scholarly journals A dual role for K63-linked ubiquitin chains in multivesicular body biogenesis and cargo sorting

2012 ◽  
Vol 23 (11) ◽  
pp. 2170-2183 ◽  
Author(s):  
Zoi Erpapazoglou ◽  
Manel Dhaoui ◽  
Marina Pantazopoulou ◽  
Francesca Giordano ◽  
Muriel Mari ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Dual Role ◽  
Yeast Cells ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Double Function ◽  
Ubiquitin Binding ◽  
Cargo Sorting

In yeast, the sorting of transmembrane proteins into the multivesicular body (MVB) internal vesicles requires their ubiquitylation by the ubiquitin ligase Rsp5. This allows their recognition by the ubiquitin-binding domains (UBDs) of several endosomal sorting complex required for transport (ESCRT) subunits. K63-linked ubiquitin (K63Ub) chains decorate several MVB cargoes, and accordingly we show that they localize prominently to the class E compartment, which accumulates ubiquitylated cargoes in cells lacking ESCRT components. Conversely, yeast cells unable to generate K63Ub chains displayed MVB sorting defects. These properties are conserved among eukaryotes, as the mammalian melanosomal MVB cargo MART-1 is modified by K63Ub chains and partly missorted when the genesis of these chains is inhibited. We show that all yeast UBD-containing ESCRT proteins undergo ubiquitylation and deubiquitylation, some being modified through the opposing activities of Rsp5 and the ubiquitin isopeptidase Ubp2, which are known to assemble and disassemble preferentially K63Ub chains, respectively. A failure to generate K63Ub chains in yeast leads to an MVB ultrastructure alteration. Our work thus unravels a double function of K63Ub chains in cargo sorting and MVB biogenesis.

scholarly journals Efficient Cargo Sorting by ESCRT-I and the Subsequent Release of ESCRT-I from Multivesicular Bodies Requires the Subunit Mvb12

2007 ◽  
Vol 18 (2) ◽  
pp. 636-645 ◽  
Author(s):  
Matt Curtiss ◽  
Charles Jones ◽  
Markus Babst
Keyword(s):  
Protein Complex ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Multivesicular Bodies ◽  
Rapid Degradation ◽  
Endosomal Sorting ◽  
Escrt Machinery ◽  
Complex Functions ◽  
Vacuolar Protein ◽  
Cargo Sorting

The endosomal sorting complex required for transport (ESCRT)-I protein complex functions in recognition and sorting of ubiquitinated transmembrane proteins into multivesicular body (MVB) vesicles. It has been shown that ESCRT-I contains the vacuolar protein sorting (Vps) proteins Vps23, Vps28, and Vps37. We identified an additional subunit of yeast ESCRT-I called Mvb12, which seems to associate with ESCRT-I by binding to Vps37. Transient recruitment of ESCRT-I to MVBs results in the rapid degradation of Mvb12. In contrast to mutations in other ESCRT-I subunits, which result in strong defects in MVB cargo sorting, deletion of MVB12 resulted in only a partial sorting phenotype. This trafficking defect was fully suppressed by overexpression of the ESCRT-II complex. Mutations in MVB12 did not affect recruitment of ESCRT-I to MVBs, but they did result in delivery of ESCRT-I to the vacuolar lumen via the MVB pathway. Together, these observations suggest that Mvb12 may function in regulating the interactions of ESCRT-I with cargo and other proteins of the ESCRT machinery to efficiently coordinate cargo sorting and release of ESCRT-I from the MVB.

scholarly journals Bro1 stimulates Vps4 to promote intralumenal vesicle formation during multivesicular body biogenesis

2021 ◽  
Vol 220 (8) ◽  
Author(s):  
Chun-Che Tseng ◽  
Shirley Dean ◽  
Brian A. Davies ◽  
Ishara F. Azmi ◽  
Natalya Pashkova ◽  
...  
Keyword(s):  
Multivesicular Body ◽  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Membrane Remodeling ◽  
Endosomal Sorting ◽  
Aaa Atpase ◽  
Ubiquitin Binding ◽  
Stimulation Of ◽  
Cargo Sorting

Endosomal sorting complexes required for transport (ESCRT-0, -I, -II, -III) execute cargo sorting and intralumenal vesicle (ILV) formation during conversion of endosomes to multivesicular bodies (MVBs). The AAA-ATPase Vps4 regulates the ESCRT-III polymer to facilitate membrane remodeling and ILV scission during MVB biogenesis. Here, we show that the conserved V domain of ESCRT-associated protein Bro1 (the yeast homologue of mammalian proteins ALIX and HD-PTP) directly stimulates Vps4. This activity is required for MVB cargo sorting. Furthermore, the Bro1 V domain alone supports Vps4/ESCRT–driven ILV formation in vivo without efficient MVB cargo sorting. These results reveal a novel activity of the V domains of Bro1 homologues in licensing ESCRT-III–dependent ILV formation and suggest a role in coordinating cargo sorting with membrane remodeling during MVB sorting. Moreover, ubiquitin binding enhances V domain stimulation of Vps4 to promote ILV formation via the Bro1–Vps4–ESCRT-III axis, uncovering a novel role for ubiquitin during MVB biogenesis in addition to facilitating cargo recognition.

scholarly journals ESCRT ubiquitin-binding domains function cooperatively during MVB cargo sorting

2009 ◽  
Vol 185 (2) ◽  
pp. 213-224 ◽  
Author(s):  
S. Brookhart Shields ◽  
Andrea J. Oestreich ◽  
Stanley Winistorfer ◽  
Doris Nguyen ◽  
Johanna A. Payne ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Structural Information ◽  
Multivesicular Bodies ◽  
Endosomal Sorting ◽  
Binding Domains ◽  
Ubiquitin Binding ◽  
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.

scholarly journals Selective Microautophagy of Proteasomes is Initiated by ESCRT-0 and Is Promoted by Proteasome Ubiquitylation

2021 ◽  
Author(s):  
Jianhui Li ◽  
Mark Hochstrasser
Keyword(s):  
Ubiquitin Ligase ◽  
Normal Growth ◽  
Ubiquitin Proteasome System ◽  
Growth Conditions ◽  
Endosomal Sorting ◽  
Glucose Depletion ◽  
Ubiquitin Binding ◽  
Ubiquitin Proteasome ◽  
Low Glucose ◽  
Amp Activated Protein Kinase

The proteasome is central to proteolysis by the ubiquitin-proteasome system under normal growth conditions but is itself degraded through macroautophagy under nutrient stress. A recently described AMPK (AMP-activated protein kinase)-regulated ESCRT (endosomal sorting complex required for transport)-dependent microautophagy pathway also regulates proteasome trafficking and degradation in low glucose conditions in yeast. Aberrant proteasomes are more prone to microautophagy, suggesting the ESCRT system fine-tunes proteasome quality control under low glucose stress. Here we uncover additional features of the selective microautophagy of proteasomes. Genetic or pharmacological induction of aberrant proteasomes is associated with increased mono- or oligo-ubiquitylation of proteasome components, which appear to be recognized by ESCRT-0. AMPK controls this pathway in part by regulating the trafficking of ESCRT-0 to the vacuole surface, which also leads to degradation of the Vps27 subunit of ESCRT-0. The Rsp5 ubiquitin ligase contributes to proteasome subunit ubiquitylation, and multiple ubiquitin-binding elements in Vps27 are involved in their recognition. We propose that ESCRT-0 at the vacuole surface recognizes ubiquitylated proteasomes and initiates their microautophagic elimination during glucose depletion.

scholarly journals Mvb12 Is a Novel Member of ESCRT-I Involved in Cargo Selection by the Multivesicular Body Pathway

2007 ◽  
Vol 18 (2) ◽  
pp. 646-657 ◽  
Author(s):  
Andrea J. Oestreich ◽  
Brian A. Davies ◽  
Johanna A. Payne ◽  
David J. Katzmann
Keyword(s):  
Normal Cell ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Eukaryotic Cells ◽  
Cell Physiology ◽  
Cellular Functions ◽  
Protein Loss ◽  
Endosomal Sorting ◽  
Vacuolar Protein ◽  
Selection Of

The multivesicular body (MVB) sorting pathway impacts a variety of cellular functions in eukaryotic cells. Perhaps the best understood role for the MVB pathway is the degradation of transmembrane proteins within the lysosome. Regulation of cargo selection by this pathway is critically important for normal cell physiology, and recent advances in our understanding of this process have highlighted the endosomal sorting complexes required for transport (ESCRTs) as pivotal players in this reaction. To better understand the mechanisms of cargo selection during MVB sorting, we performed a genetic screen to identify novel factors required for cargo-specific selection by this pathway and identified the Mvb12 protein. Loss of Mvb12 function results in differential defects in the selection of MVB cargoes. A variety of analyses indicate that Mvb12 is a stable member of ESCRT-I, a heterologous complex involved in cargo selection by the MVB pathway. Phenotypes displayed upon loss of Mvb12 are distinct from those displayed by the previously described ESCRT-I subunits (vacuolar protein sorting 23, -28, and -37), suggesting a distinct function than these core subunits. These data support a model in which Mvb12 impacts the selection of MVB cargoes by modulating the cargo recognition capabilities of ESCRT-I.

scholarly journals A Strategy for Modulation of Enzymes in the Ubiquitin System

Science ◽  
2013 ◽  
Vol 339 (6119) ◽  
pp. 590-595 ◽  
Author(s):  
Andreas Ernst ◽  
George Avvakumov ◽  
Jiefei Tong ◽  
Yihui Fan ◽  
Yanling Zhao ◽  
...  
Keyword(s):  
Ubiquitin Ligase ◽  
Combinatorial Libraries ◽  
Selection Strategy ◽  
Protein Substrates ◽  
Binding Domains ◽  
Ubiquitin System ◽  
Ubiquitin Binding ◽  
Ubiquitin Ligase E3 ◽  
Dub Inhibitor ◽  
Orthogonal Modulation

The ubiquitin system regulates virtually all aspects of cellular function. We report a method to target the myriad enzymes that govern ubiquitination of protein substrates. We used massively diverse combinatorial libraries of ubiquitin variants to develop inhibitors of four deubiquitinases (DUBs) and analyzed the DUB-inhibitor complexes with crystallography. We extended the selection strategy to the ubiquitin conjugating (E2) and ubiquitin ligase (E3) enzymes and found that ubiquitin variants can also enhance enzyme activity. Last, we showed that ubiquitin variants can bind selectively to ubiquitin-binding domains. Ubiquitin variants exhibit selective function in cells and thus enable orthogonal modulation of specific enzymatic steps in the ubiquitin system.

The role of Rsp5 ubiquitin ligase in regulation of diverse processes in yeast cells.

2008 ◽  
Vol 55 (4) ◽  
pp. 649-662 ◽  
Author(s):  
Paweł Kaliszewski ◽  
Teresa Zoładek
Keyword(s):  
Fatty Acid ◽  
Ubiquitin Ligase ◽  
Intracellular Trafficking ◽  
Phospholipid Composition ◽  
Multivesicular Body ◽  
Yeast Cells ◽  
Sterol Synthesis ◽  
Cellular Processes ◽  
Number Of Publications

Rsp5 is a conserved ubiquitin ligase involved in regulation of numerous cellular processes. A growing number of publications describing new functions of the ligase have appeared in recent years. Rsp5 was shown to be involved in the control of intracellular trafficking of proteins via endocytosis and multivesicular body sorting. Moreover, nuclear functions of Rsp5 in response to various stresses have been discovered. Rsp5 is also involved in the regulation of unsaturated fatty acid and sterol synthesis and phospholipid composition. Here, an overview of Rsp5 functions with emphasis on its involvement in the regulation of lipid biosynthesis will be presented.

scholarly journals Multivesicular Body Sorting: Ubiquitin Ligase Rsp5 Is Required for the Modification and Sorting of Carboxypeptidase S

2004 ◽  
Vol 15 (2) ◽  
pp. 468-480 ◽  
Author(s):  
David J. Katzmann ◽  
Srimonti Sarkar ◽  
Tony Chu ◽  
Anjon Audhya ◽  
Scott D. Emr
Keyword(s):  
Point Mutation ◽  
Ubiquitin Ligase ◽  
Genetic Selection ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Permissive Temperature ◽  
In Cis ◽  
Precise Role ◽  
Phosphatidylinositol 3

The multivesicular body (MVB) sorting pathway provides a mechanism for delivering transmembrane proteins into the lumen of the lysosome/vacuole. Recent studies demonstrated that ubiquitin modification acts in cis as a signal for the sorting of cargoes into this pathway. Here, we present results from a genetic selection designed to identify mutants that missort MVB cargoes. This selection identified a point mutation in ubiquitin ligase Rsp5 (Rsp5-326). At the permissive temperature, this mutant is specifically defective for ubiquitination and sorting of the ubiquitin-dependent MVB cargo precursor carboxypeptidase S (pCPS), but not ligand-induced ubiquitination of Ste2. A previous study implicated Tul1 as the ubiquitin ligase responsible for MVB sorting of pCPS. However, we detected no defect in either the sorting or ubiquitination of pCPS in tul1 mutants. We had previously shown that Fab1 phosphatidylinositol 3-phosphate 5-kinase is also required for MVB sorting of pCPS, but not Ste2. However, our analyses reveal that fab1 mutants do not exhibit a defect in ubiquitination of pCPS. Thus, both Rsp5 and Fab1 play distinct and essential roles in the targeting of biosynthetic MVB cargoes. However, whereas Rsp5 seems to be responsible for cargo ubiquitination, the precise role for Fab1 remains to be elucidated.

scholarly journals Ist1 Regulates Vps4 Localization and Assembly

2008 ◽  
Vol 19 (2) ◽  
pp. 465-474 ◽  
Author(s):  
Christian Dimaano ◽  
Charles B. Jones ◽  
Abraham Hanono ◽  
Matt Curtiss ◽  
Markus Babst
Keyword(s):  
Protein Complexes ◽  
Transmembrane Proteins ◽  
Multivesicular Body ◽  
Dual Role ◽  
Interacting Proteins ◽  
Transport Pathway ◽  
Endosomal Membrane ◽  
Escrt Machinery ◽  
Cargo Proteins ◽  
Late Step

The ESCRT protein complexes are recruited from the cytoplasm and assemble on the endosomal membrane into a protein network that functions in sorting of ubiquitinated transmembrane proteins into the multivesicular body (MVB) pathway. This transport pathway packages cargo proteins into vesicles that bud from the MVB limiting membrane into the lumen of the compartment and delivers these vesicles to the lysosome/vacuole for degradation. The dissociation of ESCRT machinery by the AAA-type ATPase Vps4 is a necessary late step in the formation of MVB vesicles. This ATP-consuming step is regulated by several Vps4-interacting proteins, including the newly identified regulator Ist1. Our data suggest that Ist1 has a dual role in the regulation of Vps4 activity: it localizes to the ESCRT machinery via Did2 where it positively regulates recruitment of Vps4 and it negatively regulates Vps4 by forming an Ist1-Vps4 heterodimer, in which Vps4 cannot bind to the ESCRT machinery. The activity of the MVB pathway might be in part determined by outcome of these two competing activities.

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