Multivesicular Body Sorting Pathway Research Articles

Monkey multi-organ cell atlas exposed to estrogen

Abstract Awareness of estrogen’s effects on health is broadening rapidly. The effects of long-term high levels of estrogen on the body involve multiple organs. Here, we used both single-cell chromatin accessibility and RNA sequencing data to analyze the potential effect of estrogen on major organs. The integrated cell map enabled in-depth dissection and comparison of molecular dynamics, cell-type compositions, and cellular heterogeneity across multiple tissues and organs under estrogen stimulation. We also inferred pseudotime cell trajectories and cell–cell communications to uncover key molecular signatures underlying their cellular processes in major organs in response to estrogen. For example, estrogen could induce the differentiation of IFIT3+ neutrophils into S100A9+ neutrophils involved in the function of endosome-to-lysosome transport and the multivesicular body sorting pathway in liver tissues. Furthermore, through integration with human genome-wide association study data, we further identified a subset of risk genes during disease development that were induced by estrogen, such as AKT1 (related to endometrial cancer), CCND1 (related to breast cancer), HSPH1 (related to colorectal cancer), and COVID-19 and asthma-related risk genes. Our work uncovers the impact of estrogen on the major organs, constitutes a useful resource, and reveals the contribution and mechanism of estrogen to related diseases.

Assessment of genotoxic chemicals using chemogenomic profiling based on gene-knockout library in Saccharomyces cerevisiae

Understanding the adverse effects of genotoxic chemicals and identifying them effectively from non-genotoxic chemicals are of great worldwide concerns. Here, Saccharomyces cerevisiae (yeast) genome-wide single-gene knockout screening approach was conducted to assess two genotoxic chemicals (4-nitroquinoline-1-oxide (4-NQO) and formaldehyde (FA)) and environmental pollutant dichloroacetic acid (DCA, genotoxicity is controversial). DNA repair was significant enriched in the gene ontology (GO) biology process (BP) terms and KEGG pathways when exposed to low concentrations of 4-NQO and FA. Higher concentrations of 4-NQO and FA influenced some RNA metabolic and biosynthesis pathways. Moreover, replication and repair associated pathways were top ranked KEGG pathways with high fold-change for low concentrations of 4-NQO and FA. The similar gene profiles perturbed by DCA with three test concentrations identified, the common GO BP terms associated with aromatic amino acid family biosynthetic process and ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway. DCA has no obvious genotoxicity as there was no enriched DNA damage and repair pathways and fold-change of replication and repair KEGG pathways were very low. Five genes (RAD18, RAD59, MUS81, MMS4, and BEM4) could serve as candidate genes for genotoxic chemicals. Overall, the yeast functional genomic profiling showed great performance for assessing the signatures and potential molecular mechanisms of genotoxic chemicals.

ESCRTing in cereals: still a long way to go.

The multivesicular body (MVB) sorting pathway provides a mechanism for the delivery of cargo destined for degradation to the vacuole or lysosome. The endosomal sorting complex required for transport (ESCRT) is essential for the MVB sorting pathway by driving the cargo sorting to its destination. Many efforts in plant research have identified the ESCRT machinery and functionally characterised the first plant ESCRT proteins. However, most studies have been performed in the model plant Arabidopsis thaliana that is genetically and physiologically different to crops. Cereal crops are important for animal feed and human nutrition and have further been utilized as promising candidates for recombinant protein production. In this review, I summarize the role of plant ESCRT components in cereals that are involved in efficient adaptation to environmental stress and grain development. A special focus is on barley (Hordeum vulgare L.) ESCRT proteins, where recent studies show their quantitative mapping during grain development, e.g. associating HvSNF7.1 with protein trafficking to protein bodies (PBs) in starchy endosperm. Thus, it is indispensable to identify the molecular key-players within the endomembrane system including ESCRT proteins to optimize and possibly enhance tolerance to environmental stress, grain yield and recombinant protein production in cereal grains.

ESCRT-III accessory proteins regulate fungal development and plant infection in Fusarium graminearum.

Ubiquitinated biosynthetic and surface proteins destined for degradation are sorted into the lysosome/vacuole via the multivesicular body sorting pathway, which depends on the function of ESCRT machinery. Fusarium head blight (FHB) caused by Fusarium graminearum is one of the most devastating diseases for wheat and barley worldwide. To better understand the role of ESCRT machinery in F. graminearum, we investigated the function of ESCRT-III accessory proteins FgVps60, FgDid2 and FgIst1 in this study. FgVps60-GFP, FgDid2-GFP and FgIst1-GFP are localized to punctate structures adjacent to the vacuolar membrane except for FgIst1-GFP that is also found in the nucleus. Then, the gene deletion mutants ΔFgvps60, ΔFgdid2 and ΔFgist1 displayed defective growth to a different extent. ΔFgvps60 and ΔFgdid2 but not ΔFgist1 also showed significant reduction in hydrophobicity on cell surface, conidiation, perithecia production and virulence. Interestingly, ΔFgist1 mutant produced a significantly higher level of DON while showing a minor reduction in pathogenicity. Microscopic analyses revealed that FgVps60 but not FgIst1 and FgDid2 is necessary for endocytosis. Moreover, spontaneous mutations were identified in the ΔFgvps60 mutant that partially rescued its defects in growth and conidiation. Taken together, we conclude that ESCRT-III accessory proteins play critical roles in growth, reproduction and plant infection in F. graminearum.

The regulation of Endosomal Sorting Complex Required for Transport and accessory proteins in multivesicular body sorting and enveloped viral budding - An overview.

ESCRT (Endosomal Sorting Complex Required for Transport) machinery drives different cellular processes such as endosomal sorting, organelle biogenesis, vesicular trafficking, maintenance of plasma membrane integrity, membrane fission during cytokinesis and enveloped virus budding. The normal cycle of assembly and disassembly of some ESCRT complexes at the membrane requires the AAA-ATPase vacuolar protein sorting 4 (Vps4p). A number of ESCRT proteins are hijacked by clinically significant enveloped viruses including Ebola, and Human Immunodeficiency Virus (HIV) to enable enveloped virus budding and Vps4p provides energy for the disassembly/recycling of these ESCRT proteins. Several years ago, the failure of the terminal budding process of HIV following Vps4 protein inhibition was published; although at that time a detailed understanding of the molecular players was missing. However, later it was acknowledged that the ESCRT machinery has a role in enveloped virus budding from cells due to its role in the multivesicular body (MVB) sorting pathway. The MVB sorting pathway facilitates several cellular activities in uninfected cells, such as the down-regulation of signaling through cell surface receptors as well as the process of viral budding from infected host cells. In this review, we focus on summarising the functional organisation of ESCRT proteins at the membrane and the role of ESCRT machinery and Vps4p during MVB sorting and enveloped viral budding.

NMR studies on the interactions between yeast Vta1 and Did2 during the multivesicular bodies sorting pathway.

As an AAA-ATPase, Vps4 is important for function of multivesicular bodies (MVB) sorting pathway, which involves in cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. The activity of Vps4 is stimulated by the interactions between Vta1 N-terminus (named as Vta1NTD) and Did2 fragment (176–204 aa) (termed as Did2176–204) or Vps60 (128–186 aa) (termed as Vps60128–186). The structural basis of how Vta1NTD binds to Did2176–204 is still unclear. To address this, in this report, the structure of Did2176–204 in complex with Vta1NTD was determined by NMR techniques, demonstrating that Did2176–204 interacts with Vta1NTD through its helix α6′ extending over the 2nd and the 3rd α-helices of Vta1NTD microtubule interacting and transport 1 (MIT1) domain. The residues within Did2176–204 helix α6′ in the interface make up of an amino acid sequence as E192′xxL195′xxR198′L199′xxL202′R203′, identical to type 1 MIT-interacting motif (MIM1) (D/E)xxLxxRLxxL(K/R) of CHMP1A180–196 observed in Vps4-CHMP1A complex structure, indicating that Did2 binds to Vta1NTD through canonical MIM1 interactions. Moreover, the Did2 binding does not result in Vta1NTD significant conformational changes, revealing that Did2, similar to Vps60, enhances Vta1 stimulation of Vps4 ATPase activity in an indirect manner.

Significantly Diverged Did2/Vps46 Orthologues from the Protozoan Parasite Giardia lamblia.

The endosomal compartment performs extensive sorting functions in most eukaryotes, some of which are accomplished with the help of the multivesicular body (MVB) sorting pathway. This pathway depends on the sequential action of complexes, termed the endosomal sorting complex required for transport (ESCRT). After successful sorting, the crucial step of recycling of the ESCRT complex components requires the activation of the AAA ATPase Vps4, and Did2/Vps46 plays an important role in this activation event. The endolysosomal system of the protozoan parasite Giardia lamblia appears to lack complexity, for instead of having distinct early endosomes, late endosomes and lysosomes, there are only peripheral vesicles (PVs) that are located close to the cell periphery. Additionally, comparative genomics studies predict the presence of only a subset of the ESCRT components in G. lamblia. Thus, it is possible that the MVB pathway is not functional in G. lamblia. To address this issue, the present study focused on the two putative orthologues of Did2/Vps46 of G. lamblia as their function is likely to be pivotal for a functional MVB sorting pathway. In spite of considerable sequence divergence, compared to other eukaryotic orthologues, the proteins encoded by both these genes have the ability to function as Did2/Vps46 in the context of the yeast ESCRT pathway. Furthermore, they also localized to the cellular periphery, where PVs are also located. Thus, this report is the first to provide experimental evidence indicating the presence of a functional ESCRT component in G. lamblia by characterizing the putative Did2/Vps46 orthologues.

Suppression of production of baboon endogenous virus by dominant negative mutants of cellular factors involved in multivesicular body sorting pathway

Baboon endogenous virus (BaEV) is an infectious endogenous gammaretrovirus isolated from a baboon placenta. BaEV-related sequences have been identified in both Old World monkeys and African apes, but not in humans or Asian apes. Recently, it was reported that BaEV-like particles were produced from Vero cells derived from African green monkeys by chemical induction, and thus BaEV-like particles may contaminate biological products manufactured using Vero cells. In this study, we constructed an infectious molecular clone of BaEV strain M7. We found two putative L-domain motifs, PPPY and PSAP, in the pp15 region of Gag. To examine the function of the L-domain motifs, we conducted virus budding assay using L-domain motif mutants. We revealed that the PPPY motif, but not the PSAP motif, plays a major role as the L-domain in BaEV budding. We also demonstrated that Vps4A/B are involved in BaEV budding. These data suggest that BaEV Gag recruits the cellular endosomal sorting complex required for transport (ESCRT) machinery through the interaction of the PPPY L-domain with cellular factors. These data will be useful for controlling contamination of BaEV-like particles in biological products in the future.

Molecular characterization of koala retroviruses isolated from koalas (Phascolarctos cinereus) reared in Japanese zoos

In northern Australia most koalas (Phascolarctos cinereus) are infected with the gammaretrovirus known as koala retrovirus (KoRV). KoRV is believed to be currently endogenizing into its host. Koalas were first introduced into three Japanese zoos in 1984 and now about 50 koalas are held in eight zoos. In 2007 KoRV was isolated from koalas reared in Japanese zoos, and, for the first time, an infectious molecular clone termed pKoRV522 was constructed. Using the molecular clone and KoRV isolates, we revealed the budding mechanism of KoRV and genomic diversity of KoRVs isolated from Japanese koalas. We found that KoRV utilizes the multivesicular body-sorting pathway. We also discovered a novel KoRV subgroup, named KoRV-J, which utilizes thiamine transport protein 1 as an entry receptor. The original KoRV, which utilizes Pit-1 as an entry receptor, is now named KoRV-A. In two Queensland koalas examined, the copy numbers of KoRV-J was less than 1 copy per cell and varied in tissues. These data, at least in these two koalas, suggest that KoRV-J is an exogenous retrovirus not an endogenous retrovirus. Miyazawa, Takayuki. 2014. Molecular characterization of koala retroviruses isolated from koalas (Phascolarctos cinereus) reared in Japanese zoos. In The Koala and its Retroviruses: Implications for Sustainability and Survival, ed. Geoffrey W. Pye, Rebecca N. Johnson and Alex D. Greenwood. Technical Reports of the Australian Museum,

CAMK1 phosphoinositide signal-mediated protein sorting and transport network in human hepatocellular carcinoma (HCC) by biocomputation.

We data-analyzed and constructed the high-expression CAMK1 phosphoinositide signal-mediated protein sorting and transport network in human hepatocellular carcinoma (HCC) compared with low-expression (fold change ≥ 2) no-tumor hepatitis/cirrhotic tissues (HBV or HCV infection) in GEO data set, using integration of gene regulatory network inference method with gene ontology (GO). Our result showed that CAMK1 transport subnetwork upstream KCNQ3, LCN2, NKX2_5, NUP62, SORT1, STX1A activated CAMK1, and downstream CAMK1-activated AFP, ENAH, KPNA2, SLC4A3; CAMK1 signal subnetwork upstream BRCA1, DKK1, GPSM2, LEF1, NR5A1, NUP62, SORT1, SSTR5, TBL3 activated CAMK1, and downstream CAMK1-activated MAP2K6, SFRP4, SSTR5, TSHB, UBE2C in HCC. We proposed that CAMK1 activated network enhanced endosome to lysosome transport, endosome transport via multivesicular body sorting pathway, Golgi to endosome transport, intracellular protein transmembrane transport, intracellular protein transport, ion transport, mRNA transport, plasma membrane to endosome transport, potassium ion transport, protein transport, vesicle-mediated transport, anion transport, intracellular transport, androgen receptor signaling pathway, cell surface receptor-linked signal transduction, hormone-mediated signaling, induction of apoptosis by extracellular signals, signal transduction by p53 class mediator resulting in transcription of p21 class mediator, signal transduction resulting in induction of apoptosis, phosphoinositide-mediated signaling, Wnt receptor signaling pathway, as a result of inducing phosphoinositide signal-mediated protein sorting, and transport in HCC. Our hypothesis was verified by CAMK1 functional regulation subnetwork containing positive regulation of calcium ion transport via voltage gated calcium channel, cell proliferation, DNA repair, exocytosis, I-kappaB kinase/NF-kappaB cascade, immunoglobulin-mediated immune response, mast cell activation, natural killer cell-mediated cytotoxicity directed against tumor cell target, protein ubiquitination, sodium ion transport, survival gene product activity, T cell-mediated cytotoxicity, transcription, transcription from RNA polymerase II promoter, transcription initiation from RNA polymerase II promoter, transcription via serum response element binding, exit from mitosis, ubiquitin ligase activity during mitotic cell cycle, regulation of angiogenesis, apoptosis, cell growth, cell proliferation, cyclin-dependent protein kinase activity, gene expression, insulin secretion, steroid biosynthesis, transcription from RNA polymerase II promoter, transcription from RNA polymerase III promoter, cell cycle, cell migration, DNA recombination, and protein metabolism; also by CAMK1 negative functional regulation subnetwork including negative regulation of apoptosis, cell proliferation, centriole replication, fatty acid biosynthesis, lipoprotein lipase activity, MAPK activity, progression through cell cycle, transcription, transcription from RNA polymerase II promoter, cell growth, phosphorylation, and ubiquitin ligase activity during mitotic cell cycle in HCC.

Identification of cellular factors required for the budding of koala retrovirus

Koala retrovirus (KoRV) is a unique gammaretrovirus that is currently endogenizing into its host and considered to be associated with leukemia, lymphoma and immunosuppression in koalas (Phascolactos cinereus). In this study, it was demonstrated that WWP2 or WWP2-like E3 ubiquitin ligases possessing the WW domain closely related to WWP2 and Vps4A/B are involved in KoRV budding. These data suggest that KoRV Gag recruits the cellular endosomal sorting complex required for transport machinery through interaction of the PPPY L-domain with the WW domain(s) of WWP2 and that progeny virions are released from cells by utilizing the multivesicular body sorting pathway.

Structural Basis of Molecular Recognition between ESCRT-III-like Protein Vps60 and AAA-ATPase Regulator Vta1 in the Multivesicular Body Pathway

The AAA-ATPase Vps4 is critical for function of the multivesicular body sorting pathway, which impacts cellular phenomena ranging from receptor down-regulation to viral budding to cytokinesis. Vps4 activity is stimulated by the interaction between Vta1 and Vps60, but the structural basis for this interaction is unclear. The fragment Vps60(128-186) was reported to display the full activity of Vps60. Vta1 interacts with Vps60 using its N-terminal domain (Vta1NTD). In this work, the structure of Vps60(128-186) in complex with Vta1NTD was determined using NMR techniques, demonstrating a novel recognition mode of the microtubule-interacting and transport (MIT) domain in which Vps60(128-186) interacts with Vta1NTD through helices α4' and α5', extending over Vta1NTD MIT2 domain helices 1-3. The Vps60 binding does not result in Vta1 conformational changes, further revealing the fact that Vps4 ATPase is enhanced by the interaction between Vta1 and Vps60 in an unanticipated manner.

Viral and cellular requirements for the budding of Feline Endogenous Retrovirus RD-114

BackgroundRD-114 virus is a feline endogenous retrovirus and produced as infectious viruses in some feline cell lines. Recently, we reported the contamination of an infectious RD-114 virus in a proportion of live attenuated vaccines for dogs and cats. It is very difficult to completely knock out the RD-114 proviruses from cells, as endogenous retroviruses are usually integrated multiply into the host genome. However, it may be possible to reduce the risk of contamination of RD-114 virus by regulating the viral release from cells.ResultsIn this study, to understand the molecular mechanism of RD-114 virus budding, we attempted to identify the viral and cellular requirements for RD-114 virus budding. Analyses of RD-114 L-domain mutants showed that the PPPY sequence in the pp15 region of Gag plays a critical role in RD-114 virus release as viral L-domain. Furthermore, we investigated the cellular factors required for RD-114 virus budding. We demonstrated that RD-114 virus release was inhibited by overexpression of dominant negative mutants of Vps4A, Vps4B, and WWP2.ConclusionsThese results strongly suggest that RD-114 budding utilizes the cellular multivesicular body sorting pathway similar to many other retroviruses.

Marburg Virus Budding: ESCRT of Progeny Virion to the Outside of the Cell

The major virion matrix protein of the Marburg virus (MARV), VP40, plays a key role in MARV assembly and budding, and its sole expression can produce enveloped virus-like particles. VP40 possesses only the PPXY motif as an L-domain critical for efficient virus budding, and interacts with the cellular ubiquitin ligase Nedd4. Functional abrogation of the cellular components of the endosomal sorting complexes required for transport complexes that participate in budding of multivesicular bodies into late endosomes by dominant-negative mutants or siRNA inhibited virus-like particle release, suggest that MARV budding utilizes the multivesicular bodies sorting pathway. In addition, tetherin/BST-2 was recently identified as an antiviral cellular factor that reduces MARV virus-like particle production. These findings may contribute to development of novel anti-MARV therapeutic strategies.

The ESCRT-Related CHMP1A and B Proteins Mediate Multivesicular Body Sorting of Auxin Carriers inArabidopsisand Are Required for Plant Development

Plasma membrane proteins internalized by endocytosis and targeted for degradation are sorted into lumenal vesicles of multivesicular bodies (MVBs) by the endosomal sorting complexes required for transport (ESCRT) machinery. Here, we show that the Arabidopsis thaliana ESCRT-related CHARGED MULTIVESICULAR BODY PROTEIN/CHROMATIN MODIFYING PROTEIN1A (CHMP1A) and CHMP1B proteins are essential for embryo and seedling development. Double homozygous chmp1a chmp1b mutant embryos showed limited polar differentiation and failed to establish bilateral symmetry. Mutant seedlings show disorganized apical meristems and rudimentary true leaves with clustered stomata and abnormal vein patterns. Mutant embryos failed to establish normal auxin gradients. Three proteins involved in auxin transport, PINFORMED1 (PIN1), PIN2, and AUXIN-RESISTANT1 (AUX1) mislocalized to the vacuolar membrane of the mutant. PIN1 was detected in MVB lumenal vesicles of control cells but remained in the limiting membrane of chmp1a chmp1b MVBs. The chmp1a chmp1b mutant forms significantly fewer MVB lumenal vesicles than the wild type. Furthermore, CHMP1A interacts in vitro with the ESCRT-related proteins At SKD1 and At LIP5. Thus, Arabidopsis CHMP1A and B are ESCRT-related proteins with conserved endosomal functions, and the auxin carriers PIN1, PIN2, and AUX1 are ESCRT cargo proteins in the MVB sorting pathway.

Regulation of Vps4 ATPase activity by ESCRT-III

MVB (multivesicular body) formation occurs when the limiting membrane of an endosome invaginates into the intraluminal space and buds into the lumen, bringing with it a subset of transmembrane cargoes. Exvagination of the endosomal membrane from the cytosol is topologically similar to the budding of retroviral particles and cytokinesis, wherein membranes bud away from the cytoplasm, and the machinery responsible for MVB sorting has been implicated in these phenomena. The AAA (ATPase associated with various cellular activities) Vps4 (vacuolar protein sorting 4) performs a critical function in the MVB sorting pathway. Vps4 appears to dissociate the ESCRTs (endosomal sorting complexes required for transport) from endosomal membranes during the course of MVB sorting, but it is unclear how Vps4 ATPase activity is synchronized with ESCRT release. We have investigated the mechanisms by which ESCRT components stimulate the ATPase activity of Vps4. These studies support a model wherein Vps4 activity is subject to spatial and temporal regulation via distinct mechanisms during MVB sorting.

The YPLGVG sequence of the Nipah virus matrix protein is required for budding

BackgroundNipah virus (NiV) is a recently emerged paramyxovirus capable of causing fatal disease in a broad range of mammalian hosts, including humans. Together with Hendra virus (HeV), they comprise the genus Henipavirus in the family Paramyxoviridae. Recombinant expression systems have played a crucial role in studying the cell biology of these Biosafety Level-4 restricted viruses. Henipavirus assembly and budding occurs at the plasma membrane, although the details of this process remain poorly understood. Multivesicular body (MVB) proteins have been found to play a role in the budding of several enveloped viruses, including some paramyxoviruses, and the recruitment of MVB proteins by viral proteins possessing late budding domains (L-domains) has become an important concept in the viral budding process. Previously we developed a system for producing NiV virus-like particles (VLPs) and demonstrated that the matrix (M) protein possessed an intrinsic budding ability and played a major role in assembly. Here, we have used this system to further explore the budding process by analyzing elements within the M protein that are critical for particle release.ResultsUsing rationally targeted site-directed mutagenesis we show that a NiV M sequence YPLGVG is required for M budding and that mutation or deletion of the sequence abrogates budding ability. Replacement of the native and overlapping Ebola VP40 L-domains with the NiV sequence failed to rescue VP40 budding; however, it did induce the cellular morphology of extensive filamentous projection consistent with wild-type VP40-expressing cells. Cells expressing wild-type NiV M also displayed this morphology, which was dependent on the YPLGVG sequence, and deletion of the sequence also resulted in nuclear localization of M. Dominant-negative VPS4 proteins had no effect on NiV M budding, suggesting that unlike other viruses such as Ebola, NiV M accomplishes budding independent of MVB cellular proteins.ConclusionThese data indicate that the YPLGVG motif within the NiV M protein plays an important role in M budding; however, involvement of any specific components of the cellular MVB sorting pathway in henipavirus budding remains to be demonstrated. Further investigation of henipavirus assembly and budding may yet reveal a novel mechanism(s) of viral assembly and release that could be applicable to other enveloped viruses or have therapeutic implications.

Hepatitis B Virus Maturation Is Sensitive to Functional Inhibition of ESCRT-III, Vps4, and γ2-Adaptin

Hepatitis B virus (HBV) is an enveloped DNA virus that presumably buds at intracellular membranes of infected cells. HBV budding involves two endocytic host proteins, the ubiquitin-interacting adaptor gamma 2-adaptin and the Nedd4 ubiquitin ligase. Here, we demonstrate that HBV release also requires the cellular machinery that generates internal vesicles of multivesicular bodies (MVBs). In order to perturb the MVB machinery in HBV-replicating liver cells, we used ectopic expression of dominant-negative mutants of different MVB components, like the ESCRT-III complex-forming CHMP proteins and the Vps4 ATPases. Upon coexpression of mutated CHMP3, CHMP4B, or CHMP4C forms, as well as of ATPase-defective Vps4A or Vps4B mutants, HBV assembly and egress were potently blocked. Each of the MVB inhibitors arrested virus particle maturation by entrapping the viral core and large and small envelope proteins in detergent-insoluble membrane structures that closely resembled aberrant endosomal class E compartments. In contrast, HBV subvirus particle release was not affected by MVB inhibitors, hinting at different export routes used by viral and subviral particles. To further define the role gamma 2-adaptin plays in HBV formation, we examined the effects of its overexpression in virus-replicating cells. Intriguingly, excess gamma 2-adaptin blocked HBV production in a manner similar to the actions of CHMP and Vps4 mutants. Moreover, overexpressed gamma 2-adaptin perturbed the endosomal morphology and diminished the budding of a retroviral Gag protein, implying that it may act as a principal inhibitor of the MVB sorting pathway. Together, these results demonstrate that HBV exploits the MVB machinery with the aid of gamma 2-adaptin.

Split-ubiquitin two-hybrid assay to analyze protein-protein interactions at the endosome: application to Saccharomyces cerevisiae Bro1 interacting with ESCRT complexes, the Doa4 ubiquitin hydrolase, and the Rsp5 ubiquitin ligase.

Targeting of membrane proteins into the lysosomal/vacuolar lumen for degradation requires their prior sorting into multivesicular bodies (MVB). The MVB sorting pathway depends on ESCRT-0, -I, -II, and -III protein complexes functioning on the endosomal membrane and on additional factors, such as Bro1/Alix and the ubiquitin ligase Rsp5/Nedd4. We used the split-ubiquitin two-hybrid assay to analyze the interaction partners of yeast Bro1 at its natural cellular location. We show that Bro1 interacts with ESCRT-I and -III components, including Vps23, the Saccharomyces cerevisiae homologue of human Tsg101. These interactions do not require the C-terminal proline-rich domain (PRD) of Bro1. Rather, this PRD interacts with the Doa4 deubiquitinating enzyme to recruit it to the endosome. This interaction is disrupted by a single amino acid substitution in the conserved ELC box motif in Doa4. The PRD of Bro1 also mediates an association with Rsp5, and this interaction appears to be conserved, as Alix, the human homologue of Bro1, coimmunoprecipitates with Nedd4 in yeast lysates. We further show that the Bro1 PRD domain is essential to MVB sorting of only cargo proteins whose sorting to the vacuolar lumen is dependent on their own ubiquitination and Doa4. The Bro1 region preceding the PRD, however, is required for MVB sorting of proteins irrespective of whether their targeting to the vacuole is dependent on their ubiquitination and Doa4. Our data indicate that Bro1 interacts with several ESCRT components and contributes via its PRD to associating ubiquitinating and deubiquitinating enzymes with the MVB sorting machinery.

Direct Binding to Rsp5p Regulates Ubiquitination-independent Vacuolar Transport of Sna3p

The sorting of integral membrane proteins such as carboxypeptidase S (Cps1p) into the luminal vesicles of multivesicular bodies (MVBs) in Saccharomyces cerevisiae requires ubiquitination of their cytosolic domains by the ubiquitin ligases Rsp5p and/or Tul1p. An exception is Sna3p, which does not require ubiquitination for entry into MVBs. The mechanism underlying this ubiquitination-independent MVB sorting pathway has not yet been characterized. Here, we show that Sna3p sorting into the MVB pathway depends on a direct interaction between a PPAY motif within its C-terminal cytosolic tail and the WW domains of Rsp5p. Disruption of this interaction inhibits vacuolar targeting of Sna3p and causes its accumulation in a compartment that overlaps only partially with MVBs. Surprisingly, Sna3p does require a functional ubiquitin-ligase HECT domain within Rsp5p; however, the dependence of Sna3p on HECT domain activity is distinct from that of Cps1p. Last, we show that Sna3p requires neither Tul1p nor the transmembrane adaptor protein Bsd2p for its MVB sorting. Our data demonstrate that Sna3p follows a novel ubiquitination-independent, but Rsp5p-mediated, sorting pathway to the vacuole.