Ubiquiton-An inducible, linkage-specific polyubiquitylation tool.

The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.

2-deoxyglucose transiently inhibits yeast AMPK signaling and triggers glucose transporter endocytosis, potentiating the drug toxicity.

2-deoxyglucose is a glucose analog that impacts many aspects of cellular physiology. After its uptake and its phosphorylation into 2-deoxyglucose-6-phosphate (2DG6P), it interferes with several metabolic pathways including glycolysis and protein N-glycosylation. Despite this systemic effect, resistance can arise through strategies that are only partially understood. In yeast, 2DG resistance is often associated with mutations causing increased activity of the yeast 5'-AMP activated protein kinase (AMPK), Snf1. Here we focus on the contribution of a Snf1 substrate in 2DG resistance, namely the alpha-arrestin Rod1 involved in nutrient transporter endocytosis. We report that 2DG triggers the endocytosis of many plasma membrane proteins, mostly in a Rod1-dependent manner. Rod1 participates in 2DG-induced endocytosis because 2DG, following its phosphorylation by hexokinase Hxk2, triggers changes in Rod1 post-translational modifications and promotes its function in endocytosis. Mechanistically, this is explained by a transient, 2DG-induced inactivation of Snf1/AMPK by protein phosphatase 1 (PP1). We show that 2DG-induced endocytosis is detrimental to cells, and the lack of Rod1 counteracts this process by stabilizing glucose transporters at the plasma membrane. This facilitates glucose uptake, which may help override the metabolic blockade caused by 2DG, and 2DG export-thus terminating the process of 2DG detoxification. Altogether, these results shed a new light on the regulation of AMPK signaling in yeast and highlight a remarkable strategy to bypass 2DG toxicity involving glucose transporter regulation.

Functional patchworking at the plasma membrane.

Lipids and proteins are not evenly distributed within the plasma membrane (PM), but instead segregate laterally into many specialized microdomains whose functional relevance is not clear. In this issue, Busto et al (2018) demonstrate that substrate flux through a nutrient transporter drives the lateral relocation of the transporter between specific microdomains at the yeast PM, suggesting that regulating the lateral plasma membrane compartmentalization for individual proteins could be a general process for cellular response to environmental conditions.

Sensitive detection of protein ubiquitylation using a protein fragment complementation assay.

Ubiquitylation is a reversible post-translational protein modification that regulates a multitude of cellular processes. Detection of ubiquitylated proteins is often challenging because of their low abundance. Here, we present NUbiCA, a sensitive protein-fragment complementation assay to facilitate the monitoring of ubiquitylation events in cultured cells and model organisms. Using yeast as a model system, we demonstrate that NUbiCA enables accurate monitoring of mono- and polyubiquitylation of proteins expressed at endogenous levels. We also show that it can be applied to decipher the topology of ubiquitin conjugates. Moreover, we assembled a genome-wide collection of yeast strains ready to investigate the ubiquitylation of proteins with this new assay. This resource will facilitate the analysis of local or transient ubiquitylation events that are difficult to detect with current methods.

Ubc13-Mms2 cooperates with a family of RING E3 proteins in budding yeast membrane protein sorting.

Polyubiquitin chains linked via lysine (K) 63 play an important role in endocytosis and membrane trafficking. Their primary source is the ubiquitin protein ligase (E3) Rsp5/NEDD4, which acts as a key regulator of membrane protein sorting. The heterodimeric ubiquitin-conjugating enzyme (E2), Ubc13-Mms2, catalyses K63-specific polyubiquitylation in genome maintenance and inflammatory signalling. In budding yeast, the only E3 proteins known to cooperate with Ubc13-Mms2 so far is a nuclear RING finger protein, Rad5, involved in the replication of damaged DNA. Here, we report a contribution of Ubc13-Mms2 to the sorting of membrane proteins to the yeast vacuole via the multivesicular body (MVB) pathway. In this context, Ubc13-Mms2 cooperates with Pib1, a FYVE-RING finger protein associated with internal membranes. Moreover, we identified a family of membrane-associated FYVE-(type)-RING finger proteins as cognate E3 proteins for Ubc13-Mms2 in several species, and genetic analysis indicates that the contribution of Ubc13-Mms2 to membrane trafficking in budding yeast goes beyond its cooperation with Pib1. Thus, our results widely implicate Ubc13-Mms2 as an Rsp5-independent source of K63-linked polyubiquitin chains in the regulation of membrane protein sorting.This article has an associated First Person interview with the first author of the paper.

The α-arrestin family of ubiquitin ligase adaptors links metabolism with selective endocytosis.

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.

Severe osmotic compression triggers a slowdown of intracellular signaling, which can be explained by molecular crowding.

Regulation of the cellular volume is fundamental for cell survival and function. Deviations from equilibrium trigger dedicated signaling and transcriptional responses that mediate water homeostasis and volume recovery. Cells are densely packed with proteins, and molecular crowding may play an important role in cellular processes. Indeed, increasing molecular crowding has been shown to modify the kinetics of biochemical reactions in vitro; however, the effects of molecular crowding in living cells are mostly unexplored. Here, we report that, in yeast, a sudden reduction in cellular volume, induced by severe osmotic stress, slows down the dynamics of several signaling cascades, including the stress-response pathways required for osmotic adaptation. We show that increasing osmotic compression decreases protein mobility and can eventually lead to a dramatic stalling of several unrelated signaling and cellular processes. The rate of these cellular processes decreased exponentially with protein density when approaching stalling osmotic compression. This suggests that, under compression, the cytoplasm behaves as a soft colloid undergoing a glass transition. Our results shed light on the physical mechanisms that force cells to cope with volume fluctuations to maintain an optimal protein density compatible with cellular functions.

A mechanism for protein monoubiquitination dependent on a trans-acting ubiquitin-binding domain.

The length of the ubiquitin chain on a substrate dictates various functional outcomes, yet little is known about its regulation in vivo. The yeast arrestin-related protein Rim8/Art9 is monoubiquitinated in vivo by the Rsp5 ubiquitin ligase. This also requires Vps23, a protein that displays an ubiquitin-E2 variant (UEV) domain. Here, we report that binding of the UEV domain to Rim8 interferes with ubiquitin chain elongation and directs Rim8 monoubiquitination. We propose that Vps23 UEV competes with Rsp5 HECT N-lobe for binding to the first conjugated ubiquitin, thereby preventing polyubiquitination. These findings reveal a novel mechanism to control ubiquitin chain length on substrates in vivo.

A perturbed ubiquitin landscape distinguishes between ubiquitin in trafficking and in proteolysis.

Any of seven lysine residues on ubiquitin can serve as the base for chain-extension, resulting in a sizeable spectrum of ubiquitin modifications differing in chain length or linkage type. By optimizing a procedure for rapid lysis, we charted the profile of conjugated cellular ubiquitin directly from whole cell extract. Roughly half of conjugated ubiquitin (even at high molecular weights) was nonextended, consisting of monoubiquitin modifications and chain terminators (endcaps). Of extended ubiquitin, the primary linkages were via Lys48 and Lys63. All other linkages were detected, contributing a relatively small portion that increased at lower molecular weights. In vivo expression of lysineless ubiquitin (K0 Ub) perturbed the ubiquitin landscape leading to elevated levels of conjugated ubiquitin, with a higher mono-to-poly ratio. Affinity purification of these trapped conjugates identified a comprehensive list of close to 900 proteins including novel targets. Many of the proteins enriched by K0 ubiquitination were membrane-associated, or involved in cellular trafficking. Prime among them are components of the ESCRT machinery and adaptors of the Rsp5 E3 ubiquitin ligase. Ubiquitin chains associated with these substrates were enriched for Lys63 linkages over Lys48, indicating that K0 Ub is unevenly distributed throughout the ubiquitinome. Biological assays validated the interference of K0 Ub with protein trafficking and MVB sorting, minimally affecting Lys48-dependent turnover of proteasome substrates. We conclude that despite the shared use of the ubiquitin molecule, the two branches of the ubiquitin machinery--the ubiquitin-proteasome system and the ubiquitin trafficking system--were unevenly perturbed by expression of K0 ubiquitin.

Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation.

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner.

Ubiquitin ligase adaptors: regulators of ubiquitylation and endocytosis of plasma membrane proteins.

The subcellular localization of plasma membrane proteins, such as receptors and transporters, must be finely tuned so that they can be readily downregulated in response to environmental cues. Some of these membrane proteins are post-translationally modified by conjugation to ubiquitin, which is used as a molecular tag to commit them to the endocytic pathway and promote their subsequent delivery to the lysosomes for degradation. This ubiquitylation step, which is performed by so-called ubiquitin ligases (or E3), appears therefore as a critical event for endocytosis and is subject to many levels of regulation. In this review, we focus on the regulation of cargo ubiquitylation by accessory proteins, or "adaptors", and discuss the various ways by which they promote the action of ubiquitin ligases toward their specific cargoes. Common features emerge on this mode of regulation, which is present from yeast to human, regardless of the type of ubiquitin ligase in charge of the ubiquitylation. Finally, because these adaptors represent an additional layer of specificity in the ubiquitylation cascade, and can themselves be subject to a complex regulation, they are essential actors in the fine-tuning of endocytosis.

Cellular toxicity of the metabolic inhibitor 2-deoxyglucose and associated resistance mechanisms.

Most malignant cells display increased glucose absorption and metabolism compared to surrounding tissues. This well-described phenomenon results from a metabolic reprogramming occurring during transformation, that provides the building blocks and supports the high energetic cost of proliferation by increasing glycolysis. These features led to the idea that drugs targeting glycolysis might prove efficient in the context of cancer treatment. One of these drugs, 2-deoxyglucose (2-DG), is a synthetic glucose analog that can be imported into cells and interfere with glycolysis and ATP generation. Its preferential targeting to sites of cell proliferation is supported by the observation that a derived molecule, 2-fluoro-2-deoxyglucose (FDG) accumulates in tumors and is used for cancer imaging. Here, we review the toxicity mechanisms of this drug, from the early-described effects on glycolysis to its other cellular consequences, including inhibition of protein glycosylation and endoplasmic reticulum stress, and its interference with signaling pathways. Then, we summarize the current data on the use of 2-DG as an anti-cancer agent, especially in the context of combination therapies, as novel 2-DG-derived drugs are being developed. We also show how the use of 2-DG helped to decipher glucose-signaling pathways in yeast and favored their engineering for biotechnologies. Finally, we discuss the resistance strategies to this inhibitor that have been identified in the course of these studies and which may have important implications regarding a medical use of this drug.

Complementary α-arrestin-ubiquitin ligase complexes control nutrient transporter endocytosis in response to amino acids.

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.

Two independent pathways traffic the intraperoxisomal peroxin PpPex8p into peroxisomes: mechanism and evolutionary implications.

Among peroxins involved in peroxisome biogenesis, only Pex8p is predominantly intraperoxisomal at steady state. Pex8p is necessary for peroxisomal matrix protein import via the PTS1 and PTS2 pathways. It is proposed to bridge two peroxisomal membrane subcomplexes comprised of the docking (Pex13p, Pex14p, Pex17p) and RING (Pex2p, Pex10p, Pex12p) peroxins and is also implicated in cargo release of PTS1 proteins in the matrix. We show that Pichia pastoris Pex8p (PpPex8p) enters the peroxisome matrix using two redundant pathways in a Pex14p-dependent, but Pex2p-independent, manner, showing that the intact importomer and RING subcomplex are not required for its import. One pathway depends on the TPR motifs in Pex5p, the C-terminal PTS1 sequence (AKL) in PpPex8p, and the intraperoxisomal presence of this peroxin. The alternative pathway uses the PTS2 receptor, Pex7p, its accessory protein, Pex20p, and a putative PTS2 motif in PpPex8p, but does not require intraperoxisomal PpPex8p. Pex20p interaction with PpPex8p is independent of Pex7p, but the interaction of PpPex8p with Pex7p requires Pex20p. These data suggest a direct interaction between PpPex8p and Pex20p. Our studies shed light on the mechanism and evolution of the dual import pathways for PpPex8p.

The induction of HAD-like phosphatases by multiple signaling pathways confers resistance to the metabolic inhibitor 2-deoxyglucose.

Anti-cancer strategies that target the glycolytic metabolism of tumors have been proposed. The glucose analog 2-deoxyglucose (2DG) is imported into cells and, after phosphorylation, becomes 2DG-6-phosphate, a toxic by-product that inhibits glycolysis. Using yeast as a model, we performed an unbiased mass spectrometry-based approach to probe the cellular effects of 2DG on the proteome and study resistance mechanisms to 2DG. We found that two phosphatases that target 2DG-6-phosphate were induced upon exposure to 2DG and participated in 2DG detoxification. Dog1 and Dog2 are HAD (haloacid dehalogenase)-like phosphatases, which are evolutionarily conserved. 2DG induced Dog2 by activating several signaling pathways, such as the stress response pathway mediated by the p38 MAPK ortholog Hog1, the unfolded protein response (UPR) triggered by 2DG-induced ER stress, and the cell wall integrity (CWI) pathway mediated by the MAPK Slt2. Loss of the UPR or CWI pathways led to 2DG hypersensitivity. In contrast, mutants impaired in the glucose-mediated repression of genes were 2DG resistant because glucose availability transcriptionally repressed DOG2 by inhibiting signaling mediated by the AMPK ortholog Snf1. The characterization and genome resequencing of spontaneous 2DG-resistant mutants revealed that DOG2 overexpression was a common strategy underlying 2DG resistance. The human Dog2 homolog HDHD1 displayed phosphatase activity toward 2DG-6-phosphate in vitro and its overexpression conferred 2DG resistance in HeLa cells, suggesting that this 2DG phosphatase could interfere with 2DG-based chemotherapies. These results show that HAD-like phosphatases are evolutionarily conserved regulators of 2DG resistance.

The yeast arrestin-related protein Bul1 is a novel actor of glucose-induced endocytosis.

Yeast cells have a remarkable ability to adapt to nutritional changes in their environment. During adaptation, nutrient-signaling pathways drive the selective endocytosis of nutrient transporters present at the cell surface. A current challenge is to understand the mechanistic basis of this regulation. Transporter endocytosis is triggered by their ubiquitylation, which involves the ubiquitin ligase Rsp5 and its adaptors of the arrestin-related family (ART). This step is highly regulated by nutrient availability. For instance, the monocarboxylate transporter Jen1 is ubiquitylated, endocytosed, and degraded upon exposure to glucose. The ART protein Rod1 is required for this overall process; yet Rod1 rather controls Jen1 trafficking later in the endocytic pathway and is almost dispensable for Jen1 internalization. Thus, how glucose triggers Jen1 internalization remains unclear. We report that another ART named Bul1, but not its paralogue Bul2, contributes to Jen1 internalization. Bul1 responds to glucose availability, and preferentially acts at the plasma membrane for Jen1 internalization. Thus, multiple ARTs can act sequentially along the endocytic pathway to control transporter homeostasis. Moreover, Bul1 is in charge of Jen1 endocytosis after cycloheximide treatment, suggesting that the functional redundancy of ARTs may be explained by their ability to interact with multiple cargoes in various conditions.

Endocytosis-mediated siderophore uptake as a strategy for Fe acquisition in diatoms.

Phytoplankton growth is limited in vast oceanic regions by the low bioavailability of iron. Iron fertilization often results in diatom blooms, yet the physiological underpinnings for how diatoms survive in chronically iron-limited waters and outcompete other phytoplankton when iron becomes available are unresolved. We show that some diatoms can use siderophore-bound iron, and exhibit a species-specific recognition for siderophore types. In Phaeodactylum tricornutum, hydroxamate siderophores are taken up without previous reduction by a high-affinity mechanism that involves binding to the cell surface followed by endocytosis-mediated uptake and delivery to the chloroplast. The affinity recorded is the highest ever described for an iron transport system in any eukaryotic cell. Collectively, our observations suggest that there are likely a variety of iron uptake mechanisms in diatoms besides the well-established reductive mechanism. We show that iron starvation-induced protein 1 (ISIP1) plays an important role in the uptake of siderophores, and through bioinformatics analyses we deduce that this protein is largely diatom-specific. We quantify expression of ISIP1 in the global ocean by querying the Tara Oceans atlas of eukaryotic genes and show a link between the abundance and distribution of diatom-associated ISIP1 with ocean provinces defined by chronic iron starvation.

Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors.

Based on earlier suggestions that peroxisomes may have arisen from endosymbionts that later lost their DNA, it was expected that protein transport into this organelle would have parallels to systems found in other organelles of endosymbiont origin, such as mitochondria and chloroplasts. This review highlights three features of peroxisomal matrix protein import that make it unique in comparison with these other subcellular compartments - the ability of this organelle to transport folded, co-factor-bound and oligomeric proteins, the dynamics of the import receptors during the matrix protein import cycle and the existence of a peroxisomal quality-control pathway, which insures that the peroxisome membrane is cleared of cargo-free receptors.