scholarly journals Constitutively active ESCRT-II suppresses the MVB-sorting phenotype of ESCRT-0 and ESCRT-I mutants

2015 ◽  
Vol 26 (3) ◽  
pp. 554-568 ◽  
Author(s):  
Shrawan Kumar Mageswaran ◽  
Natalie K. Johnson ◽  
Greg Odorizzi ◽  
Markus Babst
Keyword(s):  
Protein Complexes ◽  
Deletion Strain ◽  
Endosomal Sorting ◽  
Cargo Proteins ◽  
Redundant Functions ◽  
Cargo Sorting ◽  
Constitutively Active

The endosomal sorting complex required for transport (ESCRT) protein complexes function at the endosome in the formation of intraluminal vesicles (ILVs) containing cargo proteins destined for the vacuolar/lysosomal lumen. The early ESCRTs (ESCRT-0 and -I) are likely involved in cargo sorting, whereas ESCRT-III and Vps4 function to sever the neck of the forming ILVs. ESCRT-II links these functions by initiating ESCRT-III formation in an ESCRT-I–regulated manner. We identify a constitutively active mutant of ESCRT-II that partially suppresses the phenotype of an ESCRT-I or ESCRT-0 deletion strain, suggesting that these early ESCRTs are not essential and have redundant functions. However, the ESCRT-III/Vps4 system alone is not sufficient for ILV formation but requires cargo sorting mediated by one of the early ESCRTs.

scholarly journals Atypical parkinsonism–associated retromer mutant alters endosomal sorting of specific cargo proteins

2016 ◽  
Vol 214 (4) ◽  
pp. 389-399 ◽  
Author(s):  
Kirsty J. McMillan ◽  
Matthew Gallon ◽  
Adam P. Jellett ◽  
Thomas Clairfeuille ◽  
Frances C. Tilley ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Neurodegenerative Disease ◽  
Quantitative Proteomics ◽  
Protein Complexes ◽  
Integral Membrane Proteins ◽  
Atypical Parkinsonism ◽  
Endosomal Sorting ◽  
Retromer Complex ◽  
Cargo Proteins ◽  
Cargo Sorting

The retromer complex acts as a scaffold for endosomal protein complexes that sort integral membrane proteins to various cellular destinations. The retromer complex is a heterotrimer of VPS29, VPS35, and VPS26. Two of these paralogues, VPS26A and VPS26B, are expressed in humans. Retromer dysfunction is associated with neurodegenerative disease, and recently, three VPS26A mutations (p.K93E, p.M112V, and p.K297X) were discovered to be associated with atypical parkinsonism. Here, we apply quantitative proteomics to provide a detailed description of the retromer interactome. By establishing a comparative proteomic methodology, we identify how this interactome is perturbed in atypical parkinsonism-associated VPS26A mutants. In particular, we describe a selective defect in the association of VPS26A (p.K297X) with the SNX27 cargo adaptor. By showing how a retromer mutant leads to altered endosomal sorting of specific PDZ ligand–containing cargo proteins, we reveal a new mechanism for perturbed endosomal cargo sorting in atypical parkinsonism.

scholarly journals Did2 coordinates Vps4-mediated dissociation of ESCRT-III from endosomes

2006 ◽  
Vol 175 (5) ◽  
pp. 715-720 ◽  
Author(s):  
Daniel P. Nickerson ◽  
Matthew West ◽  
Greg Odorizzi
Keyword(s):  
Vesicle Formation ◽  
Multivesicular Bodies ◽  
Vesicle Budding ◽  
Endosomal Sorting ◽  
Essential Step ◽  
Adenosine Triphosphatases ◽  
Cargo Proteins ◽  
Endosomal Membranes ◽  
Cargo Sorting

The sorting of transmembrane cargo proteins into the lumenal vesicles of multivesicular bodies (MVBs) depends on the recruitment of endosomal sorting complexes required for transport (ESCRTs) to the cytosolic face of endosomal membranes. The subsequent dissociation of ESCRT complexes from endosomes requires Vps4, a member of the AAA family of adenosine triphosphatases. We show that Did2 directs Vps4 activity to the dissociation of ESCRT-III but has no role in the dissociation of ESCRT-I or -II. Surprisingly, vesicle budding into the endosome lumen occurs in the absence of Did2 function even though Did2 is required for the efficient sorting of MVB cargo proteins into lumenal vesicles. This uncoupling of MVB cargo sorting and lumenal vesicle formation suggests that the Vps4-mediated dissociation of ESCRT-III is an essential step in the sorting of cargo proteins into MVB vesicles but is not a prerequisite for the budding of vesicles into the endosome lumen.

scholarly journals ARFGAP1 promotes the formation of COPI vesicles, suggesting function as a component of the coat

2002 ◽  
Vol 159 (1) ◽  
pp. 69-78 ◽  
Author(s):  
Jia-Shu Yang ◽  
Stella Y. Lee ◽  
Minggeng Gao ◽  
Sylvain Bourgoin ◽  
Paul A. Randazzo ◽  
...  
Keyword(s):  
Coat Protein ◽  
Gap Functions ◽  
Vesicle Formation ◽  
Golgi Membrane ◽  
Gtpase Activating Protein ◽  
Cargo Proteins ◽  
Adp Ribosylation Factor ◽  
Soluble Components ◽  
Cargo Sorting

The role of GTPase-activating protein (GAP) that deactivates ADP-ribosylation factor 1 (ARF1) during the formation of coat protein I (COPI) vesicles has been unclear. GAP is originally thought to antagonize vesicle formation by triggering uncoating, but later studies suggest that GAP promotes cargo sorting, a process that occurs during vesicle formation. Recent models have attempted to reconcile these seemingly contradictory roles by suggesting that cargo proteins suppress GAP activity during vesicle formation, but whether GAP truly antagonizes coat recruitment in this process has not been assessed directly. We have reconstituted the formation of COPI vesicles by incubating Golgi membrane with purified soluble components, and find that ARFGAP1 in the presence of GTP promotes vesicle formation and cargo sorting. Moreover, the presence of GTPγS not only blocks vesicle uncoating but also vesicle formation by preventing the proper recruitment of GAP to nascent vesicles. Elucidating how GAP functions in vesicle formation, we find that the level of GAP on the reconstituted vesicles is at least as abundant as COPI and that GAP binds directly to the dilysine motif of cargo proteins. Collectively, these findings suggest that ARFGAP1 promotes vesicle formation by functioning as a component of the COPI coat.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2022 ◽  
Author(s):  
Javier Manzano-Lopez†* ◽  
Sofia Rodriguez-Gallardo† ◽  
Susana Sabido-Bozo† ◽  
Alejandro Cortes-Gomez ◽  
Ana Maria Perez-Linero ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to stabilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

Crosslinking assay to study a specific cargo-coat interaction through a transmembrane receptor in the secretory pathway v1

2021 ◽  
Author(s):  
Javier Manzano-Lopez † ◽  
Sofia Rodriguez-Gallardo † ◽  
Susana Sabido-Bozo ◽  
Ana Maria Perez-Linero ◽  
Rafael Lucena ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Secretory Pathway ◽  
Protein Complexes ◽  
Secretory Protein ◽  
Extracellular Proteins ◽  
Transient Nature ◽  
Transport Vesicles ◽  
System P ◽  
Cargo Receptor ◽  
Cargo Proteins

Intracellular trafficking through the secretory organelles depends on transient interactions between cargo proteins and transport machinery. Cytosolic coat protein complexes capture specific luminal cargo proteins for incorporation into transport vesicles by interacting with them indirectly through a transmembrane adaptor or cargo receptor. Due to their transient nature, it is difficult to study these specific ternary protein interactions just using conventional native co-immunoprecipitation. To overcome this technical challenge, we have applied a crosslinking assay to immobilize the transient and/or weak protein interactions. Here, we describe a protocol of protein cross-linking and co-immunoprecipitation, which was employed to prove the indirect interaction in the endoplasmic reticulum of a luminal secretory protein with a selective subunit of the cytosolic COPII coat through a specific transmembrane cargo receptor. This method can be extended to address other transient ternary interactions between cytosolic proteins and luminal or extracellular proteins through a transmembrane receptor within the endomembrane system.

ESCRT-III on endosomes: new functions, new activation pathway

2016 ◽  
Vol 473 (2) ◽  
pp. e5-e8 ◽  
Author(s):  
Philip Woodman
Keyword(s):  
Mhc Class I ◽  
Protein Tyrosine Phosphatase ◽  
Protein Complexes ◽  
Tyrosine Phosphatase ◽  
Multivesicular Body ◽  
Class I ◽  
Recent Issue ◽  
Endosomal Sorting ◽  
Histocompatibility Complex ◽  
Biochemical Journal

The multivesicular body (MVB) pathway sorts ubiquitinated membrane cargo to intraluminal vesicles (ILVs) within the endosome, en route to the lysosomal lumen. The pathway involves the sequential action of conserved protein complexes [endosomal sorting complexes required for transport (ESCRTs)], culminating in the activation by ESCRT-II of ESCRT-III, a membrane-sculpting complex. Although this linear pathway of ESCRT activation is widely accepted, a study by Luzio and colleagues in a recent issue of the Biochemical Journal suggests that there is greater complexity in ESCRT-III activation, at least for some MVB cargoes. They show that ubiquitin-dependent sorting of major histocompatibility complex (MHC) class I to the MVB requires the central ESCRT-III complex but does not involve either ESCRT-II or functional links between ESCRT-II and ESCRT-III. Instead, they propose that MHC class I utilizes histidine-domain protein tyrosine phosphatase (HD-PTP), a non-canonical ESCRT interactor, to promote ESCRT-III activation.

scholarly journals Identification of a novel NF-kappaB p50-related protein in B lymphocytes.

1996 ◽  
Vol 16 (12) ◽  
pp. 7089-7097 ◽  
Author(s):  
R J Phillips ◽  
S Gustafson ◽  
S Ghosh
Keyword(s):  
Transcription Factor ◽  
B Lymphocytes ◽  
Plasma Cells ◽  
Protein Complexes ◽  
Cell Types ◽  
Related Protein ◽  
Active Complex ◽  
Inactive Form ◽  
Nf Kappab ◽  
Constitutively Active

In most cell types other than mature B lymphocytes and macrophages, the transcription factor NF-kappaB remains in an inactive form in the cytosol by being bound to the inhibitory proteins IkappaBalpha and IkappaBbeta. To investigate the regulation of constitutively active NF-kappaB in B lymphocytes, we have examined the composition of Rel protein complexes in different mouse B-cell lines. As reported previously, the constitutively active complex in mature B cells was predominantly p50:c-Rel. However, the kappaB binding complex in the plasmacytomas that were examined lacked c-Rel and instead contained only a p50-related protein. This p50-related protein (p55) cross-reacts with three different p50 antisera, exists in both the cytosol and the nucleus, and is the protein that binds to kappaB sites in plasma cells. Transfection of reporter constructs into plasma cells indicates that the p55 complex is also transcriptionally active. The p55 protein can be detected in splenocytes from mice lacking the p105/p50 gene, and therefore it appears to be the product of a distinct gene. The implications of the existence of a NF-kappaB p50-related protein in plasma cells that is capable of binding to kappaB sites and activating transcription are discussed.

scholarly journals The alternate AP-1 adaptor subunit Apm2 interacts with the Mil1 regulatory protein and confers differential cargo sorting

2016 ◽  
Vol 27 (3) ◽  
pp. 588-598 ◽  
Author(s):  
Shawn T. Whitfield ◽  
Helen E. Burston ◽  
Björn D. M. Bean ◽  
Nandini Raghuram ◽  
Lymarie Maldonado-Báez ◽  
...  
Keyword(s):  
Protein Complexes ◽  
Regulatory Protein ◽  
Adaptor Protein ◽  
General Mechanism ◽  
Specific Expression ◽  
Membrane Recruitment ◽  
Early Endosomes ◽  
Cell Type Specific Expression ◽  
Clathrin Adaptor ◽  
Cargo Sorting

Heterotetrameric adaptor protein complexes are important mediators of cargo protein sorting in clathrin-coated vesicles. The cell type–specific expression of alternate μ chains creates distinct forms of AP-1 with altered cargo sorting, but how these subunits confer differential function is unclear. Whereas some studies suggest the μ subunits specify localization to different cellular compartments, others find that the two forms of AP-1 are present in the same vesicle but recognize different cargo. Yeast have two forms of AP-1, which differ only in the μ chain. Here we show that the variant μ chain Apm2 confers distinct cargo-sorting functions. Loss of Apm2, but not of Apm1, increases cell surface levels of the v-SNARE Snc1. However, Apm2 is unable to replace Apm1 in sorting Chs3, which requires a dileucine motif recognized by the γ/σ subunits common to both complexes. Apm2 and Apm1 colocalize at Golgi/early endosomes, suggesting that they do not associate with distinct compartments. We identified a novel, conserved regulatory protein that is required for Apm2-dependent sorting events. Mil1 is a predicted lipase that binds Apm2 but not Apm1 and contributes to its membrane recruitment. Interactions with specific regulatory factors may provide a general mechanism to diversify the functional repertoire of clathrin adaptor complexes.

scholarly journals Concurrent depletion of Vps37 proteins evokes ESCRT-I destabilization and profound cellular stress responses

2020 ◽  
Author(s):  
Krzysztof Kolmus ◽  
Purevsuren Erdenebat ◽  
Blair Stewig ◽  
Ewelina Szymańska ◽  
Krzysztof Goryca ◽  
...  
Keyword(s):  
Inflammatory Response ◽  
Stress Responses ◽  
Cell Biology ◽  
Cellular Stress ◽  
Mitogen Activated Protein Kinase ◽  
Endosomal Sorting ◽  
Mitogen Activated Protein ◽  
Transcriptional Alterations ◽  
Cellular Stress Responses ◽  
Redundant Functions

ABSTRACTMolecular details of how endocytosis contributes to oncogenesis remain elusive. Our in silico analysis of colorectal cancer (CRC) patients revealed stage-dependent alterations in the expression of 113 endocytosis-related genes. Among them transcription of the Endosomal Sorting Complex Required for Transport (ESCRT)-I component VPS37B was decreased in the advanced stages of CRC. Expression of other ESCRT-I core subunits remained unchanged in the investigated dataset. We analyzed an independent cohort of CRC patients showing also reduced VPS37A mRNA and protein abundance. Transcriptomic profiling of CRC cells revealed non-redundant functions of Vps37 proteins. Knockdown of VPS37A and VPS37B triggered p21-mediated inhibition of cell proliferation and sterile inflammatory response driven by the Nuclear Factor (NF)-κB transcription factor and associated with mitogen-activated protein kinase signaling. Co-silencing of VPS37C further potentiated activation of these independently induced processes. The type and magnitude of transcriptional alterations correlated with the differential ESCRT-I stability upon individual and concurrent Vps37 depletion. Our study provides novel insights into cancer cell biology by describing cellular stress responses that are associated with ESCRT-I destabilization, which might occur in CRC patients.SUMMARY STATEMENTEndosomal Sorting Complex Required for Transport (ESCRT)-I destabilization upon concurrent depletion of Vps37 proteins is linked to the activation of sterile inflammatory response and cell growth inhibition.

scholarly journals Mammalian retromer is an adaptable scaffold for cargo sorting from endosomes

2019 ◽  
Author(s):  
Amy K. Kendall ◽  
Boyang Xie ◽  
Peng Xu ◽  
Jue Wang ◽  
Rodger Burcham ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Core Complex ◽  
Sorting Nexin ◽  
Flat Chains ◽  
Biophysical Studies ◽  
Cargo Proteins ◽  
Golgi Network ◽  
Multi Body ◽  
Cargo Sorting

AbstractIn metazoans, retromer (VPS26/VPS35/VPS29) associates with sorting nexin (SNX) proteins to form coats on endosomal tubules and sort cargo proteins to the trans-Golgi network (TGN) or plasma membrane. This core complex is highly conserved from yeast to humans, but molecular mechanisms of metazoan retromer assembly remain undefined. Here we combine single particle cryo-electron microscopy with biophysical methods to uncover multiple oligomer structures formed by mammalian retromer. Two-dimensional class averages in ice reveal the retromer heterotrimer; dimers of trimers; tetramers of trimers; and flat chains. These species are further supported by biophysical studies in solution. We provide cryo-EM reconstructions of all species, including pseudo-atomic resolution detail for key sub-structures. Multi-body refinement demonstrates how retromer heterotrimers and dimers adopt a range of conformations. Our structures identify a flexible yet highly conserved electrostatic interface in dimers formed by interactions between VPS35 subunits. We generate a structure-based mutant to disrupt this key interface in vitro and introduce equivalent mutations into S. cerevisiae to demonstrate the mutant exhibits a cargo sorting defect. Together, structures and complementary functional data in budding yeast imply a conserved assembly interface across eukaryotes. These data further suggest mammalian retromer acts as an adaptable and plastic scaffold that accommodates interactions with different SNXs to sort multiple cargoes from endosomes their final destinations.

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