Biology subjects

Hariri, H.

Publications and source records attributed to Hariri, H..

11 recordsLinked to original sources

bioRxiv2026

Structural Organization of the Nvj3-Mdm1 Complex Reveals a Conserved Lipid-Compatible Contact Site Module

Membrane contact sites are organized by protein assemblies that physically couple organelles and coordinate lipid metabolism, yet the structural principles that enable lipid exchange across these junctions remain poorly defined. At the nuclear-vacuolar junction (NVJ) in budding yeast, the tethering protein Mdm1 and its binding partner Nvj3 form a complex that regulates lipid metabolic pathways, but the structural features underlying their interaction have not been resolved. Here, we use AlphaFold-based complex prediction and comparative structural analysis to define the organization of Nvj3-Mdm1 complex assembly. We identify a high-confidence heterodimer in which conserved PXA and PXC domains generate an extended tunnel spanning both proteins. Tunnel analysis predicts a core hydrophobic conduit traversing the Nvj3-Mdm1 interface, consistent with a lipid-compatible architecture. Evolutionary conservation is enriched at the Nvj3-Mdm1 interface. The predicted conduit shares geometric and physicochemical properties with bridge-like lipid transfer proteins, including Atg2, Fmp27, and Hob2, suggesting that heteromeric tether assemblies may contribute directly to inter-organelle lipid transfer. Notably, this conduit is predicted to arise from a heteromeric -helical assembly rather than the {beta}-sheet-rich architecture characteristic of canonical bridge-like lipid transfer proteins. Comparative phylogenetic analyses showed that Nvj3 and Mdm1 share broadly congruent evolutionary patterns across Saccharomycetes, consistent with their conserved functional association. Together, these findings define Nvj3 as a structural partner of Mdm1 and support a conduit-based model of lipid transfer at the NVJ.

bioinformatics↗

bioRxiv2026

Adaptation of white adipocytes to cooler temperatures: impacts on energy metabolism and protein acetylation

Adipocytes throughout the body reside in distinct thermal environments. Visceral adipocytes within the body core are maintained near 37 {degrees}C, whereas those in bone marrow, subcutaneous, and dermal depots occupy cooler regions within the peripheral shell. While brown and beige adipocyte responses to cold stress are well characterized, much less is known about how white adipocytes adapt to moderately reduced temperatures below 37 {degrees}C. Our recent work revealed that cultured adipocytes exposed to 31 {degrees}C, a temperature representative of distal adipose regions, exhibit enhanced mitochondrial function, including increased substrate oxidation and ATP turnover, yet the mechanisms underlying this upregulation remain unclear. Here we show that adaptation to cool temperatures leads to a widespread decrease in protein acetylation in both undifferentiated and differentiated adipocytes, independent of nutrient status, and that this change is readily reversible upon rewarming. Subcellular fractionation and immunoblotting demonstrate that the hypoacetylation coincides with a compartment-specific enrichment of acetylated proteins within mitochondria, indicating selective remodeling of the mitochondrial acetylome. Transcriptomic and biochemical analyses reveal that these temperature-dependent changes occur without alterations in acetyltransferase or deacetylase expression, NAD concentration, or acetyl-CoA availability, suggesting regulation through alternative mechanisms affecting acetyl-CoA flux or enzyme activity. Integrative acetyl-proteomic and metabolomic profiling identifies mitochondrial enzymes, including serine hydroxymethyltransferase 2 (SHMT2) and propionyl-CoA carboxylase (PCCA), whose acetylation correlates closely with changes in associated metabolite pools. Together, these findings establish physiologically relevant cooling as a cell-autonomous regulator of mitochondrial protein acetylation and metabolic adaptation in adipocytes.

cell biology↗

bioRxiv2026

Sphingolipid regulation by yeast Mdm1 supports adaptive remodeling of the methionine transporter Mup1

Membrane lipid composition influences endocytic remodeling of nutrient transporters, yet how lipid metabolism is spatially coordinated to support sustained adaptation to nutrient limitations remains unclear. Here, we investigated whether the ER-vacuole tether Mdm1 links sphingolipid homeostasis to regulation of the high-affinity methionine permease Mup1 in budding yeast. To test this, we examined Mup1 trafficking, amino acid homeostasis, and sphingolipid composition in mdm1{Delta} cells during starvation. We found that loss of Mdm1 causes persistent retention of Mup1 at the plasma membrane, accompanied by reduced intracellular methionine and broad amino acid depletion. Lipidomic analyses revealed decreased sphingoid bases and altered ceramide composition in mdm1{Delta} cells. Importantly, supplementation with the sphingolipid precursor phytosphingosine restored sphingolipid pools, rescued Mup1 endocytosis, and improved amino acid homeostasis. Consistent with a chronic amino acid restriction-like state, mdm1{Delta} cells exhibited extended chronological lifespan. Together, these findings identify Mdm1 as a spatial organizer of sphingolipid metabolism required for adaptive endocytic remodeling of Mup1, thereby linking ER-vacuole contact site function to plasma membrane proteostasis and metabolic adaptation.

cell biology↗

bioRxiv2026

Nvj3 regulates Dga1-mediated triacylglycerol synthesis and lipid droplet formation at ER contact sites

Lipid droplet (LD) biogenesis is essential for lipid homeostasis during nutrient stress, yet how lipid intermediates are spatially organized to support efficient triacylglycerol (TAG) synthesis remains unclear. Here, we identify Nvj3 as a nutrient-responsive regulator that links diacylglycerol (DAG) availability to TAG synthesis and LD formation at the endoplasmic reticulum (ER). Nvj3 is induced by glucose depletion and recruited to LD-associated ER domains. Loss of Nvj3 causes neutral lipid accumulation under steady state conditions but delays TAG synthesis under acute inducible metabolic transitions. Using controlled TAG induction systems, we show that Nvj3 is required to couple Dga1-dependent TAG synthesis to LD formation. In the absence of Nvj3, TAG accumulates but remains inefficiently packaged into LDs. Consistent with this defect, nvj3{Delta} cells exhibit altered phospholipid remodeling and mislocalization of DAG away from ER domains during starvation. Together, these findings establish Nvj3 as an organizer of lipid availability during metabolic stress and suggest that spatial control of DAG is a key determinant of LD biogenesis. SummaryThis study identifies Nvj3 as a spatial organizer of Dga1-dependent lipid droplet formation. Nvj3 promotes proper diacylglycerol positioning, and enables efficient triacylglycerol synthesis during metabolic stress. We propose that Nvj3 regulates lipid flux through spatial compartmentalization of diacylglycerol at membrane contact site-associated ER domains.

cell biology↗

bioRxiv2021

Experimental and in silico Alphafold2 derived structures of the SNX-RGS proteins suggest a new class of lipid transfer protein

Recent advances in protein structure prediction using machine learning such as AlphaFold2 and RosettaFold presage a revolution in structural biology. Genome-wide predictions of protein structures are providing unprecedented insights into their architecture and intradomain interactions, and applications have already progressed towards assessing protein complex formation. Here we present detailed analyses of the sorting nexin proteins that contain regulator of G-protein signalling domains (SNX-RGS proteins), providing a key example of the ability of AlphaFold2 to reveal novel structures with previously unsuspected biological functions. These large proteins are conserved in most eukaryotes and are known to associate with lipid droplets (LDs) and sites of LD-membrane contacts, with key roles in regulating lipid metabolism. They possess five domains, including an N-terminal transmembrane domain that anchors them to the endoplasmic reticulum, an RGS domain, a lipid interacting phox homology (PX) domain and two additional domains named the PXA and PXC domains of unknown structure and function. Here we report the crystal structure of the RGS domain of sorting nexin 25 (SNX25) and show that the AlphaFold2 prediction closely matches the experimental structure. Analysing the full-length SNX-RGS proteins across multiple homologues and species we find that the distant PXA and PXC domains in fact fold into a single unique structure that notably features a large and conserved hydrophobic pocket. The nature of this pocket strongly suggests a role in lipid or fatty acid binding, and we propose that these molecules represent a new class of conserved lipid transfer proteins.

biochemistry↗

bioRxiv2021

Vacuole-specific lipid release for tracking intracellular lipid metabolism and transport in Saccharomyces cerevisiae

Lipid metabolism is spatiotemporally regulated within cells, yet intervention into lipid functions at subcellular resolution remains difficult. Here we report a method that enables site-specific release of sphingolipids and cholesterol inside the vacuole in Saccharomyces cerevisiae. Using this approach, we monitored real-time sphingolipid metabolic flux out of the vacuole by mass spectrometry and found that the ER-vacuole tethering protein Mdm1 facilitated the metabolism of sphingoid bases into ceramides. In addition, we showed that cholesterol, once delivered into yeast using our method, could restore cell proliferation induced by ergosterol deprivation, overcoming the previously described sterol-uptake barrier under aerobic conditions. Together, these data define a new way to study intracellular lipid metabolism and transport from the vacuole in yeast.

cell biology↗

bioRxiv2020

Glucose restriction drives spatial re-organization of mevalonate metabolism and liquid-crystalline lipid droplet biogenesis

Eukaryotes compartmentalize metabolic pathways into sub-cellular domains, but the role of inter-organelle contacts in organizing metabolic reactions remains poorly understood. Here, we show that in response to acute glucose restriction (AGR) yeast undergo metabolic remodeling of their mevalonate pathway that is spatially coordinated at nucleus-vacuole junctions (NVJs). The NVJ serves as a metabolic platform by selectively retaining HMG-CoA Reductases (HMGCRs), driving mevalonate pathway flux in an Upc2-dependent manner. AGR-induced HMGCR compartmentalization enhances mevalonate metabolism and sterol-ester biosynthesis that generates lipid droplets (LDs) with liquid-crystalline sub-architecture. Loss of NVJ-dependent HMGCR partitioning affects yeast growth, but can be bypassed by artificially multimerizing HMGCRs, indicating NVJ compartmentalization enhances mevalonate pathway flux by promoting HMGCR inter-enzyme associations. We propose a non-canonical mechanism regulating mevalonate metabolism via the spatial compartmentalization of rate-limiting HMGCR enzymes, and reveal that AGR creates LDs with remarkable phase transition properties. One Sentence SummarySpatial compartmentalization of HMG-CoA Reductases at ER-lysosome contacts modulates mevalonate pathway flux

cell biology↗

bioRxiv2020

Nutrient signaling, stress response, and interorganelle communication are non-canonical determinants of cell fate

Isogenic cells can manifest distinct cellular fates for a single stress, however the nongenetic mechanisms driving such fates remain poorly understood. Here, we implement a robust multi-channel live-cell imaging approach to uncover noncanonical factors governing cell fate. We show that in response to acute glucose removal (AGR), budding yeast undergo distinct fates becoming either quiescent or senescent. Senescent cells fail to resume mitotic cycles following glucose replenishment but remain responsive to nutrient stimuli. Whereas quiescent cells manifest starvation-induced adaptation, senescent cells display perturbed endomembrane trafficking and defective nucleus-vacuole junction (NVJ) expansion. Surprisingly, we also show senescence occurs in the absence of lipid droplets. Importantly, we identify the nutrient-sensing linked kinase Rim15 as a key biomarker that predicts cell fates before AGR stress. We propose that isogenic yeast challenged with acute nutrient shortage contain determinants that influence their post-stress fate, and demonstrate that specific nutrient signaling, stress-response, endomembrane trafficking, and inter-organelle tether biomarkers are early indicators for long-term fate outcomes.

cell biology↗

bioRxiv2020

Proximity labeling of Snx14 reveals a functional interaction with delta-9 desaturase SCD1 to maintain ER homeostasis

Fatty acids (FAs) are central cellular metabolites that contribute to lipid synthesis, and can be stored or harvested for metabolic energy. Dysregulation in FA processing and storage causes toxic FA accumulation or altered membrane compositions and contributes to metabolic and neurological disorders. Saturated lipids are particularly detrimental to cells, but how lipid saturation levels are maintained remains poorly understood. Here, we identify the cerebellar ataxia SCAR20-associated protein Snx14, an endoplasmic reticulum (ER)-lipid droplet (LD) tethering protein, as a novel factor required to maintain the lipid saturation balance of cell membranes. We show that SNX14KO cells and SCAR20 disease patient-derived cells are hypersensitive to saturated FA (SFA)-mediated lipotoxic cell death that compromises ER integrity. Using APEX2-based proximity labeling, we reveal the protein composition of Snx14-associated ER-LD contacts and define a functional interaction between Snx14 and {Delta}-9 FA desaturase SCD1. Lipidomic profiling reveals that SNX14KO cells increase membrane lipid saturation following exposure to palmitate, phenocopying cells with reduced SCD1 activity. In line with this, SNX14KO cells manifest delayed FA processing and lipotoxicity, which can be rescued by SCD1 over-expression. Altogether these mechanistic insights reveal a role for Snx14 in FA and ER homeostasis, defects in which may underlie the neuropathology of SCAR20. Significance StatementSCAR20 disease is an autosomal recessive spinocerebellar ataxia primarily affecting children, and results from loss-of-function mutations in the SNX14 gene. Snx14 is an endoplasmic reticulum (ER)-localized protein that localizes to ER-lipid droplet (LD) contacts and promotes LD biogenesis following exogenous FA treatment, but why Snx14 loss causes SCAR20 is unclear. Here, we demonstrate that following exposure to saturated fatty acids, Snx14-deficient cells have defective ER homeostasis and altered lipid saturation profiles. We reveal a functional interaction between Snx14 and fatty acid (FA) desaturase SCD1. Lipidomics shows Snx14-deficient cells contain elevated saturated lipids, closely mirroring SCD1-defective cells. Furthermore, SCD1 over-expression can rescue Snx14 loss. We propose that Snx14 maintains cellular lipid homeostasis, the loss of which underlies the cellular basis for SCAR20 disease.

cell biology↗

bioRxiv2019

Diverse Species-Specific Phenotypic Consequences of Loss of Function Sorting Nexin 14 Mutations

Mutations in the SNX14 gene cause spinocerebellar ataxia, autosomal recessive 20 (SCAR20) in both humans and dogs. SCAR20 is understood to involve subcellular disruption to autophagy and lipid metabolism. Previously reported studies on the phenotypic consequences of SNX14 mutations have been limited to in vitro investigation of patient-derived dermal fibroblasts, laboratory engineered cell lines and developmental analysis of zebrafish morphants. In addition, studies have investigated the biochemical roles of SNX14 homologues Snz (Drosophila) and Mdm1 (yeast) which have demonstrated an important role during lipid biogenesis. This study investigates the impact of constitutive Snx14 mutations in laboratory species: mice and zebrafish. Loss of SNX14 in mice was found to be embryonic lethal around mid-gestation. This is due to placental pathology that involves severe disruption to syncytiotrophoblast cell differentiation. Zebrafish carrying a homozygous, maternal zygotic snx14 genetic loss-of-function mutation contrasts with other vertebrates, being both viable and anatomically normal. Whilst no obvious behavioural effects were observed, elevated levels of neutral lipids and phospholipids resemble previously reported effects on lipid homeostasis in other species. The biochemical role of SNX14 therefore appears largely conserved through evolution while the overall consequences of loss of function varies considerably between species. New mouse and zebrafish models therefore provide valuable insights into the functional importance of SNX14 with distinct opportunities for investigating its cellular and metabolic function in vivo.

developmental biology↗

bioRxiv2019

Drosophila Snazarus regulates a lipid droplet population at plasma membrane-droplet contacts in adipocytes

Adipocytes store nutrients as lipid droplets (LDs), but how they organize their LD stores to balance lipid uptake, storage, and mobilization remains poorly understood. Here, using Drosophila fat body (FB) adipocytes we characterize spatially distinct LD populations that are maintained by different lipid pools. We identify peripheral LDs (pLDs) that make close contact with the plasma membrane (PM) and are maintained by lipophorin-dependent lipid trafficking. pLDs are distinct from larger cytoplasmic medial LDs (mLDs) which are maintained by FASN1-dependent de novo lipogenesis. We find that sorting nexin CG1514/Snazarus (Snz) associates with pLDs and regulates LD homeostasis at ER-PM contact sites. Loss of SNZ perturbs pLD organization whereas Snz over-expression drives LD expansion, triacylglyceride production, starvation resistance, and lifespan extension through a DESAT1-dependent pathway. We propose that Drosophila adipocytes maintain spatially distinct LD populations and identify Snz as a novel regulator of LD organization and inter-organelle crosstalk.

cell biology↗