A metabolically controlled contact site between vacuoles and lipid droplets in yeast.

The lipid droplet (LD) organization proteins Ldo16 and Ldo45 affect multiple aspects of LD biology in yeast. They are linked to the LD biogenesis machinery seipin, and their loss causes defects in LD positioning, protein targeting, and breakdown. However, their molecular roles remained enigmatic. Here, we report that Ldo16/45 form a tether complex with Vac8 to create vacuole lipid droplet (vCLIP) contact sites, which can form in the absence of seipin. The phosphatidylinositol transfer protein (PITP) Pdr16 is a further vCLIP-resident recruited specifically by Ldo45. While only an LD subpopulation is engaged in vCLIPs at glucose-replete conditions, nutrient deprivation results in vCLIP expansion, and vCLIP defects impair lipophagy upon prolonged starvation. In summary, Ldo16/45 are multifunctional proteins that control the formation of a metabolically regulated contact site. Our studies suggest a link between LD biogenesis and breakdown and contribute to a deeper understanding of how lipid homeostasis is maintained during metabolic challenges.

Hard to handle: how lipid saturation affects the nuclear envelope.

The nuclear envelope is a unique subdomain of the endoplasmic reticulum (ER) that encapsulates the genome and mediates communication between the nucleus and the rest of the cell via nuclear pore complexes. A recent study by Romanauska and Köhler shows that balanced lipid unsaturation is critical for nuclear envelope and nuclear pore complex architecture and function.

A lysosomal biogenesis map reveals the cargo spectrum of yeast vacuolar protein targeting pathways.

The lysosome is the major catabolic organelle in the cell that has been established as a key metabolic signaling center. Mutations in many lysosomal proteins have catastrophic effects and cause neurodegeneration, cancer, and age-related diseases. The vacuole is the lysosomal analog of Saccharomyces cerevisiae that harbors many evolutionary conserved proteins. Proteins reach vacuoles via the Vps10-dependent endosomal vacuolar protein sorting pathway, via the alkaline phosphatase (ALP or AP-3) pathway, and via the cytosol-to-vacuole transport (CVT) pathway. A systematic understanding of the cargo spectrum of each pathway is completely lacking. Here, we use quantitative proteomics of purified vacuoles to generate the yeast lysosomal biogenesis map. This dataset harbors information on the cargo-receptor relationship of almost all vacuolar proteins. We map binding motifs of Vps10 and the AP-3 complex and identify a novel cargo of the CVT pathway under nutrient-rich conditions. Our data show how organelle purification and quantitative proteomics can uncover fundamental insights into organelle biogenesis.

Dual role of Mic10 in mitochondrial cristae organization and ATP synthase-linked metabolic adaptation and respiratory growth.

Invaginations of the mitochondrial inner membrane, termed cristae, are hubs for oxidative phosphorylation. The mitochondrial contact site and cristae organizing system (MICOS) and the dimeric F1Fo-ATP synthase play important roles in controlling cristae architecture. A fraction of the MICOS core subunit Mic10 is found in association with the ATP synthase, yet it is unknown whether this interaction is of relevance for mitochondrial or cellular functions. Here, we established conditions to selectively study the role of Mic10 at the ATP synthase. Mic10 variants impaired in MICOS functions stimulate ATP synthase oligomerization like wild-type Mic10 and promote efficient inner membrane energization, adaptation to non-fermentable carbon sources, and respiratory growth. Mic10's functions in respiratory growth largely depend on Mic10ATPsynthase, not on Mic10MICOS. We conclude that Mic10 plays a dual role as core subunit of MICOS and as partner of the F1Fo-ATP synthase, serving distinct functions in cristae shaping and respiratory adaptation and growth.

A move in response to starvation.

When a yeast cell runs out of fuel, it can increase the flux through a central metabolic pathway by simply changing the location of an enzyme.

Lipid Droplet Contact Sites in Health and Disease.

After having been disregarded for a long time as inert fat drops, lipid droplets (LDs) are now recognized as ubiquitous cellular organelles with key functions in lipid biology and beyond. The identification of abundant LD contact sites, places at which LDs are physically attached to other organelles, has uncovered an unexpected level of communication between LDs and the rest of the cell. In recent years, many disease factors mutated in hereditary disorders have been recognized as LD contact site proteins. Furthermore, LD contact sites are dramatically rearranged in response to infections with intracellular pathogens, as well as under pathological metabolic conditions such as hepatic steatosis. Collectively, it is emerging that LD-organelle contacts are important players in development and progression of disease.

CG32803 is the fly homolog of LDAF1 and influences lipid storage in vivo.

The Seipin protein is a conserved key component in the biogenesis of lipid droplets (LDs). Recently, a cooperation between human Seipin and the Lipid droplet assembly factor 1 (LDAF1) was described. LDAF1 physically interacts with Seipin and the holocomplex safeguards regular LD biogenesis. The function of LDAF1 proteins outside mammals is less clear. In yeast, the lipid droplet organization (LDO) proteins, which also cooperate with Seipin, are the putative homologs of LDAF1. While certain functional aspects are shared between the LDO and mammalian LDAF1 proteins, the relationship between the proteins is under debate. Here, we identify the Drosophila melanogaster protein CG32803, which we re-named to dmLDAF1, as an insect member of this protein family. dmLDAF1 decorates LDs in cultured cells and in vivo and the protein is linked to the fly and mouse Seipin proteins. Altering the dmLDAF1 abundance affects LD size, number and overall lipid storage amounts. Our results suggest that the LDAF1 proteins thus fulfill an evolutionarily conserved function in the biogenesis and biology of LDs.

Tether Me, Tether Me Not-Dynamic Organelle Contact Sites in Metabolic Rewiring.

Membrane contact sites (CSs) are specialized cellular regions where distinct organelles are actively positioned very close to each other, at a distance of just a few nanometers. Structurally, CS formation depends on tether proteins that physically link organellar membranes to each other. Functionally, these structures act as hotspots for communication and material transfer. In recent years, we are starting to understand that the cellular CS landscape is not static but instead responds dynamically to diverse metabolic cues. This review describes the interplay between cellular metabolism and CS-based organelle communication and discusses molecular mechanisms of contact modulation in cellular adaptation to changing metabolic requirements.

New friends for seipin - Implications of seipin partner proteins in the life cycle of lipid droplets.

Lipids act as building blocks for all cellular membranes and as key energy carriers. Neutral storage lipids are packaged into specialized organelles termed Lipid Droplets (LDs). LDs dynamically respond to the metabolic state of the cell, and undergo cycles of de novo biogenesis, growth, shrinkage, and consumption. How these processes are mediated on a molecular level is a key objective of the LD field. The yeast Lipid Droplet Organization (LDO) proteins and the human promethin/TMEM159/LDAF1 are newly identified molecular players involved in different aspects of the life cycle of LDs. These factors show remote homology to each other, and are physically and functionally linked to seipin, a central component in LD formation and adipogenesis.

The Sweet and Sour Taste of Phosphoinositide Signaling: Protonation of PI4P Modulates Its Function in Response to Cytoplasmic pH Changes.

Phosphoinositides are signaling lipids that recruit effector proteins to membranes. Shin et al. show that a glucose-starvation-induced drop in cytosolic pH alters the protonation state of the phosphoinositide PI4P, resulting in dissociation of its effector Osh1 from the trans-Golgi network membrane and metabolic regulation of lipid and protein sorting.

Born this way - Biogenesis of lipid droplets from specialized ER subdomains.

Both the endoplasmic reticulum (ER) and lipid droplets (LDs) are key players in lipid handling. In addition to this functional connection, the two organelles are also tightly linked due to the fact that the ER is the birthplace of LDs. LDs have an atypical architecture, consisting of a neutral lipid core that is covered by a phospholipid monolayer. LD biogenesis starts with neutral lipid synthesis in the ER membrane and formation of small neutral lipid lenses between its leaflets, followed by budding of mature LDs toward the cytosol. Several ER proteins have been identified that are required for efficient LD formation, among them seipin, Pex30, and FIT2. Recent evidence indicates that these LD biogenesis factors might cooperate with specific lipids, thus generating ER subdomains optimized for LD assembly. Intriguingly, LD biogenesis reacts dynamically to nutrient stress, resulting in a spatial reorganization of LD formation in the ER.

Come a little bit closer! Lipid droplet-ER contact sites are getting crowded.

Not so long ago, contact sites between the endoplasmic reticulum (ER) and lipid droplets (LDs) were largely unexplored on a molecular level. In recent years however, numerous proteins have been identified that are enriched or exclusively located at the interfaces between LDs and the ER. These comprise members of protein classes typically found in diverse types of contacts, such as organelle tethers and lipid transfer proteins, but also proteins that have no similarities to known contact site machineries. This structurally heterogeneous group of contact site residents might be required to fulfill unique aspects of LD-ER contact biology, such as de novo LD biogenesis, and maintenance of lipidic connections between LDs and ER. Here, we summarize the current knowledge on the molecular components of this special organelle contact site, and discuss their features and functions.

Organelle Contact Sites: Lipid Droplets Hooked by Metabolically Controlled Tethers.

Lipid droplets are physically linked to other organelles via contact sites for communication, but the underlying molecular machineries are poorly characterized. Recent studies identify metabolically controlled sorting nexin tether proteins as important players at these sites.

Promethin Is a Conserved Seipin Partner Protein.

Seipin (BSCL2/SPG17) is a key factor in lipid droplet (LD) biology, and its dysfunction results in severe pathologies, including the fat storage disease Berardinelli-Seip congenital lipodystrophy type 2, as well as several neurological seipinopathies. Despite its importance for human health, the molecular role of seipin is still enigmatic. Seipin is evolutionarily conserved from yeast to humans. In yeast, seipin was recently found to cooperate with the lipid droplet organization (LDO) proteins, Ldo16 and Ldo45, two structurally-related proteins involved in LD function and identity that display remote homology to the human protein promethin/TMEM159. In this study, we show that promethin is indeed an LD-associated protein that forms a complex with seipin, and its localization to the LD surface can be modulated by seipin expression levels. We thus identify promethin as a novel seipin partner protein.

Wrapping up the fats-a structure of the lipid droplet biogenesis protein seipin.

The lipid droplet (LD) biogenesis protein seipin is crucial for formation of normal LDs, but its exact functional role has been enigmatic. In this issue, Sui et al. (2018. J. Cell Biol. https://doi.org/10.1083/jcb.201809067) report the cryo--electron microscopy structure of seipin, which provides novel insights into how seipin might mediate LD formation.

Vps39 Interacts with Tom40 to Establish One of Two Functionally Distinct Vacuole-Mitochondria Contact Sites.

The extensive subcellular network of membrane contact sites plays central roles in organelle biogenesis and communication, yet the precise contributions of the involved machineries remain largely enigmatic. The yeast vacuole forms a membrane contact site with mitochondria, called vacuolar and mitochondrial patch (vCLAMP). Formation of vCLAMPs involves the vacuolar Rab GTPase Ypt7 and the Ypt7-interacting Vps39 subunit of the HOPS tethering complex. Here, we uncover the general preprotein translocase of the outer membrane (TOM) subunit Tom40 as the direct binding partner of Vps39 on mitochondria. We identify Vps39 mutants defective in TOM binding, but functional for HOPS. Cells that cannot form vCLAMPs show reduced growth under stress conditions and impaired survival upon starvation. Unexpectedly, our mutant analysis revealed the existence of two functionally independent vacuole-mitochondria MCSs: one formed by the Ypt7-Vps39-Tom40 tether and a second one by Vps13-Mcp1, which is redundant with ER-mitochondrial contacts formed by ERMES.

Assembly of the Mitochondrial Cristae Organizer Mic10 Is Regulated by Mic26-Mic27 Antagonism and Cardiolipin.

The multi-subunit mitochondrial contact site and cristae organizing system (MICOS) is a conserved protein complex of the inner mitochondrial membrane that is essential for maintenance of cristae architecture. The core subunit Mic10 forms large oligomers that build a scaffold and induce membrane curvature. The regulation of Mic10 oligomerization is poorly understood. We report that Mic26 exerts a destabilizing effect on Mic10 oligomers and thus functions in an antagonistic manner to the stabilizing subunit Mic27. The mitochondrial signature phospholipid cardiolipin shows a stabilizing function on Mic10 oligomers. Our findings indicate that the Mic10 core machinery of MICOS is regulated by several mechanisms, including interaction with cardiolipin and antagonistic actions of Mic26 and Mic27.

Identification of seipin-linked factors that act as determinants of a lipid droplet subpopulation.

Functional heterogeneity within the lipid droplet (LD) pool of a single cell has been observed, yet the underlying mechanisms remain enigmatic. Here, we report on identification of a specialized LD subpopulation characterized by a unique proteome and a defined geographical location at the nucleus-vacuole junction contact site. In search for factors determining identity of these LDs, we screened ∼6,000 yeast mutants for loss of targeting of the subpopulation marker Pdr16 and identified Ldo45 (LD organization protein of 45 kD) as a crucial targeting determinant. Ldo45 is the product of a splicing event connecting two adjacent genes (YMR147W and YMR148W/OSW5/LDO16). We show that Ldo proteins cooperate with the LD biogenesis component seipin and establish LD identity by defining positioning and surface-protein composition. Our studies suggest a mechanism to establish functional differentiation of organelles, opening the door to better understanding of metabolic decisions in cells.