id: Q9UK41 gene_symbol: VPS28 product_type: PROTEIN status: COMPLETE taxon: id: NCBITaxon:9606 label: Homo sapiens description: >- VPS28 is a conserved core subunit of human ESCRT-I. It pairs with TSG101, a VPS37 paralog, and an MVB12/UBAP-family subunit to organize ESCRT-I for ubiquitin-dependent endosomal cargo sorting, multivesicular body assembly, reverse-topology membrane remodeling, and phagophore/autophagosome closure. Unlike TSG101, VPS28 is not well supported as a direct ubiquitin-binding protein; its core role is ESCRT-I structural/scaffold assembly and membrane-site recruitment. alternative_products: - name: '1' id: Q9UK41-1 - name: '2' id: Q9UK41-2 sequence_note: VSP_042028 existing_annotations: - term: id: GO:0005829 label: cytosol evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: is_active_in review: summary: cytosol localization is supported but broad and non-core. action: KEEP_AS_NON_CORE reason: VPS28 is predominantly cytosolic as a soluble ESCRT-I component before membrane recruitment, but the core proteostasis function is ESCRT-I activity at endosomal/phagophore membranes. additional_reference_ids: &id018 - PMID:11134028 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id019 - reference_id: PMID:11134028 supporting_text: cytosolic protein that interacts with TSG101/mammalian VPS23 - reference_id: PMID:11134028 supporting_text: TSG101 and hVPS28 are predominantly cytosolic - term: id: GO:0043130 label: ubiquitin binding evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: enables review: summary: Direct ubiquitin binding is over-annotated for VPS28. action: MARK_AS_OVER_ANNOTATED reason: The cited paper shows the TSG101-hVPS28 complex binds ubiquitin, but isolated hVPS28 does not; the binding is attributed to TSG101 UEV-domain recognition, so VPS28 should not be curated as directly enabling ubiquitin binding. proposed_replacement_terms: &id025 - id: GO:0000813 label: ESCRT I complex - id: GO:0043162 label: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway additional_reference_ids: &id026 - PMID:11916981 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id027 - reference_id: PMID:11916981 supporting_text: Both TSG101 and hVPS28 bound specifically to ubiquitin-agarose - reference_id: PMID:11916981 supporting_text: hVPS28 did not bind to ubiquitin-agarose - reference_id: PMID:11916981 supporting_text: complex binding to ubiquitin via the Ubc domain of TSG101 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: &id001 - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id002 - &id007 reference_id: file:human/VPS28/VPS28-uniprot.txt supporting_text: Component of the ESCRT-I complex - &id008 reference_id: file:human/VPS28/VPS28-uniprot.txt supporting_text: which consists of TSG101, VPS28, a VPS37 - &id009 reference_id: PMID:18005716 supporting_text: All ESCRT-I complexes contain three - &id010 reference_id: PMID:18005716 supporting_text: TSG101, VPS28, and VPS37 - &id011 reference_id: PMID:20654576 supporting_text: comprises a heterotetramer of TSG101 - term: id: GO:0043328 label: protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway evidence_type: IBA original_reference_id: GO_REF:0000033 qualifier: involved_in review: summary: protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: &id003 - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id004 - &id012 reference_id: PMID:11134028 supporting_text: directly involved in endosomal sorting - &id013 reference_id: PMID:11916981 supporting_text: TSG101 and VPS28 are localized to ubiquitin-rich endosomes - &id014 reference_id: PMID:11916981 supporting_text: antibodies to VPS28 specifically impair EGF degradation - &id015 reference_id: PMID:21757351 supporting_text: required for sorting EGFR to the MVB - &id016 reference_id: file:human/VPS28/VPS28-uniprot.txt supporting_text: Component of the ESCRT-I complex, a regulator of vesicular - term: id: GO:0000813 label: ESCRT I complex evidence_type: IEA original_reference_id: GO_REF:0000120 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0005886 label: plasma membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: plasma membrane is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: &id005 - PMID:18005716 - PMID:20588296 - PMID:32424346 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id006 - reference_id: PMID:18005716 supporting_text: plays essential roles in HIV budding and endosomal protein sorting - reference_id: PMID:20588296 supporting_text: viral budding, cytokinesis and, probably, autophagy - reference_id: file:human/VPS28/VPS28-uniprot.txt supporting_text: Cell membrane - term: id: GO:0016236 label: macroautophagy evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in review: summary: Macroautophagy is too broad for the VPS28 ESCRT-I autophagy role. action: MODIFY reason: The direct VPS28-linked evidence concerns ESCRT-I helical assembly during phagophore/autophagosome closure, so GO:0000045 autophagosome assembly is the better term. proposed_replacement_terms: &id022 - id: GO:0000045 label: autophagosome assembly additional_reference_ids: &id023 - PMID:31519728 - PMID:32424346 - file:human/VPS28/VPS28-notes.md supported_by: &id024 - &id031 reference_id: PMID:31519728 supporting_text: ESCRT-I subunit VPS28 and CHMP2A - &id032 reference_id: PMID:31519728 supporting_text: phagophore closure in mammalian cells - &id033 reference_id: PMID:32424346 supporting_text: ESCRT-I helical interface is required for autophagosome closure - &id034 reference_id: PMID:32424346 supporting_text: blocks filament formation in vitro and autophagosome closure - term: id: GO:0031902 label: late endosome membrane evidence_type: IEA original_reference_id: GO_REF:0000044 qualifier: located_in review: summary: late endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: &id020 - PMID:11134028 - PMID:11916981 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: &id021 - reference_id: PMID:11134028 supporting_text: TSG101 and hVPS28 are predominantly cytosolic - reference_id: PMID:11134028 supporting_text: translocated to the surface of these vacuoles - reference_id: PMID:11916981 supporting_text: TSG101 and VPS28 are localized to ubiquitin-rich endosomes - reference_id: file:human/VPS28/VPS28-uniprot.txt supporting_text: "Late\nCC endosome membrane" - term: id: GO:0032509 label: endosome transport via multivesicular body sorting pathway evidence_type: IEA original_reference_id: GO_REF:0000002 qualifier: involved_in review: summary: endosome transport via multivesicular body sorting pathway is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0036258 label: multivesicular body assembly evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in review: summary: multivesicular body assembly is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0039702 label: viral budding via host ESCRT complex evidence_type: IEA original_reference_id: GO_REF:0000117 qualifier: involved_in review: summary: viral budding via host ESCRT complex is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:16189514 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: &id017 - id: GO:0000813 label: ESCRT I complex - id: GO:0036258 label: multivesicular body assembly additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:19060904 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:21516116 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:25416956 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:25959826 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:26871637 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:28514442 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:31515488 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:32814053 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:33961781 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:35271311 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005829 label: cytosol evidence_type: IEA original_reference_id: GO_REF:0000107 qualifier: located_in review: summary: cytosol localization is supported but broad and non-core. action: KEEP_AS_NON_CORE reason: VPS28 is predominantly cytosolic as a soluble ESCRT-I component before membrane recruitment, but the core proteostasis function is ESCRT-I activity at endosomal/phagophore membranes. additional_reference_ids: *id018 supported_by: *id019 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IPI original_reference_id: PMID:18005716 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IPI original_reference_id: PMID:21757351 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IPI original_reference_id: PMID:32424346 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0010008 label: endosome membrane evidence_type: NAS original_reference_id: PMID:32424346 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0036258 label: multivesicular body assembly evidence_type: NAS original_reference_id: PMID:32424346 qualifier: involved_in review: summary: multivesicular body assembly is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0043328 label: protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway evidence_type: NAS original_reference_id: PMID:32424346 qualifier: involved_in review: summary: protein transport to vacuole involved in ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0090148 label: membrane fission evidence_type: NAS original_reference_id: PMID:32424346 qualifier: involved_in review: summary: Membrane fission is supported for ESCRT-I reverse-topology membrane remodeling. action: ACCEPT reason: ESCRT-I scaffolds reverse-topology membrane remodeling; VPS28 helical-interface mutations disrupt ESCRT-I filament assembly and ESCRT-dependent sealing contexts. additional_reference_ids: - PMID:20588296 - PMID:32424346 - file:human/VPS28/VPS28-notes.md supported_by: - &id028 reference_id: PMID:20588296 supporting_text: ESCRT-I and ESCRT-II direct membrane budding away from the cytosol - &id029 reference_id: PMID:32424346 supporting_text: A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission - &id030 reference_id: PMID:32424346 supporting_text: VPS28 helical interface residues blocks filament formation in vitro and autophagosome - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:15218037 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0016236 label: macroautophagy evidence_type: TAS original_reference_id: PMID:20588296 qualifier: involved_in review: summary: Macroautophagy is too broad for the VPS28 ESCRT-I autophagy role. action: MODIFY reason: The direct VPS28-linked evidence concerns ESCRT-I helical assembly during phagophore/autophagosome closure, so GO:0000045 autophagosome assembly is the better term. proposed_replacement_terms: *id022 additional_reference_ids: *id023 supported_by: *id024 - term: id: GO:0000813 label: ESCRT I complex evidence_type: TAS original_reference_id: PMID:20588296 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0036258 label: multivesicular body assembly evidence_type: TAS original_reference_id: PMID:20588296 qualifier: involved_in review: summary: multivesicular body assembly is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0039702 label: viral budding via host ESCRT complex evidence_type: TAS original_reference_id: PMID:20588296 qualifier: involved_in review: summary: viral budding via host ESCRT complex is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IDA original_reference_id: PMID:18005716 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0005768 label: endosome evidence_type: IDA original_reference_id: PMID:17940959 qualifier: located_in review: summary: endosome localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0005886 label: plasma membrane evidence_type: IDA original_reference_id: PMID:17940959 qualifier: located_in review: summary: plasma membrane is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0045732 label: positive regulation of protein catabolic process evidence_type: IMP original_reference_id: PMID:11916981 qualifier: involved_in review: summary: Positive regulation of protein catabolic process is too broad for the VPS28 evidence. action: MODIFY reason: The PMID:11916981 evidence specifically concerns EGF/EGFR degradation through ubiquitin-dependent endosomal sorting rather than a general protein-catabolic regulatory function. proposed_replacement_terms: - id: GO:2000397 label: positive regulation of ubiquitin-dependent endocytosis - id: GO:0043162 label: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:2000397 label: positive regulation of ubiquitin-dependent endocytosis evidence_type: IMP original_reference_id: PMID:11916981 qualifier: involved_in review: summary: positive regulation of ubiquitin-dependent endocytosis is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0005769 label: early endosome evidence_type: IDA original_reference_id: PMID:11916981 qualifier: located_in review: summary: early endosome localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0043130 label: ubiquitin binding evidence_type: IDA original_reference_id: PMID:11916981 qualifier: enables review: summary: Direct ubiquitin binding is over-annotated for VPS28. action: MARK_AS_OVER_ANNOTATED reason: The cited paper shows the TSG101-hVPS28 complex binds ubiquitin, but isolated hVPS28 does not; the binding is attributed to TSG101 UEV-domain recognition, so VPS28 should not be curated as directly enabling ubiquitin binding. proposed_replacement_terms: *id025 additional_reference_ids: *id026 supported_by: *id027 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:11134028 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0010008 label: endosome membrane evidence_type: IDA original_reference_id: PMID:11134028 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0070062 label: extracellular exosome evidence_type: HDA original_reference_id: PMID:23533145 qualifier: located_in review: summary: extracellular exosome is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:14519844 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0005515 label: protein binding evidence_type: IPI original_reference_id: PMID:14505570 qualifier: enables review: summary: Protein binding is too generic to represent VPS28 function. action: MARK_AS_OVER_ANNOTATED reason: The informative function is ESCRT-I complex membership and ESCRT-I-mediated endosomal sorting/membrane remodeling, not generic protein binding from interaction assays. proposed_replacement_terms: *id017 additional_reference_ids: - PMID:18005716 - PMID:20654576 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md - PMID:11134028 - PMID:11916981 - PMID:21757351 - file:human/VPS28/VPS28-uniprot.txt - file:human/VPS28/VPS28-notes.md supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IDA original_reference_id: PMID:20654576 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IDA original_reference_id: PMID:22232651 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0043162 label: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway evidence_type: IC original_reference_id: PMID:20654576 qualifier: involved_in review: summary: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0070062 label: extracellular exosome evidence_type: HDA original_reference_id: PMID:19199708 qualifier: located_in review: summary: extracellular exosome is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0070062 label: extracellular exosome evidence_type: HDA original_reference_id: PMID:19056867 qualifier: located_in review: summary: extracellular exosome is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0070062 label: extracellular exosome evidence_type: HDA original_reference_id: PMID:20458337 qualifier: located_in review: summary: extracellular exosome is supported as a context but is not the core VPS28 proteostasis function. action: KEEP_AS_NON_CORE reason: Plasma-membrane viral budding and extracellular vesicle/exosome contexts use ESCRT machinery but are secondary to the core VPS28 ESCRT-I endosomal sorting and PN autophagophore-sealing role. additional_reference_ids: *id005 supported_by: *id006 - term: id: GO:0000813 label: ESCRT I complex evidence_type: IDA original_reference_id: PMID:21757351 qualifier: part_of review: summary: ESCRT-I complex membership is the central VPS28 cellular-component annotation. action: ACCEPT reason: VPS28 is a constitutive ESCRT-I subunit in TSG101-VPS28-VPS37-MVB12/UBAP variant complexes. additional_reference_ids: *id001 supported_by: *id002 - term: id: GO:0043162 label: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway evidence_type: IMP original_reference_id: PMID:21757351 qualifier: involved_in review: summary: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway is supported as part of the core ESCRT-I endosomal cargo-sorting role. action: ACCEPT reason: VPS28/ESCRT-I supports ubiquitin-dependent endosomal receptor/cargo sorting into MVBs and delivery toward lysosomal degradation. additional_reference_ids: *id003 supported_by: *id004 - term: id: GO:0010008 label: endosome membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-184269 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0010008 label: endosome membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-3149434 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0010008 label: endosome membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-3159232 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0010008 label: endosome membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-917696 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0010008 label: endosome membrane evidence_type: TAS original_reference_id: Reactome:R-HSA-917730 qualifier: located_in review: summary: endosome membrane localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0005737 label: cytoplasm evidence_type: IDA original_reference_id: PMID:18077552 qualifier: located_in review: summary: cytoplasm localization is supported but broad and non-core. action: KEEP_AS_NON_CORE reason: VPS28 is predominantly cytosolic as a soluble ESCRT-I component before membrane recruitment, but the core proteostasis function is ESCRT-I activity at endosomal/phagophore membranes. additional_reference_ids: *id018 supported_by: *id019 - term: id: GO:0031397 label: negative regulation of protein ubiquitination evidence_type: IDA original_reference_id: PMID:18077552 qualifier: involved_in review: summary: Negative regulation of protein ubiquitination is a broad over-annotation for VPS28. action: MARK_AS_OVER_ANNOTATED reason: The cited evidence is about VPS28/ESCRT-I occupancy limiting TSG101 access to a TSG101-associated ligase, not a general VPS28 ubiquitination-regulatory function. proposed_replacement_terms: - id: GO:0000813 label: ESCRT I complex additional_reference_ids: - PMID:18077552 - file:human/VPS28/VPS28-notes.md supported_by: - reference_id: PMID:18077552 supporting_text: VPS28 is a limiting factor - reference_id: PMID:18077552 supporting_text: presence of the other ESCRT-I proteins - term: id: GO:0005768 label: endosome evidence_type: IDA original_reference_id: PMID:11134028 qualifier: located_in review: summary: endosome localization is supported and relevant to VPS28/ESCRT-I function. action: ACCEPT reason: VPS28 is a soluble ESCRT-I component that is recruited to endosomal membranes during cargo sorting and MVB-related ESCRT activity. additional_reference_ids: *id020 supported_by: *id021 - term: id: GO:0005829 label: cytosol evidence_type: IDA original_reference_id: PMID:11134028 qualifier: located_in review: summary: cytosol localization is supported but broad and non-core. action: KEEP_AS_NON_CORE reason: VPS28 is predominantly cytosolic as a soluble ESCRT-I component before membrane recruitment, but the core proteostasis function is ESCRT-I activity at endosomal/phagophore membranes. additional_reference_ids: *id018 supported_by: *id019 - term: id: GO:0000045 label: autophagosome assembly evidence_type: IMP original_reference_id: PMID:32424346 qualifier: involved_in review: summary: VPS28 should be annotated to autophagosome assembly for ESCRT-I-dependent phagophore closure. action: NEW reason: VPS28 helical-interface mutations disrupt ESCRT-I filament assembly and cause an autophagosome closure defect; this is more precise than broad macroautophagy. additional_reference_ids: *id023 supported_by: *id024 references: - id: GO_REF:0000002 title: Gene Ontology annotation through association of InterPro records with GO terms findings: [] - id: GO_REF:0000033 title: Annotation inferences using phylogenetic trees findings: [] - id: GO_REF:0000044 title: Gene Ontology annotation based on UniProtKB/Swiss-Prot Subcellular Location vocabulary mapping, accompanied by conservative changes to GO terms applied by UniProt findings: [] - id: GO_REF:0000107 title: Automatic transfer of experimentally verified manual GO annotation data to orthologs using Ensembl Compara findings: [] - id: GO_REF:0000117 title: Electronic Gene Ontology annotations created by ARBA machine learning models findings: [] - id: GO_REF:0000120 title: Combined Automated Annotation using Multiple IEA Methods findings: [] - id: PMID:11134028 title: TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes. findings: [] - id: PMID:11916981 title: Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates. findings: [] - id: PMID:14505570 title: The protein network of HIV budding. findings: [] - id: PMID:14519844 title: Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. findings: [] - id: PMID:15218037 title: The human endosomal sorting complex required for transport (ESCRT-I) and its role in HIV-1 budding. findings: [] - id: PMID:16189514 title: Towards a proteome-scale map of the human protein-protein interaction network. findings: [] - id: PMID:17940959 title: Involvement of vacuolar protein sorting pathway in Ebola virus release independent of TSG101 interaction. findings: [] - id: PMID:18005716 title: Identification of human MVB12 proteins as ESCRT-I subunits that function in HIV budding. findings: [] - id: PMID:18077552 title: 'Regulation of Tsg101 expression by the steadiness box: a role of Tsg101-associated ligase.' findings: [] - id: PMID:19056867 title: Large-scale proteomics and phosphoproteomics of urinary exosomes. findings: [] - id: PMID:19060904 title: An empirical framework for binary interactome mapping. findings: [] - id: PMID:19199708 title: Proteomic analysis of human parotid gland exosomes by multidimensional protein identification technology (MudPIT). findings: [] - id: PMID:20458337 title: MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. findings: [] - id: PMID:20588296 title: "Membrane budding and scission by the ESCRT machinery: it's all in the neck." findings: [] - id: PMID:20654576 title: Distinct functions of human MVB12A and MVB12B in the ESCRT-I dependent on their posttranslational modifications. findings: [] - id: PMID:21516116 title: Next-generation sequencing to generate interactome datasets. findings: [] - id: PMID:21757351 title: UBAP1 is a component of an endosome-specific ESCRT-I complex that is essential for MVB sorting. findings: [] - id: PMID:22232651 title: Structural basis for membrane targeting by the MVB12-associated β-prism domain of the human ESCRT-I MVB12 subunit. findings: [] - id: PMID:23533145 title: In-depth proteomic analyses of exosomes isolated from expressed prostatic secretions in urine. findings: [] - id: PMID:25416956 title: A proteome-scale map of the human interactome network. findings: [] - id: PMID:25959826 title: Quantitative interaction proteomics of neurodegenerative disease proteins. findings: [] - id: PMID:26871637 title: Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing. findings: [] - id: PMID:28514442 title: Architecture of the human interactome defines protein communities and disease networks. findings: [] - id: PMID:31515488 title: Extensive disruption of protein interactions by genetic variants across the allele frequency spectrum in human populations. findings: [] - id: PMID:32424346 title: A helical assembly of human ESCRT-I scaffolds reverse-topology membrane scission. findings: [] - id: PMID:32814053 title: Interactome Mapping Provides a Network of Neurodegenerative Disease Proteins and Uncovers Widespread Protein Aggregation in Affected Brains. findings: [] - id: PMID:33961781 title: Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. findings: [] - id: PMID:35271311 title: 'OpenCell: Endogenous tagging for the cartography of human cellular organization.' findings: [] - id: Reactome:R-HSA-184269 title: Monoubiquitinated N-myristoyl GAG polyprotein is targeted to the late endosomal vesicle membrane by the ESCRT-I complex findings: [] - id: Reactome:R-HSA-3149434 title: Transport of GAG to the Plasma Membrane findings: [] - id: Reactome:R-HSA-3159232 title: Recruitment Of HIV Virion Budding Machinery findings: [] - id: Reactome:R-HSA-917696 title: Cargo Sequestration findings: [] - id: Reactome:R-HSA-917730 title: Cargo Recognition And Sorting findings: [] - id: PMID:31519728 title: VPS37A directs ESCRT recruitment for phagophore closure. findings: [] - id: file:human/VPS28/VPS28-uniprot.txt title: UniProtKB record for human VPS28 findings: [] - id: file:human/VPS28/VPS28-notes.md title: VPS28 review notes findings: [] core_functions: - description: VPS28 is a structural/scaffold subunit of ESCRT-I that organizes the TSG101-VPS37-MVB12/UBAP ESCRT-I assembly for endosomal cargo sorting, MVB membrane remodeling, and ESCRT-I-dependent phagophore/autophagosome closure. directly_involved_in: - id: GO:0043162 label: ubiquitin-dependent protein catabolic process via the multivesicular body sorting pathway - id: GO:0036258 label: multivesicular body assembly - id: GO:0090148 label: membrane fission - id: GO:0000045 label: autophagosome assembly locations: - id: GO:0010008 label: endosome membrane - id: GO:0031902 label: late endosome membrane - id: GO:0005768 label: endosome in_complex: id: GO:0000813 label: ESCRT I complex supported_by: - *id007 - *id008 - *id009 - *id010 - *id011 - *id012 - *id013 - *id014 - *id015 - *id016 - *id028 - *id029 - *id030 - *id031 - *id032 - *id033 - *id034 proposed_new_terms: [] suggested_questions: - question: Should VPS28 ubiquitin binding be retired or qualified because the primary evidence supports TSG101-mediated ubiquitin binding by the TSG101-VPS28 complex rather than direct VPS28 binding? experts: - GO molecular function editors - UniProt curators - question: Should broad VPS28 macroautophagy annotations be replaced with GO:0000045 autophagosome assembly to capture ESCRT-I-dependent phagophore closure? experts: - GO autophagy editors - GO ESCRT curators - question: Should viral budding, exosome, and plasma-membrane rows remain non-core in proteostasis-focused VPS28 curation? experts: - GO viral process editors - GO extracellular vesicle editors suggested_experiments: - experiment_type: VPS28 separation-of-function autophagosome closure assay hypothesis: The VPS28 helical interface is required for ESCRT-I accumulation and phagophore closure without abolishing assembly of individual ESCRT-I tetramers. description: Use endogenous VPS28 depletion/rescue with wild-type and helical-interface mutants, then measure HT-LC3 closure, ESCRT-I/ESCRT-III recruitment, and EGFR MVB sorting side by side. - experiment_type: direct ubiquitin-binding assay for purified VPS28 hypothesis: VPS28 does not independently bind ubiquitin; ubiquitin recognition by VPS28-containing ESCRT-I depends on TSG101 or other subunits. description: Compare purified VPS28, TSG101, and reconstituted ESCRT-I subcomplexes in quantitative ubiquitin-binding assays with matched positive and negative controls.