AbstractThe Golgi apparatus is essential for protein sorting, yet its quality control mechanisms are poorly understood. Here we show that the Dsc ubiquitin ligase complex uses its rhomboid pseudo-protease subunit, Dsc2, to assess the hydrophobic length of α-helical transmembrane domains (TMDs) at the Golgi. Thereby the Dsc complex likely interacts with orphaned ER and Golgi proteins that have shorter TMDs and ubiquitinates them for targeted degradation. Some Dsc substrates will be extracted by Cdc48 for endosome and Golgi associated proteasomal degradation (EGAD), while others will undergo ESCRT dependent vacuolar degradation. Some substrates are degraded by both, EGAD- or ESCRT pathways. The accumulation of Dsc substrates entails a specific increase in glycerophospholipids with shorter and asymmetric fatty acyl chains. Hence, the Dsc complex mediates the selective degradation of orphaned proteins at the sorting center of cells, which prevents their spreading across other organelles and thereby preserves cellular membrane protein and lipid composition.

Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid biosynthesis-the condensation of L-serine and palmitoyl-coenzyme A (CoA). The yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While Orm1 and Orm2 have highly similar sequences, they are differentially regulated, though the mechanistic details remain elusive. Here, we determine the cryoelectron microscopy structure of the SPT complex containing Orm2. Complementary in vitro activity assays and genetic experiments with targeted lipidomics demonstrate a lower activity of the SPT-Orm2 complex than the SPT-Orm1 complex. Our results suggest a higher inhibitory potential of Orm2, despite the similar structures of the Orm1- and Orm2-containing complexes. The high conservation of SPT from yeast to man implies different regulatory capacities for the three human ORMDL isoforms, which might be key for understanding their role in sphingolipid-mediated neurodegenerative disorders.

Sphingolipids are essential components of eukaryotic cell membranes. They typically increase the rigidity of membranes and stabilize membrane-embedded proteins. Yet, they also function as signaling molecules, and some biosynthetic intermediates are toxic at high concentrations, necessitating a tight control of cellular sphingolipid synthesis. We will discuss evolutionarily common regulatory principles, which – upon failure – can lead to devastating neurodegenerative disease in humans.

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.

Sphingolipids (SPs) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by the serine palmitoyltransferase (SPT). In budding yeast (Saccharomyces cerevisiae), SPT is negatively regulated by the two proteins, Orm1 and Orm2. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane or the lysosome (or vacuole in yeast). Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, measuring lipid metabolic enzyme activity in different cellular sub-compartments has been challenging. Combining a nanobody recruitment approach with SP flux analysis, we show that the nuclear endoplasmic reticulum (ER) localized SPT and the peripheral ER localized SPT pools are differentially active. Thus, our data add another layer to the complex network of SPT regulation. Moreover, combining lipid metabolic enzyme re-localization with flux analysis serves as versatile tool to measure lipid metabolism with subcellular resolution.

Sphingolipids (SP) are one of the three major lipid classes in eukaryotic cells and serve as structural components of the plasma membrane. The rate-limiting step in SP biosynthesis is catalyzed by serine palmitoyltransferase (SPT). In yeast, SPT consists of two catalytic subunits (Lcb1 and Lcb2), a regulatory subunit (Tsc3), negative regulators (Orm1 and Orm2), and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1, collectively known as the SPOTS complex. Regulating SPT activity enables cells to adapt SP metabolism to changing environmental conditions. Therefore, the Orm proteins are phosphorylated by two signaling pathways originating from either the plasma membrane localized target of rapamycin (TOR) complex 2 or the lysosomal/vacuolar TOR complex 1. Moreover, uptake of exogenous serine is necessary for the regulation of SP biosynthesis, which suggests the existence of differentially regulated SPT pools based on their intracellular localization. However, tools for measuring lipid metabolic enzyme activity in different cellular compartments are currently not available. We have developed a nanobody recruitment system that enables the re-localization of the SPOTS complex to the nuclear or peripheral ER. By combining this system with sphingolipid flux analysis, we have identified two distinct active SPT pools in cells. Our method thus serves as a new and versatile tool to measure lipid metabolism with sub-cellular resolution.

Target-of-rapamycin complex 1 resides on lysosomes/vacuoles and additionally on signaling endosomes. Gao et al. (2022. J. Cell Biol.https://doi.org/10.1083/jcb.202109084) set out to define the molecular identity of signaling endosomes, along with players required for the formation and maintenance of this endosomal subpopulation.

The regulation of nutrient uptake into cells is important, as it allows to either increase biomass for cell growth or to preserve homoeostasis. A key strategy to adjust cellular nutrient uptake is the reconfiguration of the nutrient transporter repertoire at the plasma membrane by the addition of nutrient transporters through the secretory pathway and by their endocytic removal. In this review, we focus on the mechanisms that regulate selective nutrient transporter endocytosis, which is mediated by the α-arrestin protein family. In the budding yeast Saccharomyces cerevisiae, 14 different α-arrestins (also named arrestin-related trafficking adaptors, ARTs) function as adaptors for the ubiquitin ligase Rsp5. They instruct Rsp5 to ubiquitinate subsets of nutrient transporters to orchestrate their endocytosis. The ART proteins are under multilevel control of the major nutrient sensing systems, including amino acid sensing by the general amino acid control and target of rapamycin pathways, and energy sensing by 5'-adenosine-monophosphate-dependent kinase. The function of the six human α-arrestins is comparably under-characterised. Here, we summarise the current knowledge about the function, regulation and substrates of yeast ARTs and human α-arrestins, and highlight emerging communalities and general principles.

How cells adjust nutrient transport across their membranes is incompletely understood. Previously, we have shown that S. cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that the selective endocytosis of four AATs during starvation required the α-arrestin family protein Art2/Ecm21, an adaptor for the ubiquitin ligase Rsp5, and its induction through the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in AATs and thereby instructs Rsp5 to ubiquitinate proximal lysine residues. When amino acids are in excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis. Thus, amino acid excess or starvation activate complementary α-arrestin-Rsp5-complexes to control selective endocytosis and adapt nutrient acquisition.

The endosomal sorting complexes required for transport (ESCRT) execute evolutionary conserved membrane remodeling processes. Here we used budding yeast to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. In response to reduced membrane tension and inhibition of the target of rapamycin complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help to maintain membrane integrity. Conversely, the growth of ESCRT mutants strongly depends on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This is caused by calcineurin phosphatase activity which causes Orm2 to accumulate at the endoplasmic reticulum (ER) in ESCRT mutants. Orm2 is a repressor of SL biosynthesis and its accumulation provokes increased membrane stress. This necessitates TORC2 signaling through its downstream kinase Ypk1 to control Orm2 protein levels and prevent a detrimental imbalance of SL metabolism. Our findings reveal new aspects of antagonistic calcineurin/TORC2 signaling for the regulation of SL biosynthesis and the maintenance of PM homeostasis, and suggest that the ESCRT machinery contributes directly and indirectly to these processes.

The endosomal sorting complexes required for transport (ESCRT) mediate evolutionarily conserved membrane remodeling processes. Here, we used budding yeast (Saccharomyces cerevisiae) to explore how the ESCRT machinery contributes to plasma membrane (PM) homeostasis. We found that in response to reduced membrane tension and inhibition of TOR complex 2 (TORC2), ESCRT-III/Vps4 assemblies form at the PM and help maintain membrane integrity. In turn, the growth of ESCRT mutants strongly depended on TORC2-mediated homeostatic regulation of sphingolipid (SL) metabolism. This was caused by calcineurin-dependent dephosphorylation of Orm2, a repressor of SL biosynthesis. Calcineurin activity impaired Orm2 export from the endoplasmic reticulum (ER) and thereby hampered its subsequent endosome and Golgi-associated degradation (EGAD). The ensuing accumulation of Orm2 at the ER in ESCRT mutants necessitated TORC2 signaling through its downstream kinase Ypk1, which repressed Orm2 and prevented a detrimental imbalance of SL metabolism. Our findings reveal compensatory cross-talk between the ESCRT machinery, calcineurin/TORC2 signaling, and the EGAD pathway important for the regulation of SL biosynthesis and the maintenance of PM homeostasis.

How cells adjust transport across their membranes is incompletely understood. Previously, we have shown that S.cerevisiae broadly re-configures the nutrient transporters at the plasma membrane in response to amino acid availability, through selective endocytosis of sugar- and amino acid transporters (AATs) (Müller et al., 2015). A genome-wide screen now revealed that Art2/Ecm21, a member of the α-arrestin family of Rsp5 ubiquitin ligase adaptors, is required for the simultaneous endocytosis of four AATs and induced during starvation by the general amino acid control pathway. Art2 uses a basic patch to recognize C-terminal acidic sorting motifs in these AATs and instructs Rsp5 to ubiquitinate proximal lysine residues. In response to amino acid excess, Rsp5 instead uses TORC1-activated Art1 to detect N-terminal acidic sorting motifs within the same AATs, which initiates exclusive substrate-induced endocytosis of individual AATs. Thus, amino acid availability activates complementary α-arrestin-Rsp5-complexes to control selective endocytosis for nutrient acquisition.

Selectable markers are indispensable for genetic engineering, yet their number and variety are limited. Most selection procedures for prototrophic cells rely on the introduction of antibiotic resistance genes. New minimally invasive tools are needed to facilitate sophisticated genetic manipulations. Here, we characterized three endogenous genes in the human fungal pathogen Aspergillus fumigatus for their potential as markers for targeted genomic insertions of DNAs of interest (DOIs). Since these genes are involved in uptake and metabolization of pyrimidines, resistance to the toxic effects of prodrugs 5-fluorocytosine and 5-fluorouracil can be used to select successfully integrated DOIs. We show that DOI integration, resulting in the inactivation of these genes, caused no adverse effects with respect to nutrient requirements, stress resistance, or virulence. Beside the individual use of markers for site-directed integration of reporter cassettes, including the 17-kb penicillin biosynthetic cluster, we demonstrate their sequential use by inserting three genes encoding fluorescent proteins into a single strain for simultaneous multicolor localization microscopy. In addition to A. fumigatus, we validated the applicability of this novel toolbox in Penicillium chrysogenum and Fusarium oxysporum Enabling multiple targeted insertions of DOIs without the necessity for exogenous markers, this technology has the potential to significantly advance genetic engineering.IMPORTANCE This work reports the discovery of a novel genetic toolbox comprising multiple, endogenous selectable markers for targeted genomic insertions of DNAs of interest (DOIs). Marker genes encode proteins involved in 5-fluorocytosine uptake and pyrimidine salvage activities mediating 5-fluorocytosine deamination as well as 5-fluorouracil phosphoribosylation. The requirement for their genomic replacement by DOIs to confer 5-fluorocytosine or 5-fluorouracil resistance for transformation selection enforces site-specific integrations. Due to the fact that the described markers are endogenously encoded, there is no necessity for the exogenous introduction of commonly employed markers such as auxotrophy-complementing genes or antibiotic resistance cassettes. Importantly, inactivation of the described marker genes had no adverse effects on nutrient requirements, growth, or virulence of the human pathogen Aspergillus fumigatus Given the limited number and distinct types of selectable markers available for the genetic manipulation of prototrophic strains such as wild-type strains, we anticipate that the proposed methodology will significantly advance genetic as well as metabolic engineering of fungal species.

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.

Eisosomes are membrane furrows at the cell surface of yeast that have been shown to function in two seemingly distinct pathways, membrane stress response and regulation of nutrient transporters. We found that many stress conditions affect both of these pathways by changing plasma membrane tension and thus the morphology and composition of eisosomes. For example, alkaline stress causes swelling of the cell and an endocytic response, which together increase membrane tension, thereby flattening the eisosomes. The flattened eisosomes affect membrane stress pathways and release nutrient transporters, which aids in their down-regulation. In contrast, glucose starvation or hyperosmotic shock causes cell shrinking, which results in membrane slack and the deepening of eisosomes. Deepened eisosomes are able to trap nutrient transporters and protect them from rapid endocytosis. Therefore, eisosomes seem to coordinate the regulation of both membrane tension and nutrient transporter stability.

Cellular homeostasis requires the ubiquitin-dependent degradation of membrane proteins. This was assumed to be mediated exclusively either by endoplasmic reticulum-associated degradation (ERAD) or by endosomal sorting complexes required for transport (ESCRT)-dependent lysosomal degradation. We identified in Saccharomyces cerevisiae an additional pathway that selectively extracts membrane proteins at Golgi and endosomes for degradation by cytosolic proteasomes. One endogenous substrate of this endosome and Golgi-associated degradation pathway (EGAD) is the ER-resident membrane protein Orm2, a negative regulator of sphingolipid biosynthesis. Orm2 degradation is initiated by phosphorylation, which triggers its ER export. Once on Golgi and endosomes, Orm2 is poly-ubiquitinated by the membrane-embedded "Defective in SREBP cleavage" (Dsc) ubiquitin ligase complex. Cdc48/VCP then extracts ubiquitinated Orm2 from membranes, which is tightly coupled to the proteasomal degradation of Orm2. Thereby, EGAD prevents the accumulation of Orm2 at the ER and in post-ER compartments and promotes the controlled de-repression of sphingolipid biosynthesis. Thus, the selective degradation of membrane proteins by EGAD contributes to proteostasis and lipid homeostasis in eukaryotic cells.

The biochemical analysis of protein phosphorylation in mitochondria lags behind that of cytosolic signaling events. One reason is the poor stability of many phosphorylation sites during common isolation procedures for mitochondria. We present here an optimized, fast protocol for the purification of yeast mitochondria that greatly increases recovery of phosphorylated mitochondrial proteins. Moreover, we describe improved protocols for the biochemical analysis of mitochondrial protein phosphorylation by Zn2+-Phos-tag electrophoresis under both denaturing and - for the first time - native conditions, and demonstrate that they outperform previously applied methods.

Rab5 GTPases are master regulators of early endosome biogenesis and transport. The genome of Saccharomyces cerevisiae encodes three Rab5 proteins: Vps21, the major isoform, Ypt52 and Ypt53. Here, we show that Vps21 is the most abundant Rab5 protein and Ypt53 is the least abundant. In stressed cells, Ypt53 levels increase but never exceed that of Vps21. Its induction requires the transcription factors Crz1 and Gis1. In growing cells, the expression of Ypt53 is suppressed by post‐transcriptional mechanisms mediated by the untranslated regions of the YPT53 mRNA. Based on genetic experiments, these sequences appear to stimulate deadenylation, Pat1‐activated decapping and Xrn1‐mediated mRNA degradation. Once this regulation is bypassed, Ypt53 protein levels surpass Vps21, and Ypt53 is sufficient to maintain endosomal function and cell growth.