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49-708: The ESCRT machinery plays a vital role in a number of cellular processes including multivesicular body (MVB) biogenesis, cellular abscission , and viral budding . Multivesicular body (MVB) biogenesis is a process in which ubiquitin -tagged proteins enter organelles called endosomes via the formation of vesicles . This process is essential for cells to destroy misfolded and damaged proteins. Without ESCRT machinery, these proteins can build up and lead to neurodegenerative disease. For example, abnormalities in ESCRT-III components can lead to neurological disorders such as hereditary spastic paraplegia (HSP). Cellular abscission,

98-430: A cell and exit from the opposite side. Also, in some circumstances, late endosomes/MVBs fuse with the plasma membrane instead of with lysosomes, releasing the lumenal vesicles, now called exosomes , into the extracellular medium. There is no consensus as to the exact nature of these pathways, and the sequential route taken by any given cargo in any given situation will tend to be a matter of debate. Vesicles pass between

147-498: A major sorting compartment of the endomembrane system in cells. Endosomes provide an environment for material to be sorted before it reaches the degradative lysosome. For example, low-density lipoprotein (LDL) is taken into the cell by binding to the LDL receptor at the cell surface. Upon reaching early endosomes, the LDL dissociates from the receptor, and the receptor can be recycled to

196-634: A nucleator of Snf7 polymer assembly. Vps24 then associates with Snf7 to cap the complex and recruit Vps2. Vps2 then brings Vps4 to the complex. All “free” cytosolic forms of each subunit are considered closed. That is, the carboxy-terminal portion of each subunit folds up onto itself in an autoinhibitory manner stabilizing the monomeric subunits. The carboxy-terminus of most ESCRT-III subunits, both essential and nonessential, contain MIMs ( M IT ( microtubule interacting and transport domain) i nteracting m otif) motifs. These motifs are responsible for binding Vps4 and

245-422: A role in all ESCRT mediated processes. During membrane abscission and viral budding, ESCRT-III forms long filaments that coil around the site of membrane constriction just prior to membrane cleavage. This mediation of abscission occurs through interactions with the centralspindlin complex. These filamentous structures are also present during multivesicular body formation and function as a ring-like fence that plugs

294-413: A role in membrane recognition and remodeling during membrane abscission by forming rings on either side of the midbody of dividing cells. ESCRT-I is also responsible for recruiting ESCRT-III, which forms the constriction zone just before the cells separate. Furthermore, ESCRT-I plays a role in viral budding by interacting with specific viral proteins, leading to recruitment of additional ESCRT machinery to

343-481: A spiral-shaped fibril adjacent to the rings formed by Vps23. The formation of this spiral-like structure deforms the membrane and the AAA-ATPase spastin is brought in by Did2 and Ist1 to cleave the microtubules formed at the midbody. Vps4 then catalyzes the disassembly of the ESCRT-III complex resulting in two newly separated daughter cells. The process of membrane abscission was described using metazoan proteins as

392-776: Is a protein that in humans is encoded by the VPS28 gene . This gene encodes a protein involved in endosomal sorting of cell surface receptors via a multivesicular body/late endosome pathway. The encoded protein is one of the three subunits of the ESCRT-I complex (endosomal complexes required for transport) involved in the sorting of ubiquitinated protein . The two other subunits of ESCRT-I are vacuolar protein sorting 23 (VPS23), also known as tumor susceptibility gene 101 (TSG101), and vacuolar protein sorting 37 (VPS37). Two alternative transcripts encoding different isoforms have been described. Additional alternative transcripts may exist but

441-406: Is a result of the concerted action of phosphoinositide kinases and phosphatases that are strategically localized There are three main compartments that have pathways that connect with endosomes. More pathways exist in specialized cells, such as melanocytes and polarized cells. For example, in epithelial cells, a special process called transcytosis allows some materials to enter one side of

490-509: Is as follows: Vps4 subunits have two functional domains, an amino-terminal MIT domain and a central AAA-ATPase domain. The MIT domain is responsible for the interaction of Vps4 with the MIM domain of Vps2. The AAA-ATPase domain hydrolyzes ATP to power disassembly of the ESCRT-III complex. This “stripping” of ESCRT-III allows all associated subunits to be recycled for further use. Vta1 is a dimeric protein containing one VSL domain (so named because it

539-400: Is contained in V ps27, H RS, and S TAM proteins). These VHS domains bind the ubiquitin on proteins the cell aims to degrade. Ubiquitin can also associate with ubiquitin interacting motifs such as the one on Hse1 or the double sided domain found on Vps27. A FYVE domain (named after the four proteins in which it was initially identified: Fab1p, YOTB, Vac1, and EEA1) is found sandwiched between

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588-574: Is found in the proteins V ps4, S BP1, and L IP5), which enables binding to Vps4, and a MIT domain for associating with ESCRT-III subunit Vps60. Though not essential, Vta1 has been shown to aid in Vps4 ring assembly, accelerate the ATPase activity of Vsp4, and encourage ESCRT-III disassembly. The main function of Bro1 is to recruit deubiquitinases to the ESCRT-III complex. This results in the removal of ubiquitin tags from proteins targeted for degradation in

637-557: Is responsible for the binding of ubiquitin, the ESCRT-0 complex, and to the PTAP ( p roline , t hreonine , a lanine , p roline) motif of viral Gag proteins . Just after this ubiquitin E2 variant domain, a proline rich motif (GPPX 3 Y) is present that directs ESCRT-I to the midbody during membrane abscission. Mvb12 can also bind ubiquitin via its carboxy-terminus . Vps28 is responsible for

686-545: The AAA-ATPase spastin . The Vps4-Vta1 proteins are required for the stripping of other ESCRT components (usually ESCRT-III) from membranes once a particular process has been completed. There is some debate as to whether Vps4 cleaves the ESCRT-III complex away or remodels the complex so one component is shed at a particular time. Vta1 is thought to act as an activator of Vps4, aiding its assembly and enhancing its AAA-ATPase activity. The manner in which these proteins function

735-570: The EGF receptor (EGFR) once it is endocytosed to endosomes. The activated EGFRs stimulate their own ubiquitination, and this directs them to lumenal vesicles (see below) and so they are not recycled to the plasma membrane. This removes the signaling portion of the protein from the cytosol and thus prevents continued stimulation of growth - in cells not stimulated with EGF, EGFRs have no EGF bound to them and therefore recycle if they reach endosomes. Transferrin also remains associated with its receptor, but, in

784-579: The centrosomal protein Cep55 is recruited to the midbody of dividing cells in association with MKLP1, a mitotic kinesin -like protein that associates with microtubules. Cep55 then recruits the Vps23 subunit of ESCRT-I and accessory protein ALIX, which form into rings on either side of the midbody. ESCRT-I and ALIX recruit ESCRT-III via its Snf7 subunit. ESCRT-III subunits Vps20, Snf7, Vps24, Vps2, and Did2 form into

833-551: The trans Golgi network . Molecules or ligands internalized from the plasma membrane can follow this pathway all the way to lysosomes for degradation or can be recycled back to the cell membrane in the endocytic cycle . Molecules are also transported to endosomes from the trans Golgi network and either continue to lysosomes or recycle back to the Golgi apparatus . Endosomes can be classified as early, sorting, or late depending on their stage post internalization. Endosomes represent

882-399: The ESCRT-III components to the cytosol and the virus is released from the cell. The mechanism described here utilizes metazoan proteins, as viral budding has been studied more extensively in metazoans. Endosome#Types Endosomes are a collection of intracellular sorting organelles in eukaryotic cells . They are parts of the endocytic membrane transport pathway originating from

931-542: The Golgi and endosomes in both directions. The GGAs and AP-1 clathrin-coated vesicle adaptors make vesicles at the Golgi that carry molecules to endosomes. In the opposite direction, retromer generates vesicles at early endosomes that carry molecules back to the Golgi. Some studies describe a retrograde traffic pathway from late endosomes to the Golgi that is mediated by Rab9 and TIP47 , but other studies dispute these findings. Molecules that follow these pathways include

980-592: The Golgi destined for the lysosome by a similar mechanism. There are three different types of endosomes: early endosomes , late endosomes , and recycling endosomes . They are distinguished by the time it takes for endocytosed material to reach them, and by markers such as Rabs . They also have different morphology. Once endocytic vesicles have uncoated, they fuse with early endosomes. Early endosomes then mature into late endosomes before fusing with lysosomes. Early endosomes mature in several ways to form late endosomes. They become increasingly acidic mainly through

1029-514: The VHS and ubiquitin interacting motif domains of Vps27. Phosphatidylinositol 3-phosphate , a common endosomal lipid, binds to this FYVE domain resulting in the recruitment of ESCRT-0 to the endosome. The role of the ESCRT-I complex is to assist in the generation of multivesicular bodies by clustering ubiquitinated proteins and acting as a bridge between the ESCRT-0 and ESCRT-II complexes. It also plays

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1078-457: The absence of ESCRT machinery. This would inevitably prevent viruses from spreading from cell to cell. Each of the ESCRT complexes and accessory proteins have unique structures that enable distinct biochemical functions. A number of synonyms exist for each protein component of the ESCRT machinery, both for yeast and metazoans . A summary table of all of these proteins is provided below. In yeast,

1127-449: The acidic endosome, iron is released from the transferrin, and then the iron-free transferrin (still bound to the transferrin receptor) returns from the early endosome to the cell surface, both directly and via recycling endosomes. Transport from late endosomes to lysosomes is, in essence, unidirectional, since a late endosome is "consumed" in the process of fusing with a lysosome (sometimes called endolysosome ).Hence, soluble molecules in

1176-469: The activity of the V-ATPase. Many molecules that are recycled are removed by concentration in the tubular regions of early endosomes. Loss of these tubules to recycling pathways means that late endosomes mostly lack tubules. They also increase in size due to the homotypic fusion of early endosomes into larger vesicles. Molecules are also sorted into smaller vesicles that bud from the perimeter membrane into

1225-508: The budding vesicle to prevent cargo proteins from escaping into the cell's cytosol. ESCRT-III exists and functions as follows: The ESCRT-III complex differs from all other ESCRT machinery in that it exists only transiently and contains both essential and nonessential components. The essential subunits must assemble in the proper order (Vps20, Snf7, Vps24 , then Vps2) for the machinery to function. Nonessential subunits include Vps60, Did2, and Ist1. Vps20 initiates assembly of ESCRT-III by acting as

1274-546: The cargo containing vesicle closed. The specific aspects of ESCRT-II are as follows: ESCRT-II is a heterotetramer (2:1:1) composed of two Vps25 subunits, one Vps22 , and one Vps36 subunit. Vps25 molecules contain PPXY motifs, which bind to winged-helix (WH) motifs of Vps22 and Vps36 creating a Y-shaped complex with Vps22 and Vps36 as the base and Vps25 molecules as arms. Vps25 molecules also contain WH motifs that are responsible for

1323-513: The cell surface. The LDL remains in the endosome and is delivered to lysosomes for processing. LDL dissociates because of the slightly acidified environment of the early endosome, generated by a vacuolar membrane proton pump V-ATPase . On the other hand, epidermal growth factor (EGF) and the EGF receptor have a pH-resistant bond that persists until it is delivered to lysosomes for their degradation. The mannose 6-phosphate receptor carries ligands from

1372-456: The endosome is thought to be enhanced by the peculiar lipid BMP or LBPA, which is only found in late endosomes, endolysosomes or lysosomes. When the endosome has matured into a late endosome/MVB and fuses with a lysosome, the vesicles in the lumen are delivered to the lysosome lumen. Proteins are marked for this pathway by the addition of ubiquitin . The endosomal sorting complexes required for transport (ESCRTs) recognise this ubiquitin and sort

1421-401: The endosome lumen, forming intraluminal vesicles (ILVs); this leads to the multivesicular appearance of late endosomes and so they are also known as multivesicular endosomes or multivesicular bodies (MVBs). Removal of recycling molecules such as transferrin receptors and mannose 6-phosphate receptors continues during this period, probably via budding of vesicles out of endosomes. Finally,

1470-611: The endosome via vesicles, forming multivesicular bodies, and are eventually delivered to the lysosome where they are degraded. This process is essential as it is the major pathway for the degradation of damaged proteins that have passed through the Golgi . The components of the ESCRT-0 complex exist as follows: The complex is a 1:1 heterodimer of Vps27 ( vacuolar protein sorting protein 27) and Hse1 . Vps27 and Hse1 dimerize through antiparallel coiled-coil GAT (so named after proteins GGA and Tom1) domains. Both Vps27 and Hse1 contain an amino-terminal VHS domain (so named because it

1519-512: The endosomes lose RAB5A and acquire RAB7A , making them competent for fusion with lysosomes. Fusion of late endosomes with lysosomes has been shown to result in the formation of a 'hybrid' compartment, with characteristics intermediate of the two source compartments. For example, lysosomes are more dense than late endosomes, and the hybrids have an intermediate density. Lysosomes reform by recondensation to their normal, higher density. However, before this happens, more late endosomes may fuse with

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1568-438: The following complexes/accessory proteins exist as follows: The ESCRT-0 complex plays a vital role in the generation of multivesicular bodies by binding and clustering ubiquitinated proteins and/or receptors on the surface of a cell. The complex is then responsible for binding to a lipid on the endosomal membrane, which recruits these tagged proteins to the endosome. Once properly localized , these proteins are then taken into

1617-442: The host cell. The process is initiated by viral Gag proteins, the major structural proteins of retroviral coats, which interact with TSG101 of the ESCRT-I complex and the ALIX accessory protein. ESCRT-III subunits (only CHMP4 and CHMP2 being essential) are recruited to the site of viral budding to constrict and sever the neck of the bud in a manner similar to that described for membrane abscission during cytokinesis. Vps4 then recycles

1666-567: The hybrid. Some material recycles to the plasma membrane directly from early endosomes, but most traffics via recycling endosomes. More subtypes exist in specialized cells such as polarized cells and macrophages . Phagosomes , macropinosomes and autophagosomes mature in a manner similar to endosomes, and may require fusion with normal endosomes for their maturation. Some intracellular pathogens subvert this process, for example, by preventing RAB7 acquisition. Late endosomes/MVBs are sometimes called endocytic carrier vesicles , but this term

1715-521: The interaction of ESCRT-I and ESCRT-II by associating with the GLUE domain ( G RAM- L ike U biquitin-binding in E AP45) of Vps36 through its carboxy-terminal four-helix bundle domain. The ESCRT-II complex functions primarily during the biogenesis of multivesicular bodies and delivery of ubiquitin tagged proteins to the endosome. Ubiquitin tagged proteins are passed from ESCRT-0 to ESCRT-I and then to ESCRT-II. ESCRT-II associates with ESCRT-III, which pinches

1764-426: The interaction of ESCRT-II with ESCRT-III. Vps36 contains a GLUE domain that binds phosphatidylinositol 3-phosphate and Vps28 of ESCRT-I. Two zinc finger domains are looped into the GLUE domain of yeast Vps36. One of these zinc finger domains binds the carboxy-terminal domain of Vps28 and the other associates with ubiquitin. The ESCRT-III complex is likely the most important of all the ESCRT machinery because it plays

1813-548: The iron transport protein transferrin. Internalization of these receptors from the plasma membrane occurs by receptor-mediated endocytosis. LDL is released in endosomes because of the lower pH, and the receptor is recycled to the cell surface. Cholesterol is carried in the blood primarily by (LDL), and transport by the LDL receptor is the main mechanism by which cholesterol is taken up by cells. EGFRs are activated when EGF binds. The activated receptors stimulate their own internalization and degradation in lysosomes. EGF remains bound to

1862-409: The lumen of endosomes will tend to end up in lysosomes, unless they are retrieved in some way. Transmembrane proteins can be delivered to the perimeter membrane or the lumen of lysosomes. Transmembrane proteins destined for the lysosome lumen are sorted into the vesicles that bud from the perimeter membrane into endosomes, a process that begins in early endosomes. The process of creating vesicles within

1911-439: The lysosome causing degradation of the cargo. A more in-depth description of the process, including associated machinery, exists as follows: Membrane abscission during cytokinesis is the process by which the membrane connecting two daughter cells is cleaved during cell division . Since it is conserved in a number of archaea , membrane abscission is considered to be the earliest role for ESCRT machinery. The process begins when

1960-428: The lysosome just prior to the generation of multivesicular bodies. It has also been speculated that Bro1 helps stabilize ESCRT-III while ubiquitin tags are cleaved from cargo proteins. Bro1 contains a Bro1 amino-terminal domain that binds to Snf7 of ESCRT-III. This binding brings Bro1 to the site of membrane abscission. Bro1 also binds the catalytic domain of Doa4, an ubiquitin hydrolase (deubiquitinase), bringing it to

2009-453: The mannose-6-phosphate receptors that carry lysosomal hydrolases to the endocytic pathway. The hydrolases are released in the acidic environment of endosomes, and the receptor is retrieved to the Golgi by retromer and Rab9. Molecules are delivered from the plasma membrane to early endosomes in endocytic vesicles. Molecules can be internalized via receptor-mediated endocytosis in clathrin -coated vesicles. Other types of vesicles also form at

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2058-443: The most important lipid signaling molecules, is found to differ as the endosomes mature from early to late. PI(4,5)P 2 is present on plasma membranes , PI(3)P on early endosomes, PI(3,5)P 2 on late endosomes and PI(4)P on the trans Golgi network . These lipids on the surface of the endosomes help in the specific recruitment of proteins from the cytosol, thus providing them an identity. The inter-conversion of these lipids

2107-440: The plasma membrane for this pathway, including ones utilising caveolin . Vesicles also transport molecules directly back to the plasma membrane, but many molecules are transported in vesicles that first fuse with recycling endosomes. Molecules following this recycling pathway are concentrated in the tubules of early endosomes. Molecules that follow these pathways include the receptors for LDL , epidermal growth factor (EGF), and

2156-406: The potential site of viral release. Details of the ESCRT-I machinery are described below. The ESCRT-I complex is a heterotetramer (1:1:1:1) of Vps23, Vps28 , Vps37, and Mvb12. The assembled heterotetramer appears as a rod-shaped stalk composed of Vps23, Vps37, and Mvb12 with a fanned cap composed of single helices of Vps23, Vps28, and Vps37. Vps23 contains one ubiquitin E2 variant domain, which

2205-435: The process by which the membrane connecting two daughter cells is cleaved, is also mediated by ESCRT machinery. Without the ESCRT complexes, daughter cells could not separate and abnormal cells containing twice the amount of DNA would be generated. These cells would inevitably be destroyed through a process known as apoptosis . Lastly, viral budding, or the process by which specific types of viruses exit cells, may not occur in

2254-465: The process has been studied to a greater extent in metazoans. The release of viral particles, also known as viral budding , is a process by which free virions are released from within cells via the hijacking of host cell ESCRT machinery. Retroviruses , such as HIV-1 and human T-lymphotropic virus , as well as a number of enveloped viruses , including the Ebola virus , require ESCRT machinery to exit

2303-777: The protein into the forming lumenal vesicles. Molecules that follow these pathways include LDL and the lysosomal hydrolases delivered by mannose-6-phosphate receptors. These soluble molecules remain in endosomes and are therefore delivered to lysosomes. Also, the transmembrane EGFRs, bound to EGF, are tagged with ubiquitin and are therefore sorted into lumenal vesicles by the ESCRTs. VPS28 51160 66914 ENSG00000160948 ENSG00000285339 ENSMUSG00000115987 Q9UK41 Q548N1 Q9D1C8 NM_016208 NM_183057 NM_025842 NM_001305668 NP_057292 NP_898880 NP_057292.1 NP_001292597 NP_080118 Vacuolar protein sorting-associated protein 28 homolog

2352-418: The site of abscission. Doa4 removes ubiquitin from cargo proteins being targeted to the lysosome. Multivesicular bodies play a large role in the transport of ubiquitinated proteins and receptors to a lysosome. ESCRT complexes transport ubiquitinated cargo to cellular vesicles that bud directly into the cell’s endosomal compartment, forming multivesicular bodies. These multivesicular bodies eventually fuse with

2401-475: Was used to describe vesicles that bud from early endosomes and fuse with late endosomes. However, several observations (described above) have now demonstrated that it is more likely that transport between these two compartments occurs by a maturation process, rather than vesicle transport. Another unique identifying feature that differs between the various classes of endosomes is the lipid composition in their membranes. Phosphatidyl inositol phosphates (PIPs), one of

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