Active-site directed probes are powerful in studies of enzymatic function. We report an active-site directed probe based on a warhead so far considered unreactive. By replacing the C-terminal carboxylate of ubiquitin (Ub) with an alkyne functionality, a selective reaction with the active-site cysteine residue of de-ubiquitinating enzymes was observed. The resulting product was shown to be a quaternary vinyl thioether, as determined by X-ray crystallography. Proteomic analysis of proteins bound to an immobilized Ub alkyne probe confirmed the selectivity toward de-ubiquitinating enzymes. The observed reactivity is not just restricted to propargylated Ub, as highlighted by the selective reaction between caspase-1 (interleukin converting enzyme) and a propargylated peptide derived from IL-1β, a caspase-1 substrate.

Phenotypic plasticity and the exposure of hidden genetic variation both affect the survival and evolution of new traits, but their contributing molecular mechanisms are largely unknown. A single factor, the yeast prion [PSI(+)], may exert a profound effect on both. [PSI(+)] is a conserved, protein-based genetic element that is formed by a change in the conformation and function of the translation termination factor Sup35p, and is transmitted from mother to progeny. Curing cells of [PSI(+)] alters their survival in different growth conditions and produces a spectrum of phenotypes in different genetic backgrounds. Here we show, by examining three plausible explanations for this phenotypic diversity, that all traits tested involved [PSI(+)]-mediated read-through of nonsense codons. Notably, the phenotypes analysed were genetically complex, and genetic re-assortment frequently converted [PSI(+)]-dependent phenotypes to stable traits that persisted in the absence of [PSI(+)]. Thus, [PSI(+)] provides a temporary survival advantage under diverse conditions, increasing the likelihood that new traits will become fixed by subsequent genetic change. As an epigenetic mechanism that globally affects the relationship between genotype and phenotype, [PSI(+)] expands the conceptual framework for phenotypic plasticity, provides a one-step mechanism for the acquisition of complex traits and affords a route to the genetic assimilation of initially transient epigenetic traits.

SummaryThrough a network of progressively maturing vesicles, the endosomal system connects the cell’s interior with extracellular space. Intriguingly, this network exhibits a bilateral architecture, comprised of a relatively immobile perinuclear vesicle “cloud” and a highly dynamic peripheral contingent. How this spatiotemporal organization is achieved and what function(s) it curates is unclear. Here, we reveal the endoplasmic reticulum (ER)-located ubiquitin ligase Ring finger protein 26 (RNF26) as the global architect of the entire endosomal system, including the trans-Golgi network (TGN). To specify perinuclear vesicle coordinates, catalytically competent RNF26 recruits and ubiquitinates the scaffold p62/sequestosome 1 (p62/SQSTM1), in turn attracting ubiquitin-binding domains (UBDs) of various vesicle adaptors. Consequently, RNF26 restrains fast transport of diverse vesicles through a common molecular mechanism operating at the ER membrane, until the deubiquitinating enzyme USP15 opposes RNF26 activity to allow vesicle release into the cell’s periphery. By drawing the endosomal system’s architecture, RNF26 orchestrates endosomal maturation and trafficking of cargoes, including signaling receptors, in space and time.

Autophagy is the main homeostatic pathway guiding cytosolic materials for degradation by the lysosome. Maturation of autophagosomes requires their transport towards the perinuclear region of the cell, with key factors underlying both processes still poorly understood. Here we show that transport and positioning of late autophagosomes depends on cholesterol by way of the cholesterol-sensing Rab7 effector ORP1L. ORP1L localizes to late autophagosomes and—under low-cholesterol conditions—contacts the ER protein VAP-A, forming ER-autophagosome contact sites, which prevent minus-end transport by the Rab7–RILP–dynein complex. ORP1L-mediated contact sites also inhibit localization of PLEKHM1 to Rab7. PLEKHM1, together with RILP, then recruits the homotypic fusion and vacuole protein-sorting (HOPS) complex for fusion of autophagosomes with late endosomes and lysosomes. Thus, ORP1L, via its liganding by lipids and the formation of contacts between autophagic vacuoles and the ER, governs the last steps in autophagy that lead to the lysosomal degradation of cytosolic material.

When cell surface receptors engage their cognate ligands in the extracellular space, they become competent to transmit potent signals to the inside of the cell, thereby instigating growth, differentiation, motility and many other processes. In order to control these signals, activated receptors are endocytosed and thoroughly curated by the endosomal network of intracellular vesicles and proteolytic organelles. In this Review, we follow the epidermal growth factor (EGF) receptor (EGFR) from ligand engagement, through its voyage on endosomes and, ultimately, to its destruction in the lysosome. We focus on the spatial and temporal considerations underlying the molecular decisions that govern this complex journey and discuss how additional cellular organelles - particularly the ER - play active roles in the regulation of receptor lifespan. In summarizing the functions of relevant molecules on the endosomes and the ER, we cover the order of molecular events in receptor activation, trafficking and downregulation, and provide an overview of how signaling is controlled at the interface between these organelles.

Post-translational modifications of proteins with ubiquitin (Ub) and ubiquitin-like (Ubl) modifiers, orchestrated by a cascade of specialized E1, E2 and E3 enzymes, control a staggering breadth of cellular processes. To monitor catalysis along these complex reaction pathways, we developed a cascading activity-based probe, UbDha. Akin to the native Ub, upon ATP-dependent activation by the E1, UbDha can travel downstream to the E2 (and subsequently E3) enzymes through sequential trans-thioesterifications. Unlike the native Ub, at each step along the cascade UbDha has the option to react irreversibly with active site cysteine residues of target enzymes, thus enabling their detection. We show that our cascading probe ‘hops’ and ‘traps’ catalytically active ubiquitin-modifying enzymes (but not their substrates) by a mechanism diversifiable to Ubls. Our founder methodology, amenable to structural studies, proteome-wide profiling and monitoring of enzymatic activities in living cells, presents novel and versatile tools to interrogate the Ub/Ubl cascades.

Reversible ubiquitination of activated receptor complexes signals their sorting between recycling and degradation and thereby dictates receptor fate. The deubiquitinating enzyme ubiquitin-specific protease 8 (USP8/UBPy) has been previously implicated in the regulation of the epidermal growth factor receptor (EGFR); however, the molecular mechanisms governing its recruitment and activity in this context remain unclear. Herein, we investigate the role of USP8 in countering ligandinduced ubiquitination and down-regulation of EGFR and characterize a subset of protein-protein interaction determinants critical for this function. USP8 depletion accelerates receptor turnover, whereas loss of hepatocyte growth factor-regulated substrate (Hrs) rescues this phenotype, indicating that USP8 protects EGFR from degradation via an Hrs-dependent pathway. Catalytic inactivation of USP8 incurs EGFR hyperubiquitination and promotes receptor localization to endosomes marked by high ubiquitin content. These phenotypes require the central region of USP8, containing three extended Arg-X-XLys (RXXK) motifs that specify direct low affinity interactions with the SH3 domain(s) of ESCRT-0 proteins, STAM1/2. The USP8⅐STAM complex critically impinges on receptor ubiquitination status and modulates ubiquitin dynamics on EGFR-positive endosomes. Consequently, USP8-mediated deubiquitination slows progression of EGFR past the earlyto-recycling endosome circuit in a manner dependent upon the RXXK motifs. Collectively, these findings demonstrate a role for the USP8⅐STAM complex as a protective mechanism regulating early endosomal sorting of EGFR between pathways destined for lysosomal degradation and recycling.

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