needed to flip the heptasaccharide lipid intermediate across the ER membrane. However, other studies indicated that Rft1 is not required for heptasaccharide lipid flipping in microsomes or unilamellar vesicles reconstituted with ER membrane proteins, nor is it required for the viability of at least one eukaryote. It is therefore not known what essential role Rft1 plays in N-glycosylation. Here, we present a molecular characterization of human Rft1, using yeast cells as a reporter system. We show that it is a multi-spanning membrane protein located in the ER, with its N and C-termini facing the cytoplasm. It is not N-glycosylated. The majority of RFT1-CDG mutations map to highly conserved regions of the protein. We identify key residues that are important for Rft1s ability to support N-glycosylation and cell viability. Our results provide a necessary platform for future work on this enigmatic protein.

cell biology↗

bioRxiv2023

A cholesterol switch controls phospholipid scrambling by G protein-coupled receptors

Class A G protein-coupled receptors (GPCRs), a superfamily of cell membrane signaling receptors, moonlight as constitutively active phospholipid scramblases. The plasma membrane of metazoan cells is replete with GPCRs, yet has a strong resting trans-bilayer phospholipid asymmetry, with the signaling lipid phosphatidylserine confined to the cytoplasmic leaflet. To account for the persistence of this lipid asymmetry in the presence of GPCR scramblases, we hypothesized that GPCR-mediated lipid scrambling is regulated by cholesterol, a major constituent of the plasma membrane. We now present a technique whereby synthetic vesicles reconstituted with GPCRs can be supplemented with cholesterol to a level similar to that of the plasma membrane and show that the scramblase activity of two prototypical GPCRs, opsin and the {beta}1-adrenergic receptor, is impaired upon cholesterol loading. Our data suggest that cholesterol acts as a switch, inhibiting scrambling above a receptor-specific threshold concentration to disable GPCR scramblases at the plasma membrane.

biochemistry↗

bioRxiv2023

Insertases Scramble Lipids: Molecular Simulations of MTCH2

Scramblases play a pivotal role in facilitating bidirectional lipid transport across cell membranes, thereby influencing lipid metabolism, membrane homeostasis, and cellular signaling. MTCH2, a mitochondrial outer membrane protein insertase, has a membrane-spanning hydrophilic groove resembling those that form the lipid transit pathway in known scramblases. Employing both coarse-grained and atomistic molecular dynamics simulations, we show that MTCH2 significantly reduces the free energy barrier for lipid movement along the groove and therefore can indeed function as a scramblase. Notably, the scrambling rate of MTCH2 in silico is similar to that of VDAC, a recently discovered scramblase of the outer mitochondrial membrane, suggesting a potential complementary physiological role for these mitochondrial proteins. Finally, our findings suggest that other insertases which possess a hydrophilic path across the membrane like MTCH2, can also function as scramblases. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=183 SRC="FIGDIR/small/553169v2_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@e78668org.highwire.dtl.DTLVardef@292ce0org.highwire.dtl.DTLVardef@1413077org.highwire.dtl.DTLVardef@d4e29b_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIScrambling activity of MTCH2 identified using computer simulations C_LIO_LIMTCH2 may act redundantly with VDAC as outer mitochondrial membrane scram-blase C_LIO_LIInsertases and scramblases may share a common functional mechanism C_LI

biophysics↗

bioRxiv2022

Mitochondrial phospholipid import mediated by VDAC, a dimeric beta barrel scramblase

Mitochondria are double-membrane-bounded organelles that depend critically on phospholipids supplied by the endoplasmic reticulum. These lipids must cross the outer membrane to support mitochondrial function, but how they do this is unclear. We identified the voltage-dependent ion channel (VDAC), an abundant outer membrane protein, as a scramblase-type lipid transporter that catalyzes lipid entry. On reconstitution into membrane vesicles, dimers of human VDAC1 and VDAC2 catalyze rapid transbilayer translocation of phospholipids by a mechanism that is unrelated to their channel activity. Coarse-grained molecular dynamics simulations of VDAC1 reveal that lipid scrambling occurs at a specific dimer interface where polar residues induce large water defects and bilayer thinning. The rate of phospholipid import into yeast mitochondria is an order of magnitude lower in the absence of VDAC homologs, indicating that VDACs provide the main pathway for lipid entry. Thus, VDAC isoforms, members of a superfamily of beta barrel proteins, moonlight as a new class of phospholipid scramblases - distinct from alpha-helical scramblase proteins - that act by an unprecedented mechanism to import lipids into mitochondria.

biochemistry↗

bioRxiv2022

A single vesicle fluorescence-bleaching assay for multi-parameter analysis of proteoliposomes by total internal reflection fluorescence microscopy

Reconstitution of membrane proteins into model membranes is an essential approach for their functional analysis under chemically defined conditions. Established model-membrane systems used in ensemble average measurements are limited by sample heterogeneity and insufficient knowledge of lipid and protein content at the single vesicle level, which limits quantitative analysis of vesicle properties and prevents their correlation with protein activity. Here, we describe a versatile total internal reflection fluorescence microscopy-based bleaching protocol that permits parallel analyses of multiple parameters (physical size, tightness, unilamellarity, membrane protein content and orientation) of individual proteoliposomes prepared with fluorescently tagged membrane proteins and lipid markers. The approach makes use of commercially available fluorophores including the commonly used nitrobenzoxadiazole (NBD) dye and may be applied to deduce functional molecular characteristics of many types of reconstituted fluorescently tagged membrane proteins.

biochemistry↗

bioRxiv2021

CLPTM1L is a lipid scramblase involved in glycosylphosphatidylinositol biosynthesis

Glycosylphosphatidylinositols (GPIs) are membrane anchors of many eukaryotic cell surface proteins. Biosynthesis of GPIs is initiated at the cytosolic face of the endoplasmic reticulum (ER) and the second intermediate, glucosaminyl-phosphatidylinositol (GlcN-PI), is translocated across the membrane to the lumenal face for later biosynthetic steps and attachment to proteins. The mechanism of the lumenal translocation of GlcN-PI is unclear. We report that Cleft lip and palate transmembrane protein 1-like protein (CLPTM1L), an ER membrane protein of unknown function, is a lipid scramblase involved in GPI biosynthesis. Purified CLPTM1L scrambles GlcN-PI, PI, and several other phospholipids in vitro. Knockout of CLPTM1L gene in mammalian cultured cells partially decreased GPI-anchored proteins due to impaired usage of GlcN-PI, suggesting a major role of CLPTM1L in lumenal translocation of GlcN-PI. One-Sentence SummaryCLPTM1L translocates glucosaminyl-phosphatidylinositol across the membrane during glycosylphosphatidylinositol biosynthesis.

biochemistry↗

bioRxiv2021

Endoplasmic reticulum phospholipid scramblase activity revealed after protein reconstitution into giant unilamellar vesicles containing a photostable lipid reporter

Transbilayer movement of phospholipids in biological membranes is mediated by a diverse set of lipid transporters. Among them are scramblases that facilitate a rapid bi-directional movement of lipids without metabolic energy input. Here, we established a new fluorescence microscopy-based assay for detecting phospholipid scramblase activity of membrane proteins upon their reconstitution into giant unilamellar vesicles formed from proteoliposomes by electroformation. The assay is based on chemical bleaching of fluorescence of a photostable ATTO-dye labeled phospholipid with the membrane-impermeant reductant sodium dithionite. We demonstrate that this new methodology is suitable for the study of the scramblase activity of the yeast endoplasmic reticulum at single vesicle level.

biochemistry↗

bioRxiv2021

Elimination of GPI2 suppresses glycosylphosphatidylinositol GlcNAc transferase activity and alters GPI glycan modification in Trypanosoma brucei

The biosynthesis of glycosylphosphatidylinositol (GPI) membrane protein anchors is initiated in the endoplasmic reticulum by transfer of GlcNAc from the sugar nucleotide UDP-GlcNAc to phosphatidylinositol. The reaction is catalyzed by GPI GlcNAc transferase, a multi-subunit complex comprising the catalytic subunit Gpi3/PIG-A, as well as at least five other subunits including the hydrophobic protein Gpi2 which is essential for activity in yeast and mammals, but whose function is not known. Here we exploited Trypanosoma brucei (Tb), an early diverging eukaryote and important model organism, to investigate the function of Gpi2. We generated trypanosomes that lack TbGPI2 and found that in TbGPI2-null parasites (i) GPI GlcNAc transferase activity is reduced but not lost, in contrast with the situation in yeast and human cells, (ii) the GPI GlcNAc transferase complex persists, but its architecture is affected, with loss of at least the TbGPI1 subunit, and (iii) the GPI anchors of the major surface proteins are underglycosylated when compared with their wild-type counterparts, indicating the importance of TbGPI2 for reactions that are expected to occur in the Golgi apparatus. Additionally, TbGPI2-null parasites were unable to perform social motility, a form of collective migration on agarose plates. Immunofluorescence microscopy localized TbGPI2 to the endoplasmic reticulum as expected, but also to the Golgi apparatus, suggesting that in addition to its expected function as a subunit of the GPI GlcNAc transferase complex, TbGPI2 may have an enigmatic non-canonical role in Golgi-localized GPI anchor modification in trypanosomes.

biochemistry↗

bioRxiv2020

Unusual mode of dimerization of retinitis pigmentosa-associated F220C rhodopsin

Mutations in the G protein-coupled receptor (GPCR) rhodopsin are a common cause of autosomal dominant retinitis pigmentosa, a blinding disease. Rhodopsin self-associates in the membrane, and the purified monomeric apo-protein opsin dimerizes in vitro as it transitions from detergent micelles to reconstitute into a lipid bilayer. We previously reported that the retinitis pigmentosa-linked F220C opsin mutant fails to dimerize in vitro, reconstituting as a monomer. Using fluorescence-based assays and molecular dynamics simulations we now report that whereas wildtype and F220C opsin display distinct dimerization propensities in vitro as previously shown, they both dimerize in the plasma membrane of HEK293 cells. Unexpectedly, molecular dynamics simulations show that F220C opsin forms an energetically favored dimer in the membrane when compared with the wild-type protein. The conformation of the F220C dimer is unique, with transmembrane helices 5 and 6 splayed apart, promoting widening of the intracellular vestibule of each protomer and influx of water into the protein interior. FRET experiments with SNAP-tagged wild-type and F220C opsin expressed in HEK293 cells are consistent with this conformational difference. We speculate that the unusual mode of dimerization of F220C opsin in the membrane may have physiological consequences.

biophysics↗

bioRxiv2020

On identifying the eukaryotic N-glycosylation scramblase by activity correlation profiling

The canonical pathway of N-linked protein glycosylation in yeast and humans involves transfer of the oligosaccharide moiety from the glycolipid Glc3Man9GlcNAc2-PP-dolichol to select asparagine residues in proteins that have been translocated into the lumen of the endoplasmic reticulum (ER). Synthesis of Glc3Man9GlcNAc2-PP-dolichol occurs in two stages, producing first the key intermediate Man5GlcNAc2-PP-dolichol (M5-DLO) on the cytoplasmic face of the ER, followed by translocation of M5-DLO across the ER membrane to the luminal leaflet where the remaining glycosyltransfer reactions occur to complete the structure. Despite its critical importance for N-glycosylation, the scramblase protein that mediates the translocation of M5-DLO across the ER membrane has not been identified. Building on our ability to recapitulate scramblase activity in large unilamellar proteoliposomes reconstituted with a crude mixture of ER membrane proteins, we developed a mass spectrometry-based activity correlation profiling approach to identify scramblase candidates in the yeast Saccharomyces cerevisiae. Curation of the activity correlation profiling data prioritized six polytopic ER membrane proteins as scramblase candidates, but reconstitution-based assays and gene disruption in the protist Trypanosoma brucei revealed, unexpectedly, that none of these proteins were necessary for M5-DLO scramblase activity. Our results instead suggest the possibility that the M5-DLO scramblase may be a protein, or protein complex, whose activity is regulated at the level of quaternary structure. This key insight will aid future attempts to identify the scramblase.

biochemistry↗

bioRxiv2019

Water for sterol: an unusual mechanism of sterol egress from a StARkin domain

Previously we identified a new family of endoplasmic reticulum membrane proteins that possess sterol-binding StARkin domains (Gatta et al. eLife 2015). These Lam/GramD1 proteins are implicated in intracellular sterol homeostasis, a function that requires them to be able to bind sterols. Here we show how these proteins exchange sterol molecules with membranes. An aperture at one end of the StARkin domain enables sterol to enter/exit the binding pocket. Strikingly, the wall of the pocket is fractured along its length, exposing bound sterol to solvent. We considered whether hydration of the pocket could mediate sterol entry/exit. Large-scale atomistic molecular dynamics simulations reveal that sterol egress involves widening of the fracture, penetration of water into the cavity and consequent destabilization of the bound sterol. The simulations also identify polar residues along the fracture that are important for sterol release. Their replacement with alanine affects the ability of the StARkin domain to bind sterol, catalyze inter-vesicular sterol exchange and alleviate the nystatin-sensitivity of lam2{Delta} yeast cells. These data suggest an unprecedented, water-controlled mechanism of sterol acquisition and discharge from a StARkin domain.

biochemistry↗

bioRxiv2017

Phylogenetic profiling identifies glucosyl-phosphoryl dolichol scramblase candidates

1. Background 1. Background 2. Results 3. Summary Appendix A:. List of... Appendix B:. Ranked top... References Protein glycosylation is essential for all eukaryotes, from disease-causing protists such as malaria and trypanosomes, to yeast and mammals. Secretory proteins are almost invariably N-glycosylated, O- and C-mannosylated, and/or GPI-anchored as they enter the lumen of the endoplasmic reticulum (ER).\n\nAll ER protein glycosylation reactions occur in the lumen and often involve lumenal mannosylation and glucosylation steps in which mannose and glucose residues are sourced from the glycolipids mannosyl- and glucosyl-phosphoryl dolichol (MPD and GPD, respectively) [1, 2]. Paradoxically, these two lipids are synthesized on the cytoplasmic ...

cell biology↗