The ESCRT‐III protein CeVPS‐32 is enriched in domains distinct from CeVPS‐27 and CeVPS‐23 at the endosomal membrane of epithelial cells

Background information. Within the endocytic pathway, the ESCRT (endosomal sorting complex required for transport) machinery is essential for the biogenesis of MVBs (multivesicular bodies). In yeast, ESCRTs are recruited at the endosomal membrane and are involved in cargo sorting into intralumenal vesicles of the MVBs. Results. In the present study, we characterize the ESCRT‐III protein CeVPS‐32 (Caenorhabditis elegans vacuolar protein sorting 32) and its interactions with CeVPS‐27, CeVPS‐23 and CeVPS‐4. In contrast with other CevpsE (class E vps) genes, depletion of Cevps‐32 is embryonic lethal with severe defects in the remodelling of epithelial cell shape during organogenesis. Furthermore, Cevps‐32 animals display an accumulation of enlarged early endosomes in epithelial cells and an accumulation of autophagosomes. The CeVPS‐32 protein is enriched in epithelial tissues and in residual bodies during spermatid maturation. We show that CeVPS‐32 and CeVPS‐27/Hrs (hepatocyte‐growth‐factor‐regulated tyrosine kinase substrate) are enriched in distinct subdomains at the endosomal membrane. CeVPS‐27‐positive subdomains are also enriched for the ESCRT‐I protein CeVPS‐23/TSG101 (tumour susceptibility gene 101). The formation of CeVPS‐27 subdomains is not affected by the depletion of CeVPS‐23, CeVPS‐32 or the ATPase CeVPS‐4. Conclusion. Our results suggest that the formation of membrane subdomains is essential for the maturation of endosomes.     

ESCRTing autophagic clearance of aggregating proteins

Autophagy has recently been found to play an important role in the degradation of damaged macromolecules, in particular misfolded, aberrant proteins that can disrupt neuronal function and cause neurodegeneration if not removed. Mutations in the Endosomal Sorting Complex Required for Transport (ESCRT)-III subunit CHMP2B were recently associated with frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), neurodegenerative diseases characterized by abnormal ubiquitin-positive protein deposits in affected neurons. The ESCRT proteins are known to sort ubiquitinated integral membrane proteins into intralumenal vesicles of the multivesicular body (MVB), but it was not known how ESCRT mutations could cause neurodegenerative disease. We found autophagic degradation to be inhibited in cells depleted of ESCRT subunits or expressing CHMP2B mutants and in Drosphila melanogaster lacking ESCRTs. In addition to accumulation of autophagic vesicles, we also found increased levels of membrane-free ubiquitin-positive protein aggregates in ESCRT-depleted cells. Using cellular and Drosophila models for Huntington’s disease, we showed that reduced ESCRT levels inhibit clearance of expanded polyglutamine aggregates and aggravate their neurotoxic effect. Together, our data indicate that efficient autophagic degradation requires functional MVBs and provide a possible explanation to the observed neurodegenerative phenotype seen in patients with CHMP2B mutations. In this addendum we discuss models to explain the functions of ESCRTs and MVBs in autophagic degradation.

Addendum to: Filimonenko M, Stuffers S, Raiborg C, Yamamoto A, Malerod L, Fisher EM, Isaacs A, Brech A, Stenmark H, Simonsen A. Functional multivesicular bodies are required for autophagic clearance of protein aggregates associated with neurodegenerative disease. J Cell Biol 2007; 179;485-500.

and

Rusten TE, Vaccari T, Lindmo K, Rodahl LM, Nezis IP, Sem-Jacobsen C, Wendler F, Vincent JP, Brech A, Bilder D, Stenmark H. ESCRTs and Fab1 regulate distinct steps of autophagy. Curr Biol 2007; 17;1817-25.     

The Endosomal Sorting Complex Required for Transport Pathway Mediates Chemokine Receptor CXCR4-promoted Lysosomal Degradation of the Mammalian Target of Rapamycin Antagonist DEPTOR

G protein-coupled receptor (GPCR) signaling mediates many cellular functions, including cell survival, proliferation, and cell motility. Many of these processes are mediated by GPCR-promoted activation of Akt signaling by mammalian target of rapamycin complex 2 (mTORC2) and the phosphatidylinositol 3-kinase (PI3K)/phosphoinositide-dependent kinase 1 (PDK1) pathway. However, the molecular mechanisms by which GPCRs govern Akt activation by these kinases remain poorly understood. Here, we show that the endosomal sorting complex required for transport (ESCRT) pathway mediates Akt signaling promoted by the chemokine receptor CXCR4. Pharmacological inhibition of heterotrimeric G protein Gα_i or PI3K signaling and siRNA targeting ESCRTs blocks CXCR4-promoted degradation of DEPTOR, an endogenous antagonist of mTORC2 activity. Depletion of ESCRTs by siRNA leads to increased levels of DEPTOR and attenuated CXCR4-promoted Akt activation and signaling, consistent with decreased mTORC2 activity. In addition, ESCRTs likely have a broad role in Akt signaling because ESCRT depletion also attenuates receptor tyrosine kinase-promoted Akt activation and signaling. Our data reveal a novel role for the ESCRT pathway in promoting intracellular signaling, which may begin to identify the signal transduction pathways that are important in the physiological roles of ESCRTs and Akt.     

HIV-1 Gag release from yeast reveals ESCRT interaction with the Gag N-terminal protein region

The HIV-1 protein Gag assembles at the plasma membrane and drives virion budding, assisted by the cellular endosomal complex required for transport (ESCRT) proteins. Two ESCRT proteins, TSG101 and ALIX, bind to the Gag C-terminal p6 peptide. TSG101 binding is important for efficient HIV-1 release, but how ESCRTs contribute to the budding process and how their activity is coordinated with Gag assembly is poorly understood. Yeast, allowing genetic manipulation that is not easily available in human cells, has been used to characterize the cellular ESCRT function. Previous work reported Gag budding from yeast spheroplasts, but Gag release was ESCRT-independent. We developed a yeast model for ESCRT-dependent Gag release. We combined yeast genetics and Gag mutational analysis with Gag-ESCRT binding studies and the characterization of Gag-plasma membrane binding and Gag release. With our system, we identified a previously unknown interaction between ESCRT proteins and the Gag N-terminal protein region. Mutations in the Gag-plasma membrane–binding matrix domain that reduced Gag-ESCRT binding increased Gag-plasma membrane binding and Gag release. ESCRT knockout mutants showed that the release enhancement was an ESCRT-dependent effect. Similarly, matrix mutation enhanced Gag release from human HEK293 cells. Release enhancement partly depended on ALIX binding to p6, although binding site mutation did not impair WT Gag release. Accordingly, the relative affinity for matrix compared with p6 in GST-pulldown experiments was higher for ALIX than for TSG101. We suggest that a transient matrix-ESCRT interaction is replaced when Gag binds to the plasma membrane. This step may activate ESCRT proteins and thereby coordinate ESCRT function with virion assembly.     

Binding to Any ESCRT Can Mediate Ubiquitin‐Independent Cargo Sorting

The ESCRT (endosomal sorting complex required for transport) machinery is known to sort ubiquitinated transmembrane proteins into vesicles that bud into the lumen of multivesicular bodies (MVBs). Although the ESCRTs themselves are ubiquitinated they are excluded from the intraluminal vesicles and recycle back to the cytoplasm for further rounds of sorting. To obtain insights into the rules that distinguish ESCRT machinery from cargo we analyzed the trafficking of artificial ESCRT‐like protein fusions. These studies showed that lowering ESCRT‐binding affinity converts a protein from behaving like ESCRT machinery into cargo of the MVB pathway, highlighting the close relationship between machinery and the cargoes they sort. Furthermore, our findings give insights into the targeting of soluble proteins into the MVB pathway and show that binding to any of the ESCRTs can mediate ubiquitin‐independent MVB sorting.     

古菌ESCRT系统研究进展

内体分拣转运复合体（ESCRT,endosomal sorting complex required for transport）曾被认为是真核生物特有的系统,涉及膜重塑、泛素化蛋白质分拣等重要细胞生命过程。近年的研究显示,TACK（包括Thaumarchaeota、Aigarchaeota、Crenarchaeota和Korarchaeota门）古菌超门中存在着一类与分泌膜囊泡、古菌病毒出胞以及细胞分裂过程等膜重塑过程相关的细胞分裂（Cdv,cell division）系统,该系统中的CdvB和CdvC是真核生物ESCRT-III和Vps4的同源蛋白,提示真核生物ESCRT系统可能起源自古菌。然而,由于TACK古菌中缺少真核生物ESCRT系统的其他关键成分,这一假设仍有争议。最近发现的阿斯加德（Asgard）古菌是一类被认为与真核生物最近缘的古菌,其基因组具有较完整的ESCRT相关蛋白的编码基因,提示真核生物的ESCRT很可能起源于阿斯加德古菌。本文首先简要介绍真核生物ESCRT系统的组成及生物学功能,然后分别总结TACK古菌的Cdv系统和阿斯加德古菌的ESCRT系统的研究进展,重点讨论它们的组成及生物学功能,为进一步了解古菌ESCRT系统与真核生物起源的关系提供参考。     

VHS domains of ESCRT‐0 cooperate in high‐avidity binding to polyubiquitinated cargo

VHS (Vps27, Hrs, and STAM) domains occur in ESCRT‐0 subunits Hrs and STAM, GGA adapters, and other trafficking proteins. The structure of the STAM VHS domain–ubiquitin complex was solved at 2.6 Å resolution, revealing that determinants for ubiquitin recognition are conserved in nearly all VHS domains. VHS domains from all classes of VHS‐domain containing proteins in yeast and humans, including both subunits of ESCRT‐0, bound ubiquitin in vitro. ESCRTs have been implicated in the sorting of Lys63‐linked polyubiquitinated cargo. Intact human ESCRT‐0 binds Lys63‐linked tetraubiquitin 50‐fold more tightly than monoubiquitin, though only 2‐fold more tightly than Lys48‐linked tetraubiquitin. The gain in affinity is attributed to the cooperation of flexibly connected VHS and UIM motifs of ESCRT‐0 in avid binding to the polyubiquitin chain. Mutational analysis of all the five ubiquitin‐binding sites in yeast ESCRT‐0 shows that cooperation between them is required for the sorting of the Lys63‐linked polyubiquitinated cargo Cps1 to the vacuole.     

ESCRT proteins in physiology and disease

As a mechanism of signal attenuation, receptors for growth factors, peptide hormones and cytokines are internalized in response to ligand binding, followed by degradation in lysosomes. Receptor ubiquitination is a key signal for such downregulation, and four protein complexes known as endosomal sorting complex required for transport (ESCRT)-0, -I, -II and -III have been identified as the machinery required for degradative endosomal sorting of ubiquitinated membrane proteins in yeast and metazoans. Three of these complexes contain ubiquitin-binding domains whereas ESCRT-III instead recruits deubiquitinating enzymes. The concerted action of the ESCRTs not only serves to sort ubiquitinated cargo but is also thought to cause inward vesiculation of endosomal membranes, thereby mediating biogenesis of multivesicular endosomes (MVEs). Because ligand-mediated receptor downregulation plays an important role in signal attenuation, it is not surprising that dysfunction of ESCRT components is associated with disease. In this review we discuss the possible roles of ESCRTs in protection against cancer, neurodegenerative diseases and bacterial infections, and we highlight the fact that many RNA viruses exploit the ESCRT machinery for the final abscission step of their budding from cells. We also review the additional functions of ESCRT proteins in cytokinesis and discuss how these may be related to ESCRT-associated pathologies.     

ESCRT-Independent Budding of HIV-1 Gag Virus-Like Particles from Saccharomyces cerevisiae Spheroplasts

Heterologous expression of HIV-1 Gag in a variety of host cells results in its packaging into virus-like particles (VLPs) that are subsequently released into the extracellular milieu. This phenomenon represents a useful tool for probing cellular factors required for viral budding and has contributed to the discovery of roles for ubiquitin ligases and the endosomal sorting complexes required for transport (ESCRTs) in viral budding. These factors are highly conserved throughout eukaryotes and have been studied extensively in the yeast Saccharomyces cerevisiae, a model eukaryote previously utilized as a host for the production of VLPs. We used heterologous expression of HIV Gag in yeast spheroplasts to examine the role of ESCRTs and associated factors (Rsp5, a HECT ubiquitin ligase of the Nedd4 family; Bro1, a homolog of Alix; and Vps4, the AAA-ATPase required for ESCRT function in all contexts/organisms investigated) in the generation of VLPs. Our data reveal: 1) characterized Gag-ESCRT interaction motifs (late domains) are not required for VLP budding, 2) loss of function alleles of the essential HECT ubiquitin ligase Rsp5 do not display defects in VLP formation, and 3) ESCRT function is not required for VLP formation from spheroplasts. These results suggest that the egress of HIV Gag from yeast cells is distinct from the most commonly described mode of exit from mammalian cells, instead mimicking ESCRT-independent VLP formation observed in a subset of mammalian cells. As such, budding of Gag from yeast cells appears to represent ESCRT-independent budding relevant to viral replication in at least some situations. Thus the myriad of genetic and biochemical tools available in the yeast system may be of utility in the study of this aspect of viral budding.     

Chm7 and Heh1 collaborate to link nuclear pore complex quality control with nuclear envelope sealing

The integrity of the nuclear envelope barrier relies on membrane remodeling by the ESCRTs, which seal nuclear envelope holes and contribute to the quality control of nuclear pore complexes (NPCs); whether these processes are mechanistically related remains poorly defined. Here, we show that the ESCRT‐II/III chimera, Chm7, is recruited to a nuclear envelope subdomain that expands upon inhibition of NPC assembly and is required for the formation of the storage of improperly assembled NPCs (SINC) compartment. Recruitment to sites of NPC assembly is mediated by its ESCRT‐II domain and the LAP2‐emerin‐MAN1 (LEM) family of integral inner nuclear membrane proteins, Heh1 and Heh2. We establish direct binding between Heh2 and the “open” forms of both Chm7 and the ESCRT‐III, Snf7, and between Chm7 and Snf7. Interestingly, Chm7 is required for the viability of yeast strains where double membrane seals have been observed over defective NPCs; deletion of CHM7 in these strains leads to a loss of nuclear compartmentalization suggesting that the sealing of defective NPCs and nuclear envelope ruptures could proceed through similar mechanisms.     

Differential requirements of mammalian ESCRTs in multivesicular body formation, virus budding and cell division

The endosomal sorting complexes required for transport (ESCRTs) mediate membrane fission from the cytoplasmic face of the bud neck. ESCRTs were originally identified as factors involved in multivesicular body vesicle biogenesis in yeast but have since been shown to function in other membrane fission events in mammalian cells, including enveloped virus budding and the abscission step of cytokinesis. Several recent studies have revealed that not all ESCRT factors are required for each of these biological processes, and this review summarizes our current understanding of the different requirements for ESCRT factors in these three different ESCRT-mediated mammalian membrane fission processes.     

ESCRT‐dependent protein sorting is required for the viability of yeast clathrin‐mediated endocytosis mutants

Endocytosis regulates many processes, including signaling pathways, nutrient uptake, and protein turnover. During clathrin‐mediated endocytosis (CME), adaptors bind to cytoplasmic regions of transmembrane cargo proteins, and many endocytic adaptors are also directly involved in the recruitment of clathrin. This clathrin‐associated sorting protein family includes the yeast epsins, Ent1/2, and AP180/PICALM homologs, Yap1801/2. Mutant strains lacking these four adaptors, but expressing an epsin N‐terminal homology (ENTH) domain necessary for viability (4Δ+ENTH), exhibit endocytic defects, such as cargo accumulation at the plasma membrane (PM). This CME‐deficient strain provides a sensitized background ideal for revealing cellular components that interact with clathrin adaptors. We performed a mutagenic screen to identify alleles that are lethal in 4Δ+ENTH cells using a colony‐sectoring reporter assay. After isolating candidate synthetic lethal genes by complementation, we confirmed that mutations in VPS4 led to inviability of a 4Δ+ENTH strain. Vps4 mediates the final step of endosomal sorting complex required for transport (ESCRT)‐dependent trafficking, and we found that multiple ESCRTs are also essential in 4Δ+ENTH cells, including Snf7, Snf8 and Vps36. Deletion of VPS4 from an end3Δ strain, another CME mutant, similarly resulted in inviability, and upregulation of a clathrin‐independent endocytosis pathway rescued 4Δ+ENTH vps4Δ cells. Loss of Vps4 from an otherwise wild‐type background caused multiple cargoes to accumulate at the PM because of an increase in Rcy1‐dependent recycling of internalized protein to the cell surface. Additionally, vps4Δ rcy1Δ mutants exhibited deleterious growth phenotypes. Together, our findings reveal previously unappreciated effects of disrupted ESCRT‐dependent trafficking on endocytic recycling and the PM.     

Endosomal sorting complexes required for ESCRTing cells toward death during neurogenesis,neurodevelopment and neurodegeneration

The endosomal sorting complexes required for transport (ESCRT) proteins help in the recognition, sorting and degradation of ubiquitinated cargoes from the cell surface, long‐lived proteins or aggregates, and aged organelles present in the cytosol. These proteins take part in the endo‐lysosomal system of degradation. The ESCRT proteins also play an integral role in cytokinesis, viral budding and mRNA transport. Many neurodegenerative diseases are caused by toxic accumulation of cargo in the cell, which causes stress and ultimately leads to neuronal death. This accumulation of cargo occurs because of defects in the endo‐lysosomal degradative pathway—loss of function of ESCRTs has been implicated in this mechanism. ESCRTs also take part in many survival processes, lack of which can culminate in neuronal cell death. While the role played by the ESCRT proteins in maintaining healthy neurons is known, their role in neurodegenerative diseases is still poorly understood. In this review, we highlight the importance of ESCRTs in maintaining healthy neurons and then suggest how perturbations in many of the survival mechanisms governed by these proteins could eventually lead to cell death; quite often these correlations are not so obviously laid out. Extensive neuronal death eventually culminates in neurodegeneration.      

Functional assignment of multiple ESCRT‐III homologs in cell division and budding in Sulfolobus islandicus

The archaea Sulfolobus utilizes the ESCRT‐III‐based machinery for cell division. This machinery comprises three proteins: CdvA, Eukaryotic‐like ESCRT‐III and Vps4. In addition to ESCRT‐III, Sulfolobus cells also encode three other ESCRT‐III homologs termed ESCRT‐III‐1, ?2 and ?3. Herein, we show that ESCRT‐III‐1 and ?2 in S. islandicus REY15A are localized at midcell between segregating chromosomes, indicating that both are involved in cell division. Genetic analysis reveals that escrt‐III‐2 is indispensable for cell viability and cells with reduced overall level of ESCRT‐III‐1 exhibit growth retardation and cytokinesis defect with chain‐like cell morphology. In contrast, escrt‐III‐3 is dispensable for cell division. We show that S. islandicus REY15A cells generate buds when infected with S. tengchongensis spindle shaped‐virus 2 (STSV2) or when ESCRT‐III‐3 is over‐expressed. Interestingly, Δescrt‐III‐3 cells infected with STSV2 do not produce buds. These results suggest that ESCRT‐III‐3 plays an important role in budding. In addition, cells over‐expressing the C‐terminal truncated mutants of ESCRT‐III, ESCRT‐III‐1 and ESCRT‐III‐2 are maintained predominantly at the early, late, and membrane abscission stages of cell division respectively, suggesting a crucial role of the ESCRTs at different stages of membrane ingression. Intriguingly, intercellular bridge and midbody‐like structures are observed in cells over‐expressing MIM2‐truncated mutant of ESCRT‐III‐2.     

Expression of NYV1 encoding the negative regulator of Pmc1 is repressed by two transcriptional repressors,Nrg1 and Mig1

ESCRT components function to form multivesicular bodies for sorting of proteins destined to the yeast vacuole. The calcium hypersensitivity of ESCRT mutants is mainly due to repressed expression of PMR1 through the Rim101/Nrg1 pathway in budding yeast. Here, we show that overexpression of PMC1 and its negative regulator gene NYV1 suppresses and increases calcium hypersensitivity of ESCRT mutants, respectively. Consistently, deletion of NYV1 suppresses their calcium hypersensitivity. Expression of NYV1 is dramatically reduced in ESCRT mutants. Promoter analysis demonstrates that both Nrg1 and Mig1 repress NYV1 expression. Deletion of ESCRTs increases Nrg1 binding, but not Mig1-binding, to the NYV1 promoter. Deletion of MIG1 increases calcium sensitivity of ESCRT mutants due to derepression of NYV1 expression.     

ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

Ubiquitin (Ub) sorting receptors facilitate the targeting of ubiquitinated membrane proteins into multivesicular bodies (MVBs). Ub-binding domains (UBDs) have been described in several endosomal sorting complexes required for transport (ESCRT). Using available structural information, we have investigated the role of the multiple UBDs within ESCRTs during MVB cargo selection. We found a novel UBD within ESCRT-I and show that it contributes to MVB sorting in concert with the known UBDs within the ESCRT complexes. These experiments reveal an unexpected level of coordination among the ESCRT UBDs, suggesting that they collectively recognize a diverse set of cargo rather than act sequentially at discrete steps.     

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.     

ESCRT proteins and cell signalling

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.     

How the vacuole ESCRTs its own proteins to their final destination

Lysosomes (vacuoles in yeast) are master regulators of metabolism and protein turnover, but how they degrade their own resident proteins is unclear. Recently, multiple models have been proposed explaining yeast vacuole protein sorting, but the role of the ESCRT pathway was unclear. In this JCB issue, work from Yang et al. (https://doi.org/10.1083/jcb.202012104) highlights how the ESCRT pathway localizes to the vacuole surface to execute protein sorting of its resident proteins.

Lysosomes are key metabolic organelles that influence nutrient sensing, protein trafficking, lipid homeostasis, and catabolic metabolism (1). Because of their many roles, how lysosomes receive and degrade proteins has been a pervasive question in cell biology, and has driven the discovery of multiple trafficking pathways that deliver proteins from different regions of the cell to the lysosome for turnover. In budding yeast, the vacuole (functionally equivalent to the lysosome) is a superb model to dissect the mechanisms of endolysosomal trafficking. However, how the vacuole/lysosome senses and degrades its own resident proteins has remained mysterious. This is a critical question, since the vacuole/lysosome surface is home to many nutrient and lipid transporters, which must be selectively maintained or degraded in response metabolic cues to enable cell homeostasis.Recently, a flurry of papers were published with models explaining how the turnover of resident vacuole proteins is achieved. Two opposing models emerged (Fig. 1). In one, the ESCRT (endosomal sorting complexes required for transport) proteins, which are traditionally known to sort cargoes on the endosome surface into intralumenal vesicles that push into the endosome, were proposed to localize to the vacuole surface and execute a topologically similar protein sorting mechanism (albeit now on a different organelle; 2, 3, 4). However, another model called the intralumenal fragment (ILF) model proposed a radically different mechanism. Here, the homotypic fusion of the vacuole with itself could create a bubble-like fragment inside, containing vacuole surface proteins to be degraded. Critically, the formation of this fragment would be ESCRT-independent but require other membrane trafficking machinery like Rab7 (5, 6, 7). Paradoxically, both models were implicated in sorting the same vacuole proteins. Whether these two models were mutually exclusive, and how they related to one another, was unclear.Open in a separate windowFigure 1.Cartoon schematic of the models for resident vacuole protein turnover. Left: ESCRT-dependent sorting of vacuole proteins via recruitment of ESCRTs to ubiquitinated surface proteins, which are sorted into a vesicle that protrudes into the vacuole lumen and is degraded. Right: ILF pathway showing an ESCRT-independent selective sorting of proteins into a fragment, which via homotypic vacuole fusion is deposited into the vacuole lumen and then degraded.In a recent paper published by JCB, Yang and colleagues (8) shed new light on the mystery of how resident vacuole proteins are degraded and resolve several of the issues between these two contrasting models. To begin, Yang et al. used Western blotting and a microfluidics-based imaging system to monitor GFP-tagged vacuole proteins. Capitalizing on a tetracycline (TET)-OFF system that enabled them to essentially conduct pulse-chase assays, they could monitor each GFP-tagged protein and its turnover kinetics following treatment with rapamycin, which initiated the degradation process. They learned that some proteins like Zrt3-GFP (a zinc transporter) were first sorted into foci on the vacuole surface, then a short time later accumulated in the vacuole lumen, consistent with a sorting process that enabled Zrt3-GFP breakdown by vacuolar proteases. Indeed, Western blotting revealed free GFP accumulation over time as the Zrt3-GFP was degraded, indicating the fusion protein was being broken down in the vacuole lumen, leaving behind soluble GFP.Using this experimental setup, they next examined a vacuole protein previously reported to be a substrate of the ILF pathway, Fth1 (5). Fth1 had been proposed to be selectively sorted via the ILF pathway, whereas its binding partner Fet5 was not. However, Yang et al. were able to show that these proteins coimmunoprecipitated together, suggesting they exist in a complex and were unlikely to be separated. Indeed, deletion of Fth1 caused Fet5 to be trapped at the ER, consistent with it needing Fth1 for stability. Furthermore, time-lapse imaging indicated that Fth1 turnover was very slow, in contrast to earlier reports that Fth1 was constitutively turned over via the ILF pathway. Motivated by these observations, they next examined other ILF cargoes reported to be degraded by the ILF pathway following exposure of yeast to heat or the drug cycloheximide. Again capitalizing on their time-lapse imaging system and Western blotting, they failed to observe significant turnover of these proteins, in contrast to previous work.A key difference between the two models is dependence on the ESCRT pathway. To further dissect, Yang et al. used imaging to interrogate how loss of ESCRT machinery impacted vacuole protein dynamics and degradation. By monitoring vacuole proteins whose turnover was stimulated by rapamycin, they observed that yeast lacking the ESCRT component Vps4 failed to sort Zrt3 and other cargoes into the vacuole. This argued that their degradation was ESCRT dependent, and likely not through the ILF pathway. Serendipitously, time-lapse imaging experiments also detected occasional ILF-like structures within the vacuole. However, imaging revealed that ILF structures were rare, and often contained the protein Zrc1-mCherry, which is stable and not degraded. Collectively, this argued that ESCRT machinery is required for the turnover of several vacuole proteins, and that the ILF system is likely not the predominant mechanism of resident vacuole protein degradation.Finally, Yang et al. used their imaging platform to interrogate how ubiquitin and the ESCRTs influence vacuole protein turnover. They took advantage of an inducible degradation system called RapIDeg that uses an FK506-binding protein–FKBP Rapamycin-binding domain system to inducibly attach ubiquitin to a GFP-tagged vacuole protein by adding rapamycin. Strikingly, this revealed that within 10–30 min of ubiquitination, the vacuole protein was sorted into bright foci on the membrane surface that contained ESCRT machinery, strongly suggesting this protein sorting was ESCRT dependent.Collectively, the data argue that many vacuole proteins are degraded via a ubiquitin and ESCRT-dependent pathway that operates on the vacuole surface in a manner topologically similar to the formation of multi-vesicular bodies at endosomes (Fig. 1). In this model, ubiquitination of vacuole proteins attracts ESCRT machinery, which bend the membrane away from the cytoplasm to create a vesicle within the vacuole, which is subsequently degraded by vacuolar proteases. Although ILFs were observed in this and previous studies, these structures are rare, and appear to lack the ability to selectively sort proteins.It should be noted that this study and the earlier ILF pathway studies used very different methodologies. The ILF studies relied on purified vacuoles in a cell-free system (5, 7), whereas Yang et al. employed in vivo time-lapse imaging and blotting (8). Since purified vacuoles lack the full cellular ubiquitination machinery, it is feasible that ESCRT-independent membrane remodeling may occur in vitro. Relatedly, it is possible that the ILF system may sort other proteins not examined by Yang et al. However, Yang clearly establishes that several vacuole proteins are sorted via an ESCRT-dependent pathway that acts on the vacuole surface. Second, it is worth noting that ILF structures are observed in several studies of vacuole remodeling. Although likely not a major mechanism for protein sorting, these ILF structures may provide means for the vacuole to control its membrane composition and even size. Third, the work of Yang et al. adds to the continually growing list of roles for ESCRTs in cellular homeostasis. ESCRTs now have established roles at endosomes, the plasma membrane, the nuclear envelope, and yeast vacuole. This versatile machinery is continually used to execute its topologically unique membrane remodeling. This is important to understand, as experiments that perturb or block ESCRT function likely impact multiple cellular processes and have pleiotropic effects.Given these insights, questions still remain. Although Yang et al. shed light on how the yeast vacuole consumes its proteins, how this system relates to mammalian lysosomes and how distinct resident proteins are selectively degraded or retained on the surface during metabolic cues remains to be explored. It is also unclear whether the ESCRT pathway, which utilizes several distinct complexes, operates completely the same on vacuoles as it does at other organelles. How ESCRTs function on vacuoles/lysosomes is of growing importance since ESCRTs have also been shown to facilitate micro-autophagy, delivering substrates such as lipid droplets into the vacuole lumen (9). These and other questions will no doubt drive further studies of the amazing yeast vacuole and mammalian lysosome, and how they organize and govern the lives of their many resident proteins.     

Genetic interactions with mutations affecting septin assembly reveal ESCRT functions in budding yeast cytokinesis

Membrane trafficking via targeted exocytosis to the Saccharomyces cerevisiae bud neck provides new membrane and membrane-associated factors that are critical for cytokinesis. It remains unknown whether yeast plasma membrane abscission, the final step of cytokinesis, occurs spontaneously following extensive vesicle fusion, as in plant cells, or requires dedicated membrane fission machinery, as in cultured mammalian cells. Components of the endosomal sorting complexes required for transport (ESCRT) pathway, or close relatives thereof, appear to participate in cytokinetic abscission in various cell types, but roles in cell division had not been documented in budding yeast, where ESCRTs were first characterized. By contrast, the septin family of filament-forming cytoskeletal proteins were first identified by their requirement for yeast cell division. We show here that mutations in ESCRT-encoding genes exacerbate the cytokinesis defects of cla4Δ or elm1Δ mutants, in which septin assembly is perturbed at an early stage in cell division, and alleviate phenotypes of cells carrying temperature-sensitive alleles of a septin-encoding gene, CDC10. Elevated chitin synthase II (Chs2) levels coupled with aberrant morphogenesis and chitin deposition in elm1Δ cells carrying ESCRT mutations suggest that ESCRTs normally enhance the efficiency of cell division by promoting timely endocytic turnover of key cytokinetic enzymes.