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.

The endosomal sorting complex ESCRT-II mediates the assembly and architecture of ESCRT-III helices.

The endosomal sorting complexes required for transport (ESCRTs) constitute hetero-oligomeric machines that mediate topologically similar membrane-sculpting processes, including cytokinesis, retroviral egress, and multivesicular body (MVB) biogenesis. Although ESCRT-III drives membrane remodeling that creates MVBs, its structure and the mechanism of vesicle formation are unclear. Using electron microscopy, we visualize an ESCRT-II:ESCRT-III supercomplex and propose how it mediates vesicle formation. We define conformational changes that activate ESCRT-III subunit Snf7 and show that it assembles into spiraling ~9 nm protofilaments on lipid monolayers. A high-content flow cytometry assay further demonstrates that mutations halting ESCRT-III assembly block ESCRT function. Strikingly, the addition of Vps24 and Vps2 transforms flat Snf7 spirals into membrane-sculpting helices. Finally, we show that ESCRT-II and ESCRT-III coassemble into ~65 nm diameter rings indicative of a cargo-sequestering supercomplex. We propose that ESCRT-III has distinct architectural stages that are modulated by ESCRT-II to mediate cargo capture and vesicle formation by ordered assembly.

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.

Osh proteins regulate phosphoinositide metabolism at ER-plasma membrane contact sites.

Sac1 phosphoinositide (PI) phosphatases are essential regulators of PI-signaling networks. Yeast Sac1, an integral endoplasmic reticulum (ER) membrane protein, controls PI4P levels at the ER, Golgi, and plasma membrane (PM). Whether Sac1 can act in trans and turn over PI4P at the Golgi and PM from the ER remains a paradox. We find that Sac1-mediated PI4P metabolism requires the oxysterol-binding homology (Osh) proteins. The PH domain-containing family member, Osh3, localizes to PM/ER membrane contact sites dependent upon PM PI4P levels. We reconstitute Osh protein-stimulated Sac1 PI phosphatase activity in vitro. We also show that the ER membrane VAP proteins, Scs2/Scs22, control PM PI4P levels and Sac1 activity in vitro. We propose that Osh3 functions at ER/PM contact sites as both a sensor of PM PI4P and an activator of the ER Sac1 phosphatase. Our findings further suggest that the conserved Osh proteins control PI metabolism at additional membrane contact sites.

The Hob proteins are novel and conserved lipid-binding proteins at ER-PM contact sites.

Membrane contact sites are critical junctures for organelle signaling and communication. Endoplasmic reticulum-plasma membrane (ER-PM) contact sites were the first membrane contact sites to be described; however, the protein composition and molecular function of these sites is still emerging. Here, we leverage yeast and Drosophila model systems to uncover a novel role for the Hobbit (Hob) proteins at ER-PM contact sites. We find that Hobbit localizes to ER-PM contact sites in both yeast cells and the Drosophila larval salivary glands, and this localization is mediated by an N-terminal ER membrane anchor and conserved C-terminal sequences. The C-terminus of Hobbit binds to plasma membrane phosphatidylinositols, and the distribution of these lipids is altered in hobbit mutant cells. Notably, the Hobbit protein is essential for viability in Drosophila, providing one of the first examples of a membrane contact site-localized lipid binding protein that is required for development.

Functional reconstitution of ESCRT-III assembly and disassembly.

Receptor downregulation in the MVB pathway is mediated by the ESCRT complexes. ESCRT-III is composed of four protein subunits that are monomeric in the cytosol and oligomerize into a protein lattice only upon membrane binding. Recent studies have shown that the ESCRT-III protein Snf7 can form a filament by undergoing homo-oligomerization. To examine the role of membrane binding and of interactions with other ESCRT components in initiating Snf7 oligomerization, we used fluorescence spectroscopy to directly detect and characterize the assembly of the Snf7 oligomer on liposomes using purified ESCRT components. The observed fluorescence changes reveal an obligatory sequence of membrane-protein and protein-protein interactions that generate the active conformation of Snf7. Also, we demonstrate that ESCRT-III assembly drives membrane deformation. Furthermore, using an in vitro disassembly assay, we directly demonstrate that Vps24 and Vps2 function as adaptors in the ATP-dependent membrane disassembly of the ESCRT-III complex by recruiting the AAA ATPase Vps4.

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.

Ubiquitin-dependent lysosomal membrane protein sorting and degradation.

As an essential organelle in the cell, the lysosome is responsible for digestion and recycling of intracellular components, storage of nutrients, and pH homeostasis. The lysosome is enclosed by a special membrane to maintain its integrity, and nutrients are transported across the membrane by numerous transporters. Despite their importance in maintaining nutrient homeostasis and regulating signaling pathways, little is known about how lysosomal membrane protein lifetimes are regulated. We identified a yeast vacuolar amino acid transporter, Ypq1, that is selectively sorted and degraded in the vacuolar lumen following lysine withdrawal. This selective degradation process requires a vacuole anchored ubiquitin ligase (VAcUL-1) complex composed of Rsp5 and Ssh4. We propose that after ubiquitination, Ypq1 is selectively sorted into an intermediate compartment. The ESCRT machinery is then recruited to sort the ubiquitinated Ypq1 into intraluminal vesicles (ILVs). Finally, the compartment fuses with the vacuole and delivers ILVs into the lumen for degradation.

Phosphoinositide [PI(3,5)P2] lipid-dependent regulation of the general transcriptional regulator Tup1.

Transcriptional activity of a gene is governed by transcriptional regulatory complexes that assemble/disassemble on the gene and control the chromatin architecture. How cytoplasmic components influence the assembly/disassembly of transcriptional regulatory complexes is poorly understood. Here we report that the budding yeast Saccharomyces cerevisiae has a chromatin architecture-modulating mechanism that is dependent on the endosomal lipid PI(3,5)P(2). We identified Tup1 and Cti6 as new, highly specific PI(3,5)P(2) interactors. Tup1--which associates with multiple transcriptional regulators, including the HDAC (histone deacetylase) and SAGA complexes--plays a crucial role in determining an activated or repressed chromatin state of numerous genes, including GAL1. We show that, in the context that the Gal4 activation pathway is compromised, PI(3,5)P(2) plays an essential role in converting the Tup1-driven repressed chromatin structure into a SAGA-containing activated chromatin structure at the GAL1 promoter. Biochemical and cell biological experiments suggest that PI(3,5)P(2) recruits Cti6 and the Cyc8-Tup1 corepressor complex to the late endosomal/vacuolar membrane and mediates the assembly of a Cti6-Cyc8-Tup1 coactivator complex that functions to recruit the SAGA complex to the GAL1 promoter. Our findings provide important insights toward understanding how the chromatin architecture and epigenetic status of a gene are regulated by cytoplasmic components.

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.

The phosphatidylinositol 3,5-bisphosphate (PI(3,5)P2)-dependent Tup1 conversion (PIPTC) regulates metabolic reprogramming from glycolysis to gluconeogenesis.

Glucose/carbon metabolism is a fundamental cellular process in living cells. In response to varying environments, eukaryotic cells reprogram their glucose/carbon metabolism between aerobic or anaerobic glycolysis, oxidative phosphorylation, and/or gluconeogenesis. The distinct type of glucose/carbon metabolism that a cell carries out has significant effects on the cell's proliferation and differentiation. However, it is poorly understood how the reprogramming of glucose/carbon metabolism is regulated. Here, we report a novel endosomal PI(3,5)P2 lipid-dependent regulatory mechanism that is required for metabolic reprogramming from glycolysis to gluconeogenesis in Saccharomyces cerevisiae. Certain gluconeogenesis genes, such as FBP1 (encoding fructose-1,6-bisphosphatase 1) and ICL1 (encoding isocitrate lyase 1) are under control of the Mig1 repressor and Cyc8-Tup1 corepressor complex. We previously identified the PI(3,5)P2-dependent Tup1 conversion (PIPTC), a mechanism to convert Cyc8-Tup1 corepressor to Cti6-Cyc8-Tup1 coactivator. We demonstrate that the PIPTC plays a critical role for transcriptional activation of FBP1 and ICL1. Furthermore, without the PIPTC, the Cat8 and Sip4 transcriptional activators cannot be efficiently recruited to the promoters of FBP1 and ICL1, suggesting a key role for the PIPTC in remodulating the chromatin architecture at the promoters. Our findings expand our understanding of the regulatory mechanisms for metabolic reprogramming in eukaryotes to include key regulation steps outside the nucleus. Given that Tup1 and the metabolic enzymes that control PI(3,5)P2 are highly conserved among eukaryotes, our findings may provide important insights toward understanding glucose/carbon metabolic reprogramming in other eukaryotes, including humans.

ESCRT-II coordinates the assembly of ESCRT-III filaments for cargo sorting and multivesicular body vesicle formation.

The sequential action of five distinct endosomal-sorting complex required for transport (ESCRT) complexes is required for the lysosomal downregulation of cell surface receptors through the multivesicular body (MVB) pathway. On endosomes, the assembly of ESCRT-III is a highly ordered process. We show that the length of ESCRT-III (Snf7) oligomers controls the size of MVB vesicles and addresses how ESCRT-II regulates ESCRT-III assembly. The first step of ESCRT-III assembly is mediated by Vps20, which nucleates Snf7/Vps32 oligomerization, and serves as the link to ESCRT-II. The ESCRT-II subunit Vps25 induces an essential conformational switch that converts inactive monomeric Vps20 into the active nucleator for Snf7 oligomerization. Each ESCRT-II complex contains two Vps25 molecules (arms) that generate a characteristic Y-shaped structure. Mutant 'one-armed' ESCRT-II complexes with a single Vps25 arm are sufficient to nucleate Snf7 oligomerization. However, these oligomers cannot execute ESCRT-III function. Both Vps25 arms provide essential geometry for the assembly of a functional ESCRT-III complex. We propose that ESCRT-II serves as a scaffold that nucleates the assembly of two Snf7 oligomers, which together are required for cargo sequestration and vesicle formation during MVB sorting.

Crystal structure of the yeast Sac1: implications for its phosphoinositide phosphatase function.

Sac family phosphoinositide (PI) phosphatases are an essential family of CX(5)R(T/S)-based enzymes, involved in numerous aspects of cellular function such as PI homeostasis, cellular signalling, and membrane trafficking. Genetic deletions of several Sac family members result in lethality in animal models and mutations of the Sac3 gene have been found in human hereditary diseases. In this study, we report the crystal structure of a founding member of this family, the Sac phosphatase domain of yeast Sac1. The 2.0 A resolution structure shows that the Sac domain comprises of two closely packed sub-domains, a novel N-terminal sub-domain and the PI phosphatase catalytic sub-domain. The structure further shows a striking conformation of the catalytic P-loop and a large positively charged groove at the catalytic site. These findings suggest an unusual mechanism for its dephosphorylation function. Homology structural modeling of human Fig4/Sac3 allows the mapping of several disease-related mutations and provides a framework for the understanding of the molecular mechanisms of human diseases.

Cargo ubiquitination is essential for multivesicular body intralumenal vesicle formation.

The efficient formation of a variety of transport vesicles is influenced by the presence of cargo, suggesting that cargo itself might have a defining role in vesicle biogenesis. However, definitive in vivo experiments supporting this concept are lacking, as it is difficult to eliminate endogenous cargo. The Endosomal Sorting Complexes Required for Transport (ESCRT) apparatus sorts ubiquitinated membrane proteins into endosomal intralumenal vesicles (ILVs) that accumulate within multivesicular bodies. Here we show that cargo ubiquitination is required for effective recruitment of the ESCRT machinery onto endosomal membranes and for the subsequent formation of ILVs.

A role for Vps13-mediated lipid transfer at the ER-endosome contact site in ESCRT-mediated sorting.

Endosomes are specialized organelles that function in the secretory and endocytic protein sorting pathways. Endocytosed cell surface receptors and transporters destined for lysosomal degradation are sorted into intraluminal vesicles (ILVs) at endosomes by endosomal sorting complexes required for transport (ESCRT) proteins. The endosomes (multivesicular bodies, MVBs) then fuse with the lysosome. During endosomal maturation, the number of ILVs increases, but the size of endosomes does not decrease despite the consumption of the limiting membrane during ILV formation. Vesicle-mediated trafficking is thought to provide lipids to support MVB biogenesis. However, we have uncovered an unexpected contribution of a large bridge-like lipid transfer protein, Vps13, in this process. Here, we reveal that Vps13-mediated lipid transfer at ER-endosome contact sites is required for the ESCRT pathway. We propose that Vps13 may play a critical role in supplying lipids to the endosome, ensuring continuous ESCRT-mediated sorting during MVB biogenesis.

TRAPPII subunits are required for the specificity switch of a Ypt-Rab GEF.

Ypt-Rab GTPases are key regulators of the various steps of intracellular trafficking. Guanine nucleotide-exchange factors (GEFs) regulate the conversion of Ypt-Rabs to the GTP-bound state, in which they interact with effectors that mediate all the known aspects of vesicular transport. An interesting possibility is that Ypt-Rabs coordinate separate steps of the transport pathways. The conserved modular complex TRAPP is a GEF for the Golgi gatekeepers Ypt1 and Ypt31/32 (Refs 5-7). However, it is not known how Golgi entry and exit are coordinated. TRAPP comes in two configurations: the seven-subunit TRAPPI is required for endoplasmic reticulum-to-Golgi transport, whereas the ten-subunit TRAPPII functions in late Golgi. The two essential TRAPPII-specific subunits Trs120 and Trs130 have been identified as Ypt31/32 genetic interactors. Here, we show that they are required for switching the GEF specificity of TRAPP from Ypt1 to Ypt31. Moreover, a trs130ts mutation confers opposite effects on the intracellular localization of these GTPases. We suggest that the Trs120-Trs130 subcomplex joins TRAPP in the late Golgi to switch its GEF activity from Ypt1 to Ypt31/32. Such a 'switchable' GEF could ensure sequential activation of these Ypts, thereby coordinating Golgi entry and exit.

Structural basis for selective recognition of ESCRT-III by the AAA ATPase Vps4.

The AAA+ ATPases are essential for various activities such as membrane trafficking, organelle biogenesis, DNA replication, intracellular locomotion, cytoskeletal remodelling, protein folding and proteolysis. The AAA ATPase Vps4, which is central to endosomal traffic to lysosomes, retroviral budding and cytokinesis, dissociates ESCRT complexes (the endosomal sorting complexes required for transport) from membranes. Here we show that, of the six ESCRT--related subunits in yeast, only Vps2 and Did2 bind the MIT (microtubule interacting and transport) domain of Vps4, and that the carboxy-terminal 30 residues of the subunits are both necessary and sufficient for interaction. We determined the crystal structure of the Vps2 C terminus in a complex with the Vps4 MIT domain, explaining the basis for selective ESCRT-III recognition. MIT helices alpha2 and alpha3 recognize a (D/E)xxLxxRLxxL(K/R) motif, and mutations within this motif cause sorting defects in yeast. Our crystal structure of the amino-terminal domain of an archaeal AAA ATPase of unknown function shows that it is closely related to the MIT domain of Vps4. The archaeal ATPase interacts with an archaeal ESCRT-III-like protein even though these organisms have no endomembrane system, suggesting that the Vps4/ESCRT-III partnership is a relic of a function that pre-dates the divergence of eukaryotes and Archaea.

Pheromone-induced anisotropy in yeast plasma membrane phosphatidylinositol-4,5-bisphosphate distribution is required for MAPK signaling.

During response of budding yeast to peptide mating pheromone, the cell becomes markedly polarized and MAPK scaffold protein Ste5 localizes to the resulting projection (shmoo tip). We demonstrated before that this recruitment is essential for sustained MAPK signaling and requires interaction of a pleckstrin homology (PH) domain in Ste5 with phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] in the plasma membrane. Using fluorescently tagged high-affinity probes specific for PtdIns(4,5)P(2), we have now found that this phosphoinositide is highly concentrated at the shmoo tip in cells responding to pheromone. Maintenance of this strikingly anisotropic distribution of PtdIns(4,5)P(2), stable tethering of Ste5 at the shmoo tip, downstream MAPK activation, and expression of a mating pathway-specific reporter gene all require continuous function of the plasma membrane-associated PtdIns 4-kinase Stt4 and the plasma membrane-associated PtdIns4P 5-kinase Mss4 (but not the Golgi-associated PtdIns 4-kinase Pik1). Our observations demonstrate that PtdIns(4,5)P(2) is the primary determinant for restricting localization of Ste5 within the plasma membrane and provide direct evidence that an extracellular stimulus-evoked self-reinforcing mechanism generates a spatially enriched pool of PtdIns(4,5)P(2) necessary for the membrane anchoring and function of a signaling complex.

The Fab1 phosphatidylinositol kinase pathway in the regulation of vacuole morphology.

Yeast vacuoles are very dynamic structures that must respond to changes in extracellular osmolarity by rapidly altering their size, thereby releasing or taking up water and ions. Further, the need to accommodate a constant biosynthetic influx of membrane and to partition vacuoles during cell division necessitates precise regulation of the size and shape of the vacuole. While it is has been shown that the lipid kinase Fab1p and its product phosphatidylinositol 3,5-bisphosphate, and not the mitogen-activated protein kinase Hog1p, are central to this regulatory pathway, key effectors still await identification. Atg18p is the most recently identified candidate for a Fab1p effector mediating the largely uncharacterized processes of vesicle fission and membrane recycling at the vacuole.