Affinage

VPS28

Vacuolar protein sorting-associated protein 28 homolog · UniProt Q9UK41

Length
221 aa
Mass
25.4 kDa
Annotated
2026-06-11
24 papers in source corpus 16 papers cited in narrative 16 extracted findings
Cross-family judge vs UniProt: Affinage preferred faithfulness: 6/6 claims corpus-supported (100%)

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

VPS28 is a core subunit of the ESCRT-I complex that drives endosomal protein sorting and the membrane-remodeling events of multivesicular body biogenesis (PMID:8817003, PMID:11134028). The protein is recruited into ESCRT-I through a direct interaction with TSG101/VPS23 mediated by the conserved C-terminal portion of TSG101, and the assembled complex localizes to endosomes (PMID:11134028). Its C-terminal domain folds independently into a four-helical bundle that acts as an adaptor module recruiting the ESCRT-III component Vps20 downstream of ESCRT-I, coupling cargo sorting to membrane scission (PMID:16749904). Loss of VPS28 collapses transport out of the prevacuolar/late endosome and produces an aberrant class E compartment, establishing its requirement for forming transport intermediates (PMID:8817003). Through this ESCRT-I activity VPS28 controls multivesicular body formation and extracellular vesicle secretion, including release of VEGF-A-loaded neuronal EVs that pattern brain vasculature and maintain blood-brain barrier integrity (PMID:35330682), and is required for phagophore closure during autophagy (PMID:36907049). Under the alias CIIA, VPS28 additionally binds SOS1 and acts as a molecular switch, promoting SOS1-dependent Rac1 GEF activity to drive TGF-β-induced migration while inhibiting SOS1-mediated Ras–Erk1/2 signaling (PMID:22042618, PMID:24522193), and it negatively regulates ASK1-dependent apoptotic signaling by blocking ASK1–TRAF2 interaction in neuronal stress models (PMID:25018698, PMID:25098452).

Mechanistic history

Synthesis pass · year-by-year structured walk · 13 steps
  1. 1996 High

    Established that Vps28 is required for transport out of the prevacuolar endosome, defining its place in the endosomal sorting machinery before the ESCRT concept existed.

    Evidence Gene disruption with EM immunolocalization and trafficking/sorting assays in yeast

    PMID:8817003

    Open questions at the time
    • Did not identify direct protein partners
    • Molecular mechanism of intermediate formation unresolved
  2. 2000 High

    Showed that human VPS28 directly binds TSG101 to form part of the multiprotein ESCRT-I complex and localizes to endosomes, placing VPS28 in mammalian endosomal sorting.

    Evidence Co-IP, chemical cross-linking, and dominant-negative VPS4-induced relocalization in human cells

    PMID:11134028

    Open questions at the time
    • Stoichiometry and full subunit composition not defined
    • How the complex engages cargo not addressed
  3. 2006 High

    Defined the structural basis for VPS28 function, showing its C-terminal four-helix bundle is an adaptor that recruits ESCRT-III (Vps20) downstream of ESCRT-I.

    Evidence X-ray crystallography of yeast Vps28-CTD, pulldown mutagenesis, and EIAV Gag late-domain rescue

    PMID:16749904

    Open questions at the time
    • Structure of full-length VPS28 within assembled ESCRT-I not determined
    • Dynamics of ESCRT-I to ESCRT-III handoff not resolved
  4. 2005 Medium

    Extended VPS28/ESCRT-I function to signal transduction, linking it to the Rim101 pH-sensing pathway and fungal virulence.

    Evidence Gene deletion, transcriptional reporters, RIM101 epistasis, and mouse virulence model in Candida albicans

    PMID:16299290

    Open questions at the time
    • RIM101-independent branch unexplained
    • No direct biochemical link to pathway components
  5. 2006 Medium

    Tested whether VPS28 supports viral budding, finding influenza M1 binds VPS28 via an L-domain motif and that VPS28 depletion lowers viral output.

    Evidence Co-IP and siRNA knockdown with viral titer assay

    PMID:16474136

    Open questions at the time
    • Whether binding is functionally required for budding not settled here
  6. 2009 Medium

    Re-examined the VPS28 requirement for influenza budding and concluded budding proceeds independently of VPS28 and VPS4, qualifying the earlier dependence.

    Evidence Dominant-negative VPS4, overexpression, and siRNA depletion with morphology/titer readouts

    PMID:19524996

    Open questions at the time
    • Reason for discrepancy with the 2006 M1-binding result unresolved
    • Physiological role of M1–VPS28 binding unknown
  7. 2011 Medium

    Revealed a moonlighting role: VPS28/CIIA binds SOS1 and biases its GEF activity toward Rac1, driving TGF-β-induced migration.

    Evidence Co-IP, RNAi, GTPase pull-down activity assays, and migration assays

    PMID:22042618

    Open questions at the time
    • Relationship to ESCRT-I function not addressed
    • Single lab, no structural basis for the switch
  8. 2014 Medium

    Completed the SOS1 switch model by showing CIIA inhibits SOS1 Ras-GEF activity and suppresses EGF–Erk1/2 signaling, and fails to inhibit Noonan-associated SOS1 mutants.

    Evidence Co-IP, Ras GTPase pull-down, RNAi, and effector Western blots with SOS1 mutant analysis

    PMID:24522193

    Open questions at the time
    • Mechanism distinguishing Rac1 vs Ras outcomes unresolved
    • In vivo relevance not established
  9. 2009 Medium

    Linked CIIA to epithelial-mesenchymal transition, showing it remodels adhesion markers and promotes migration/invasion.

    Evidence Overexpression, RNAi, 3D culture, and migration/invasion assays with EMT marker blots

    PMID:19615336

    Open questions at the time
    • No direct biochemical mechanism for marker changes
    • Connection to SOS1/Rac1 axis not tested
  10. 2010 Low

    Implicated CIIA as a negative regulator of anoikis, suggesting a pro-survival role in detached cancer cells.

    Evidence RNAi knockdown with anoikis, caspase, and soft-agar assays

    PMID:20670956

    Open questions at the time
    • Single-method RNAi with no molecular pathway placement beyond caspase activation
    • No interacting partner identified
  11. 2014 Medium

    Defined a neuroprotective mechanism in which CIIA negatively regulates ASK1 signaling, shown across ALS and ischemia models by blocking ASK1–TRAF2 and ASK1 oligomerization.

    Evidence RNAi, Co-IP, ASK1 kinase assays, and mitochondrial/caspase readouts in SOD1(G93A) cells, neuroblastoma lines, and primary cortical neurons

    PMID:25018698 PMID:25098452

    Open questions at the time
    • Direct CIIA–ASK1 binding vs indirect effect not distinguished
    • Link to ESCRT-I or SOS1 roles unexplored
  12. 2022 Medium

    Connected VPS28's ESCRT-I/MVB function to a neurovascular signaling axis, showing Vps28 controls neuronal EV secretion of VEGF-A required for brain vascular sprouting and BBB integrity.

    Evidence Zebrafish genetic disruption, live imaging, EV isolation and rescue with VEGF-A detection, and BBB permeability assay

    PMID:35330682

    Open questions at the time
    • Mechanism of VEGF-A loading into EVs not defined
    • Mammalian conservation not tested
  13. 2023 Medium

    Provided chemical validation of VPS28 as a druggable ESCRT-I node, showing arctigenin binds VPS28 and triggers its proteasomal degradation to block phagophore closure.

    Evidence Chemoproteomic photo-crosslinking, MS target ID, proteasome inhibitor assays, and phagophore closure assay in PANC-1 cells

    PMID:36907049

    Open questions at the time
    • Binding site on VPS28 not mapped
    • Whether degradation acts only through autophagy unclear

Open questions

Synthesis pass · forward-looking unresolved questions
  • How VPS28's canonical ESCRT-I/MVB function mechanistically relates to its SOS1-switch and ASK1-regulatory activities, and whether these are separable molecular pools, remains unresolved.
  • No structural or interaction study reconciling ESCRT-I and CIIA roles
  • No mammalian disease-causing mutation in VPS28 reported in the corpus
  • Substrate/cargo specificity of mammalian ESCRT-I VPS28 not detailed

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0098772 molecular function regulator activity 4 GO:0060090 molecular adaptor activity 2
Localization
GO:0005768 endosome 2 GO:0005829 cytosol 2
Pathway
R-HSA-162582 Signal Transduction 4 R-HSA-5653656 Vesicle-mediated transport 3 R-HSA-9612973 Autophagy 1
Partners
SOS1TSG101VPS20
Complex memberships
ESCRT-I

Evidence

Reading pass · 16 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1996 Yeast Vps28p is a cytoplasmic 28 kDa hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar endosome; its loss causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar class E compartment, indicating Vps28p facilitates formation of transport intermediates at the prevacuolar endosome. Gene disruption, immunofluorescence, electron microscopy with immunolocalization, FM 4-64 endocytic trafficking assay, carboxypeptidase Y sorting assay Molecular biology of the cell High 8817003
2000 Human VPS28 (hVPS28) is a 221-amino acid cytosolic protein that directly interacts with TSG101/mammalian VPS23 via the conserved C-terminal portion of TSG101 to form part of a multiprotein ESCRT-I complex; upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates to the surface of enlarged endosomal vacuoles, implicating the complex directly in endosomal sorting. Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/localization The Journal of biological chemistry High 11134028
2006 The crystal structure of the C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Mutagenesis of its conserved surface abolishes interaction with the ESCRT-III component Vps20 in vitro and prevents rescue of an EIAV Gag late-domain deletion, demonstrating that Vps28-CTD acts as an adaptor module recruiting ESCRT-III (Vps20) downstream of ESCRT-I. X-ray crystallography, co-expression pulldown, site-directed mutagenesis, EIAV Gag late-domain rescue assay Traffic (Copenhagen, Denmark) High 16749904
2005 In Candida albicans, Vps28p (ESCRT-I) is required for signal transduction along the Rim101 pH-sensing pathway; VPS28 deletion impairs transcriptional regulation of Rim101 targets (PHR1, PHR2), and the growth defect at alkaline pH is only partially rescued by constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways. Gene deletion, transcriptional reporter assays, epistasis with constitutively active RIM101, in vivo mouse virulence model Infection and immunity Medium 16299290
2006 Influenza A virus M1 protein interacts with VPS28 (ESCRT-I component) via its YRKL L-domain motif; co-immunoprecipitation confirmed M1–VPS28 binding, and siRNA depletion of VPS28 reduced influenza virus production. Co-immunoprecipitation, Western blotting, siRNA knockdown, viral titer assay Journal of virology Medium 16474136
2009 Despite M1 binding VPS28 in vitro, overexpression or dominant-negative VPS4 and siRNA depletion of VPS28 had no significant effect on influenza virus replication or filamentous virion production, demonstrating that influenza budding occurs via a VPS4- and VPS28-independent mechanism. Confocal microscopy, dominant-negative VPS4 overexpression, VPS28 overexpression, siRNA knockdown, viral titer and morphology assay Virology Medium 19524996
2011 CIIA (VPS28) is a binding partner of SOS1 that functions as a molecular switch: it promotes the SOS1–Rac1 interaction and SOS1–EPS8 complex formation, thereby stimulating SOS1-mediated Rac1 GEF activity, while simultaneously inhibiting SOS1-mediated Ras activation. TGF-β upregulates CIIA expression, driving CIIA–SOS1 association and consequent Rac1-dependent cell migration; CIIA knockdown blocks these TGF-β-induced effects. Co-immunoprecipitation, RNAi knockdown, Rac1/Ras activity assays (GTPase pull-down), cell migration assay The Journal of cell biology Medium 22042618
2014 CIIA (VPS28) physically associates with SOS1 and inhibits its Ras-specific GEF activity, suppressing EGF-induced Ras–Erk1/2 pathway activation, cyclin D1 expression, and DNA synthesis. CIIA failed to inhibit Ras-GEF activity of Noonan-syndrome-associated SOS1 mutants (M269R, R552G, W729L, E846K). Co-immunoprecipitation, Ras activity (GTPase pull-down) assay, RNAi, Western blotting for pathway effectors Journal of cell science Medium 24522193
2009 CIIA (VPS28) promotes epithelial-mesenchymal transition (EMT): ectopic expression in MDCK cells downregulates E-cadherin and claudin-1 and upregulates N-cadherin, disrupts 3D epithelial morphology, and increases migration and invasion; endogenous CIIA depletion inhibits HeLa cell migration/invasion in a claudin-1-dependent manner. Ectopic overexpression, RNAi knockdown, 3D Matrigel culture, migration/invasion assay, Western blotting for EMT markers Biochemical and biophysical research communications Medium 19615336
2010 CIIA (VPS28) acts as a negative regulator of anoikis (detachment-induced apoptosis): CIIA knockdown in SW620 and KM12SM colon cancer cells promotes cell death after detachment through caspase activation, and inhibits anchorage-independent growth. RNAi knockdown, anoikis assay, caspase activity assay, soft-agar colony formation Cancer research Low 20670956
2014 CIIA (VPS28) negatively modulates ASK1-mediated cytotoxic signaling in a SOD1(G93A) ALS cell model: CIIA knockdown enhances ASK1–TRAF2 interaction, ASK1 activity, loss of mitochondrial membrane potential, cytochrome c release, and caspase activation induced by the ALS-linked SOD1 G93A mutant. RNAi knockdown, Co-immunoprecipitation (ASK1–TRAF2), ASK1 kinase activity assay, mitochondrial membrane potential assay, cytochrome c release assay, caspase assay Frontiers in cellular neuroscience Medium 25018698
2014 CIIA (VPS28) suppresses neuronal cell death from oxygen-glucose deprivation/reoxygenation by inhibiting ASK1 homo-oligomerization, blocking ASK1–TRAF2 binding, and suppressing downstream JNK and p38 kinase activation and caspase-3 activation. RNAi knockdown, OGD/R ischemia model in neuroblastoma lines and primary cortical neurons, Co-immunoprecipitation, kinase activity assays, caspase-3 assay Molecular and cellular biochemistry Medium 25098452
2019 Drosophila Vps28 (ESCRT-I component) is required for maintaining normal intracellular levels of Awd (NME1/2 homolog) in larval adipocytes; Vps28 loss reduces Awd intracellular levels, placing Vps28 upstream in the endosomal trafficking pathway that controls intracellular Awd abundance. Drosophila genetic loss-of-function, immunofluorescence, endosomal marker co-localization, confocal microscopy Frontiers in physiology Low 31427986
2022 Zebrafish Vps28 is essential for brain vascular sprouting (central arteries) and blood-brain barrier integrity by controlling MVB formation and thereby extracellular vesicle (EV) secretion from neurons; neuronal EVs containing VEGF-A rescued brain vasculature defects caused by Vps28 disruption, establishing a Vps28→MVB→EV→VEGF-A neurovascular signaling axis. Zebrafish genetic disruption (morpholino/mutant), in vivo live imaging, EV isolation and rescue experiments, VEGF-A detection in EVs, BBB permeability assay iScience Medium 35330682
2023 Arctigenin directly binds VPS28 (identified by chemoproteomic photo-crosslinking in living cells) and induces VPS28 degradation via the ubiquitin-proteasome pathway, causing a phagophore closure-blockade phenotype in PANC-1 cells, consistent with VPS28's role as an ESCRT-I subunit required for phagophore closure. Chemoproteomic photo-crosslinking with arctigenin probes, target identification by MS, ubiquitin-proteasome pathway inhibitor assays, autophagy/phagophore closure assay Bioorganic chemistry Medium 36907049
2019 LRSAM1 deregulation significantly reduces VPS28 protein levels in both CMT2P patient lymphoblastoid cells and LRSAM1-knockdown SH-SY5Y cells; TSG101 downregulation also reduces VPS28 levels, indicating VPS28 abundance is regulated downstream of the LRSAM1–TSG101 axis. LRSAM1 and TSG101 siRNA knockdown, protein expression analysis in patient cell lines and neuronal cells PloS one Low 30726272

Source papers

Stage 0 corpus · 24 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
1996 Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Molecular biology of the cell 251 8817003
2000 TSG101/mammalian VPS23 and mammalian VPS28 interact directly and are recruited to VPS4-induced endosomes. The Journal of biological chemistry 170 11134028
2005 Deletions of endocytic components VPS28 and VPS32 affect growth at alkaline pH and virulence through both RIM101-dependent and RIM101-independent pathways in Candida albicans. Infection and immunity 58 16299290
2009 Budding of filamentous and non-filamentous influenza A virus occurs via a VPS4 and VPS28-independent pathway. Virology 54 19524996
2006 The crystal structure of the C-terminal domain of Vps28 reveals a conserved surface required for Vps20 recruitment. Traffic (Copenhagen, Denmark) 51 16749904
2004 Multiple roles of CLAN (caspase-associated recruitment domain, leucine-rich repeat, and NAIP CIIA HET-E, and TP1-containing protein) in the mammalian innate immune response. Journal of immunology (Baltimore, Md. : 1950) 33 15528373
2006 Deletions of the endocytic components VPS28 and VPS32 in Candida albicans lead to echinocandin and azole hypersensitivity. Antimicrobial agents and chemotherapy 29 17005841
2006 YRKL sequence of influenza virus M1 functions as the L domain motif and interacts with VPS28 and Cdc42. Journal of virology 24 16474136
2022 VPS28 regulates brain vasculature by controlling neuronal VEGF trafficking through extracellular vesicle secretion. iScience 13 35330682
2019 Identification and functional analysis of candidate gene VPS28 for milk fat in bovine mammary epithelial cells. Biochemical and biophysical research communications 13 30739790
2011 CIIA functions as a molecular switch for the Rac1-specific GEF activity of SOS1. The Journal of cell biology 13 22042618
2019 Vps28 Is Involved in the Intracellular Trafficking of Awd, the Drosophila Homolog of NME1/2. Frontiers in physiology 6 31427986
2018 [Regulation of VPS28 gene knockdown on the milk fat synthesis in Chinese Holstein dairy]. Yi chuan = Hereditas 6 30559098
2014 CIIA negatively regulates the Ras-Erk1/2 signaling pathway through inhibiting the Ras-specific GEF activity of SOS1. Journal of cell science 6 24522193
2014 CIIA prevents SOD1(G93A)-induced cytotoxicity by blocking ASK1-mediated signaling. Frontiers in cellular neuroscience 6 25018698
2010 CIIA is a novel regulator of detachment-induced cell death. Cancer research 6 20670956
2021 Deciphering the Oncogenic Role of VPS28 Modulated by miR-491-5p in Breast Cancer Cells Using In Silico and Functional Analysis. Frontiers in molecular biosciences 5 34395516
2020 Comparative proteome analysis reveals VPS28 regulates milk fat synthesis through ubiquitylation in bovine mammary epithelial cells. PeerJ 5 33194328
2009 CIIA induces the epithelial-mesenchymal transition and cell invasion. Biochemical and biophysical research communications 4 19615336
2019 Deregulation of LRSAM1 expression impairs the levels of TSG101, UBE2N, VPS28, MDM2 and EGFR. PloS one 3 30726272
2014 CIIA negatively regulates neuronal cell death induced by oxygen-glucose deprivation and reoxygenation. Molecular and cellular biochemistry 3 25098452
2023 Chemoproteomics reveals arctigenin as a phagophore-closure blocker via targeting ESCRT-I subunit VPS28. Bioorganic chemistry 2 36907049
2024 Mechanism to disrupt ESCRT-mediated intracellular trafficking through Vps28-small molecules interaction: an in silico approach. Journal of biomolecular structure & dynamics 1 39668793
2024 VPS28 regulates triglyceride synthesis via ubiquitination in bovine mammary epithelial cells. Scientific reports 1 39732879

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