Hanaa Hariri

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 α-helical assembly rather than the β-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.

S-adenosylmethionine (AdoMet) is a central metabolite required for methylation and sulfur metabolism, synthesized in Saccharomyces cerevisiae by the cytosolic enzymes Sam1 and Sam2. While transcriptional and metabolic regulation of AdoMet biosynthesis have been extensively studied, how metabolic state influences the spatial organization of AdoMet synthases remains incompletely understood. Herein, we show that Sam1 undergoes robust and reversible relocalization into cytosolic assemblies under low-glucose metabolic states. These assemblies are dynamic and dissolve rapidly upon glucose refeeding in a manner that requires metabolizable glucose, indicating regulation by metabolic flux. While Sam1 assemblies associate with stress granules during prolonged starvation, a substantial fraction form independently under acute low-glucose conditions, indicating that Sam1 relocalization is not fully coordinated with canonical stress granule formation. Untargeted metabolomic analysis indicates that AdoMet pools are broadly maintained, while a targeted fluorescence-based enzymatic assay detects a modest but reproducible reduction in AdoMet under low-glucose conditions. Together, these findings define a metabolically gated mechanism for reversible Sam1 sequestration and support a model in which spatial organization of metabolic enzymes is coupled to cellular metabolic state.

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. Cophylogenetic analysis reveals coordinated coevolution of Nvj3 and Mdm1 across Saccharomycetes. Together, these findings define Nvj3 as a structural partner of Mdm1 and support a conduit-based model of lipid transfer at the NVJ.

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 Δ 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 Δ 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 Δ 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.

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, nvj3Δ 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.

Sphingolipids are essential for cell membrane structure and the regulation of organelle functions. Sphingolipid synthesis requires the coordinated activity of multiple organelles, including the endoplasmic reticulum, Golgi, lysosomes and mitochondria, which are connected via membrane contact sites. Metabolic remodeling of sphingolipid pathways is observed in aging and numerous age-related disorders. However, numerous studies have highlighted the complex and species-specific roles of sphingolipid metabolism in aging. In budding yeast, inhibition of sphingolipid synthesis extends lifespan by a mechanism that is poorly understood. Recent findings suggest that inhibition of sphingolipid synthesis in cells mimics methionine restriction, a condition known to extend lifespan across different experimental models. However, how sphingolipid remodeling alters cellular methionine levels, and whether this directly influences aging, remains unclear. In this Review, we explore the roles of sphingolipids in organelle function, highlighting their metabolic connections to methionine restriction and aging.

Lipid droplets (LDs) are fat storage organelles that are conserved from bacteria to humans. LDs are broken down to supply cells with fatty acids (FAs) that can be used as an energy source or membrane synthesis. An overload of FAs disrupts cellular functions and causes lipotoxicity. Thus, by acting as hubs for storing excess fat, LDs prevent lipotoxicity and preserve cellular homeostasis. LD synthesis and turnover have to be precisely regulated to maintain a balanced lipid distribution and allow for cellular adaptation during stress. Here, we discuss how prolonged exposure to excess lipids affects cellular functions, and the roles of LDs in buffering cellular stress focusing on lipotoxicity.

SNX-RGS proteins are molecular tethers localized to multiple interorganelle contact sites that exhibit roles in cellular metabolism. Here, we highlight recent findings on these proteins and discuss their emerging roles in metabolism, human disease, and lipid trafficking.

Summary: 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.