Ubiquitin and membrane protein turnover: from cradle to grave.

From the moment of cotranslational insertion into the lipid bilayer of the endoplasmic reticulum (ER), newly synthesized integral membrane proteins are subject to a complex series of sorting, trafficking, quality control, and quality maintenance systems. Many of these processes are intimately controlled by ubiquitination, a posttranslational modification that directs trafficking decisions related to both the biosynthetic delivery of proteins to the plasma membrane (PM) via the secretory pathway and the removal of proteins from the PM via the endocytic pathway. Ubiquitin modification of integral membrane proteins (or "cargoes") generally acts as a sorting signal, which is recognized, captured, and delivered to a specific cellular destination via specialized trafficking events. By affecting the quality, quantity, and localization of integral membrane proteins in the cell, defects in these processes contribute to human diseases, including cystic fibrosis, circulatory diseases, and various neuropathies. This review summarizes our current understanding of how ubiquitin modification influences cargo trafficking, with a special emphasis on mechanisms of quality control and quality maintenance in the secretory and endocytic pathways.

TORC1 regulates endocytosis via Npr1-mediated phosphoinhibition of a ubiquitin ligase adaptor.

The TORC1 kinase signaling complex is a key determinant of cell growth that senses nutritional status and responds by coordinating diverse cellular processes including transcription, translation, and autophagy. Here, we demonstrate that TORC1 modulates the composition of plasma membrane (PM) proteins by regulating ubiquitin-mediated endocytosis. The mechanism involves the Npr1 kinase, a negative regulator of endocytosis that is itself negatively regulated by TORC1. We show that Npr1 inhibits the activity of Art1, an arrestin-like adaptor protein that promotes endocytosis by targeting the Rsp5 ubiquitin ligase to specific PM cargoes. Npr1 antagonizes Art1-mediated endocytosis via N-terminal phosphorylation, a modification that prevents Art1 association with the PM. Thus, our study adds ubiquitin ligase targeting and control of endocytosis to the known effector mechanisms of TORC1, underscoring how TORC1 coordinates ubiquitin-mediated endocytosis with protein synthesis and autophagy in order to regulate cell growth.

Art2 mediates selective endocytosis of methionine transporters during adaptation to sphingolipid depletion.

Accumulating evidence in several model organisms indicates that reduced sphingolipid biosynthesis promotes longevity, although underlying mechanisms remain unclear. In yeast, sphingolipid depletion induces a state resembling amino acid restriction, which we hypothesized might be due to altered stability of amino acid transporters at the plasma membrane. To test this, we measured surface abundance for a diverse panel of membrane proteins in the presence of myriocin, a sphingolipid biosynthesis inhibitor, in Saccharomyces cerevisiae. Unexpectedly, we found that surface levels of most proteins examined were either unaffected or increased during myriocin treatment, consistent with an observed decrease in bulk endocytosis. In contrast, sphingolipid depletion triggered selective endocytosis of the methionine transporter Mup1. Unlike methionine-induced Mup1 endocytosis, myriocin triggered Mup1 endocytosis that required the Rsp5 adaptor Art2, C-terminal lysine residues of Mup1 and the formation of K63-linked ubiquitin polymers. These findings reveal cellular adaptation to sphingolipid depletion by ubiquitin-mediated remodeling of nutrient transporter composition at the cell surface.

Coupling Conjugation and Deconjugation Activities to Achieve Cellular Ubiquitin Dynamics.

In eukaryotic cells, proteome remodeling is mediated by the ubiquitin-proteasome system, which regulates protein degradation, trafficking, and signaling events in the cell. Interplay between the cellular proteome and ubiquitin is complex and dynamic and many regulatory features that support this system have only recently come into focus. An unexpected recurring feature in this system is the physical interaction between E3 ubiquitin ligases and deubiquitylases (DUBs). Recent studies have reported on the regulatory significance of DUB-E3 interactions and it is becoming clear that they play important but complicated roles in the regulation of diverse cellular processes. Here, we summarize the current understanding of interactions between ubiquitin conjugation and deconjugation machineries and we examine the regulatory logic of these enigmatic complexes.

Arrestin-related ubiquitin-ligase adaptors regulate endocytosis and protein turnover at the cell surface.

The diversity of plasma membrane (PM) proteins presents a challenge for the achievement of cargo-specific regulation of endocytosis. Here, we describe a family of proteins in yeast (ARTs, for arrestin-related trafficking adaptors) that function by targeting specific PM proteins to the endocytic system. Two members (Art1 and Art2) of the family were discovered in chemical-genetic screens, and they direct downregulation of distinct amino acid transporters triggered by specific stimuli. Sequence analysis revealed a total of nine ART family members in yeast. In addition to similarity to arrestins, the ARTs each contain multiple PY motifs. These motifs are required for recruitment of the Rsp5/Nedd4-like ubiquitin ligase, which modifies the cargoes as well as the ARTs. As a result, ubiquitinated cargoes are internalized and targeted to the vacuole (lysosome) for degradation. We propose that ARTs provide a cargo-specific quality-control pathway that mediates endocytic downregulation by coupling Rsp5/Nedd4 to diverse plasma membrane proteins.

A Snf1-related nutrient-responsive kinase antagonizes endocytosis in yeast.

Endocytosis is regulated in response to changing environmental conditions to adjust plasma membrane (PM) protein composition for optimal cell growth. Protein networks involved in cargo capture and sorting, membrane sculpting and deformation, and vesicle scission have been well-characterized, but less is known about the networks that sense extracellular cues and relay signals to trigger endocytosis of specific cargo. Hal4 and Hal5 are yeast Snf1-related kinases that were previously reported to regulate nutrient transporter stability by an unknown mechanism. Here we demonstrate that loss of Hal4 and Hal5 activates endocytosis of many different kinds of PM proteins, including Art1-mediated and Art1-independent endocytic events. Acute inhibition of Hal5 in the absence of Hal4 triggers rapid endocytosis, suggesting that Hal kinases function in a nutrient-sensing relay upstream of the endocytic response. Interestingly, Hal5 localizes to the PM, but shifts away from the cell surface in response to stimulation with specific nutrients. We propose that Hal5 functions as a nutrient-responsive regulator of PM protein stability, antagonizing endocytosis and promoting stability of endocytic cargos at the PM in nutrient-limiting conditions.

The dual PH domain protein Opy1 functions as a sensor and modulator of PtdIns(4,5)P2 synthesis.

Phosphatidylinositol-4,5-bisphosphate, PtdIns(4,5)P(2), is an essential signalling lipid that regulates key processes such as endocytosis, exocytosis, actin cytoskeletal organization and calcium signalling. Maintaining proper levels of PtdIns(4,5)P(2) at the plasma membrane (PM) is crucial for cell survival and growth. We show that the conserved PtdIns(4)P 5-kinase, Mss4, forms dynamic, oligomeric structures at the PM that we term PIK patches. The dynamic assembly and disassembly of Mss4 PIK patches may provide a mechanism to precisely modulate Mss4 kinase activity, as needed, for localized regulation of PtdIns(4,5)P(2) synthesis. Furthermore, we identify a tandem PH domain-containing protein, Opy1, as a novel Mss4-interacting protein that partially colocalizes with PIK patches. Based upon genetic, cell biological, and biochemical data, we propose that Opy1 functions as a coincidence detector of the Mss4 PtdIns(4)P 5-kinase and PtdIns(4,5)P(2) and serves as a negative regulator of PtdIns(4,5)P(2) synthesis at the PM. Our results also suggest that additional conserved tandem PH domain-containing proteins may play important roles in regulating phosphoinositide signalling.

Use of Deubiquitinase Fusion Proteins to Characterize Endocytic Trafficking in Yeast.

Ubiquitin modification is known to regulate endocytic trafficking of many different types of cargo in eukaryotic cells, but it can be challenging to determine what role, if any, ubiquitin plays in the trafficking of a novel or uncharacterized endocytic cargo. Here, we describe a useful approach that leverages fusion to deubiquitinase (DUB) catalytic domains to explore the role ubiquitin plays in endocytic trafficking. This approach can be applied to the analysis of many different endocytic cargos in different cell types, and it can also be used to study linkage specificity in endocytic trafficking. Several different trafficking assays are described to illustrate the broad utility of this "DUB fusion" approach.

Lysosomal trafficking of the glucose transporter GLUT1 requires sequential regulation by TXNIP and ubiquitin.

Glucose transporters are gatekeepers of cellular glucose metabolism. Understanding how their activity is regulated can provide insight into mechanisms of glucose homeostasis and diseases arising from dysregulation of glucose transport. Glucose stimulates endocytosis of the human glucose transporter GLUT1, but several important questions remain surrounding the intracellular trafficking itinerary of GLUT1. Here, we report that increased glucose availability triggers lysosomal trafficking of GLUT1 in HeLa cells, with a subpopulation of GLUT1 routed through ESCRT-associated late endosomes. This itinerary requires the arrestin-like protein TXNIP, which interacts with both clathrin and E3 ubiquitin ligases to promote GLUT1 lysosomal trafficking. We also find that glucose stimulates GLUT1 ubiquitylation, which promotes its lysosomal trafficking. Our results suggest that excess glucose first triggers TXNIP-mediated endocytosis of GLUT1 and, subsequently, ubiquitylation to promote lysosomal trafficking. Our findings underscore how complex coordination of multiple regulators is required for fine-tuning of GLUT1 stability at the cell surface.

Reduced sphingolipid biosynthesis modulates proteostasis networks to enhance longevity.

As the elderly population increases, chronic, age-associated diseases are challenging healthcare systems around the world. Nutrient limitation is well known to slow the aging process and improve health. Regrettably, practicing nutrient restriction to improve health is unachievable for most people. Alternatively, pharmacological strategies are being pursued including myriocin which increases lifespan in budding yeast. Myriocin impairs sphingolipid synthesis, resulting in lowered amino acid pools which promote entry into a quiescent, long-lived state. Here we present transcriptomic data during the first 6 hours of drug treatment that improves our mechanistic understanding of the cellular response to myriocin and reveals a new role for ubiquitin in longevity. Previously we found that the methionine transporter Mup1 traffics to the plasma membrane normally in myriocin-treated cells but is not active and undergoes endocytic clearance. We now show that UBI4, a gene encoding stressed-induced ubiquitin, is vital for myriocin-enhanced lifespan. Furthermore, we show that Mup1 fused to a deubiquitinase domain impairs myriocin-enhanced longevity. Broader effects of myriocin treatment on ubiquitination are indicated by our finding of a significant increase in K63-linked ubiquitin polymers following myriocin treatment. Although proteostasis is broadly accepted as a pillar of aging, our finding that ubiquitination of an amino acid transporter promotes longevity in myriocin-treated cells is novel. Addressing the role of ubiquitination/deubiquitination in longevity has the potential to reveal new strategies and targets for promoting healthy aging.

Detection, visualization and quantification of protein complexes in human Alzheimer's disease brains using proximity ligation assay.

Examination of healthy and diseased human brain is essential to translational neuroscience. Protein-protein interactions play a pivotal role in physiological and pathological processes, but their detection is difficult, especially in aged and fixed human brain tissue. We used the in-situ proximity ligation assay (PLA) to broaden the range of molecular interactions assessable in-situ in the human neuropathology. We adapted fluorescent in-situ PLA to detect ubiquitin-modified proteins in human brains with Alzheimer's disease (AD), including approaches for the management of autofluorescence and quantification using a high-content image analysis system. We confirmed that phosphorylated microtubule-associated protein tau (Serine202, Threonine205) aggregates were modified by ubiquitin and that phospho-tau-ubiquitin complexes were increased in hippocampal and frontal cortex regions in AD compared to non-AD brains. Overall, we refined PLA for use in human neuropathology, which has revealed a profound change in the distribution of ubiquitin in AD brain and its association with characteristic tau pathologies.

Regulation of ubiquitin and ubiquitin-like modifiers by phosphorylation.

The regulatory influence of ubiquitin is vast, encompassing all cellular processes, by virtue of its central roles in protein degradation, membrane trafficking, and cell signaling. But how does ubiquitin, a 76 amino acid peptide, carry out such diverse, complex functions in eukaryotic cells? Part of the answer is rooted in the high degree of complexity associated with ubiquitin polymers, which can be 'read' and processed differently depending on topology and cellular context. However, recent evidence indicates that post-translational modifications on ubiquitin itself enhance the complexity of the ubiquitin code. Here, we review recent discoveries related to the regulation of the ubiquitin code by phosphorylation. We summarize what is currently known about phosphorylation of ubiquitin at Ser65, Ser57, and Thr12, and we discuss the potential for phosphoregulation of ubiquitin at other sites. We also discuss accumulating evidence that ubiquitin-like modifiers, such as SUMO, are likewise regulated by phosphorylation. A complete understanding of these regulatory codes and their complex lexicon will require dissection of mechanisms that govern phosphorylation of ubiquitin and ubiquitin-like proteins, particularly in the context of cellular stress and disease.

Ubiquitination drives COPI priming and Golgi SNARE localization.

Deciphering mechanisms controlling SNARE localization within the Golgi complex is crucial to understanding protein trafficking patterns within the secretory pathway. SNAREs are also thought to prime coatomer protein I (COPI) assembly to ensure incorporation of these essential cargoes into vesicles, but the regulation of these events is poorly understood. Here, we report roles for ubiquitin recognition by COPI in SNARE trafficking and in stabilizing interactions between Arf, COPI, and Golgi SNAREs in Saccharomyces cerevisiae. The ability of COPI to bind ubiquitin, but not the dilysine motif, through its N-terminal WD repeat domain of ß'-COP or through an unrelated ubiquitin-binding domain is essential for the proper localization of Golgi SNAREs Bet1 and Gos1. We find that COPI, the ArfGAP Glo3, and multiple Golgi SNAREs are ubiquitinated. Notably, the binding of Arf and COPI to Gos1 is markedly enhanced by ubiquitination of these components. Glo3 is proposed to prime COPI-SNARE interactions; however, Glo3 is not enriched in the ubiquitin-stabilized SNARE-Arf-COPI complex but is instead enriched with COPI complexes that lack SNAREs. These results support a new model for how posttranslational modifications drive COPI priming events crucial for Golgi SNARE localization.

Enhancing lifespan of budding yeast by pharmacological lowering of amino acid pools.

The increasing prevalence of age-related diseases and resulting healthcare insecurity and emotional burden require novel treatment approaches. Several promising strategies seek to limit nutrients and promote healthy aging. Unfortunately, the human desire to consume food means this strategy is not practical for most people but pharmacological approaches might be a viable alternative. We previously showed that myriocin, which impairs sphingolipid synthesis, increases lifespan in Saccharomyces cerevisiae by modulating signaling pathways including the target of rapamycin complex 1 (TORC1). Since TORC1 senses cellular amino acids, we analyzed amino acid pools and identified 17 that are lowered by myriocin treatment. Studying the methionine transporter, Mup1, we found that newly synthesized Mup1 traffics to the plasma membrane and is stable for several hours but is inactive in drug-treated cells. Activity can be restored by adding phytosphingosine to culture medium thereby bypassing drug inhibition, thus confirming a sphingolipid requirement for Mup1 activity. Importantly, genetic analysis of myriocin-induced longevity revealed a requirement for the Gtr1/2 (mammalian Rags) and Vps34-Pib2 amino acid sensing pathways upstream of TORC1, consistent with a mechanism of action involving decreased amino acid availability. These studies demonstrate the feasibility of pharmacologically inducing a state resembling amino acid restriction to promote healthy aging.

Identification of ubiquitin Ser57 kinases regulating the oxidative stress response in yeast.

Ubiquitination regulates many different cellular processes, including protein quality control, membrane trafficking, and stress responses. The diversity of ubiquitin functions in the cell is partly due to its ability to form chains with distinct linkages that can alter the fate of substrate proteins in unique ways. The complexity of the ubiquitin code is further enhanced by post-translational modifications on ubiquitin itself, the biological functions of which are not well understood. Here, we present genetic and biochemical evidence that serine 57 (Ser57) phosphorylation of ubiquitin functions in stress responses in Saccharomyces cerevisiae, including the oxidative stress response. We also identify and characterize the first known Ser57 ubiquitin kinases in yeast and human cells, and we report that two Ser57 ubiquitin kinases regulate the oxidative stress response in yeast. These studies implicate ubiquitin phosphorylation at the Ser57 position as an important modifier of ubiquitin function, particularly in response to proteotoxic stress.

Cargo Release from Myosin V Requires the Convergence of Parallel Pathways that Phosphorylate and Ubiquitylate the Cargo Adaptor.

Cellular function requires molecular motors to transport cargoes to their correct intracellular locations. The regulated assembly and disassembly of motor-adaptor complexes ensures that cargoes are loaded at their origin and unloaded at their destination. In Saccharomyces cerevisiae, early in the cell cycle, a portion of the vacuole is transported into the emerging bud. This transport requires a myosin V motor, Myo2, which attaches to the vacuole via Vac17, the vacuole-specific adaptor protein. Vac17 also binds to Vac8, a vacuolar membrane protein. Once the vacuole is brought to the bud cortex via the Myo2-Vac17-Vac8 complex, Vac17 is degraded and the vacuole is released from Myo2. However, mechanisms governing dissociation of the Myo2-Vac17-Vac8 complex are not well understood. Ubiquitylation of the Vac17 adaptor at the bud cortex provides spatial regulation of vacuole release. Here, we report that ubiquitylation alone is not sufficient for cargo release. We find that a parallel pathway, which initiates on the vacuole, converges with ubiquitylation to release the vacuole from Myo2. Specifically, we show that Yck3 and Vps41, independent of their known roles in homotypic fusion and protein sorting (HOPS)-mediated vesicle tethering, are required for the phosphorylation of Vac17 in its Myo2 binding domain. These phosphorylation events allow ubiquitylated Vac17 to be released from Myo2 and Vac8. Our data suggest that Vps41 is regulating the phosphorylation of Vac17 via Yck3, a casein kinase I, and likely another unknown kinase. That parallel pathways are required to release the vacuole from Myo2 suggests that multiple signals are integrated to terminate organelle inheritance.

Roles for a lipid phosphatase in the activation of its opposing lipid kinase.

Fig4 is a phosphoinositide phosphatase that converts PI3,5P2 to PI3P. Paradoxically, mutation of Fig4 results in lower PI3,5P2, indicating that Fig4 is also required for PI3,5P2 production. Fig4 promotes elevation of PI3,5P2, in part, through stabilization of a protein complex that includes its opposing lipid kinase, Fab1, and the scaffold protein Vac14. Here we show that multiple regions of Fig4 contribute to its roles in the elevation of PI3,5P2: its catalytic site, an N-terminal disease-related surface, and a C-terminal region. We show that mutation of the Fig4 catalytic site enhances the formation of the Fab1-Vac14-Fig4 complex, and reduces the ability to elevate PI3,5P2. This suggests that independent of its lipid phosphatase function, the active site plays a role in the Fab1-Vac14-Fig4 complex. We also show that the N-terminal disease-related surface contributes to the elevation of PI3,5P2 and promotes Fig4 association with Vac14 in a manner that requires the Fig4 C-terminus. We find that the Fig4 C-terminus alone interacts with Vac14 in vivo and retains some functions of full-length Fig4. Thus, a subset of Fig4 functions are independent of its phosphatase domain and at least three regions of Fig4 play roles in the function of the Fab1-Vac14-Fig4 complex.

A mycobacterium ESX-1-secreted virulence factor with unique requirements for export.

Specialized secretion systems of pathogenic bacteria commonly transport multiple effectors that act in concert to control and exploit the host cell as a replication-permissive niche. Both the Mycobacterium marinum and the Mycobacterium tuberculosis genomes contain an extended region of difference 1 (extRD1) locus that encodes one such pathway, the early secretory antigenic target 6 (ESAT-6) system 1 (ESX-1) secretion apparatus. ESX-1 is required for virulence and for secretion of the proteins ESAT-6, culture filtrate protein 10 (CFP-10), and EspA. Here, we show that both Rv3881c and its M. marinum homolog, Mh3881c, are secreted proteins, and disruption of RD1 in either organism blocks secretion. We have renamed the Rv3881c/Mh3881c gene espB for ESX-1 substrate protein B. Secretion of M. marinum EspB (EspBM) requires both the Mh3879c and Mh3871 genes within RD1, while CFP-10 secretion is not affected by disruption of Mh3879c. In contrast, disruption of Mh3866 or Mh3867 within the extRD1 locus prevents CFP-10 secretion without effect on EspBM. Mutants that fail to secrete only EspBM or only CFP-10 are less attenuated in macrophages than mutants failing to secrete both substrates. EspBM physically interacts with Mh3879c; the M. tuberculosis homolog, EspBT, physically interacts with Rv3879c; and mutants of EspBM that fail to bind Mh3879c fail to be secreted. We also found interaction between Rv3879c and Rv3871, a component of the ESX-1 machine, suggesting a mechanism for the secretion of EspB. The results establish EspB as a substrate of ESX-1 that is required for virulence and growth in macrophages and suggests that the contribution of ESX-1 to virulence may arise from the secretion of multiple independent substrates.

M. tuberculosis Ser/Thr protein kinase D phosphorylates an anti-anti-sigma factor homolog.

Receptor Ser/Thr protein kinases are candidates for sensors that govern developmental changes and disease processes of Mycobacterium tuberculosis (Mtb), but the functions of these kinases are not established. Here, we show that Mtb protein kinase (Pkn) D overexpression alters transcription of numerous bacterial genes, including Rv0516c, a putative anti-anti-sigma factor, and genes regulated by sigma factor F. The PknD kinase domain directly phosphorylated Rv0516c, but no other sigma factor regulator, in vitro. In contrast, the purified PknB and PknE kinase domains phosphorylated distinct sigma regulators. Rather than modifying a consensus site, PknD phosphorylated Rv0516c in vitro and in vivo on Thr2 in a unique N-terminal extension. This phosphorylation inhibited Rv0516c binding in vitro to a homologous anti-anti-sigma factor, Rv2638. These results support a model in which signals transmitted through PknD alter the transcriptional program of Mtb by stimulating phosphorylation of a sigma factor regulator at an unprecedented control site.

Methionine triggers Ppz-mediated dephosphorylation of Art1 to promote cargo-specific endocytosis.

Regulation of plasma membrane (PM) protein abundance by selective endocytosis is critical for cellular adaptation to stress or changing nutrient availability. One example involves rapid endocytic turnover of Mup1, a yeast methionine transporter, in response to increased methionine availability. Here, we report that methionine triggers rapid translocation of the ubiquitin ligase adaptor Art1 to the PM and dephosphorylation of Art1 at specific threonine residues. This methionine-induced dephosphorylation of Art1 is mediated by Ppz phosphatases, and analysis of phosphomimetic and phosphorylation-defective variants of Art1 indicates that these events toggle Art1 recognition of Mup1 at the PM. Importantly, we find that Ppz phosphatases are dispensable for Art1 PM translocation, but are required for Art1 interaction with Mup1. Based on our findings, we propose that methionine influx triggers Art1 translocation to the PM, followed by Ppz-mediated dephosphorylation which promotes cargo recognition at the PM.