Affinage

VPS28

Vacuolar protein sorting-associated protein 28 homolog · UniProt Q9UK41

Round 2 corrected
Length
221 aa
Mass
25.4 kDa
Annotated
2026-04-28
54 papers in source corpus 13 papers cited in narrative 14 extracted findings

Mechanistic narrative

Synthesis pass · prose summary of the discoveries below

VPS28 is a core subunit of the ESCRT-I complex that functions as a central adaptor linking ubiquitinated cargo recognition to downstream membrane remodeling during multivesicular body (MVB) biogenesis, viral budding, phagophore closure, and cytokinesis. Its N-terminal region binds directly to TSG101/VPS23 to assemble ESCRT-I, while its independently folded C-terminal four-helical-bundle domain recruits ESCRT-III via direct interaction with Vps20, a connection essential for membrane scission events including retroviral budding (PMID:16749904, PMID:11134028). VPS28-dependent endosomal sorting is required for degradation of ubiquitinated receptors such as EGFR (PMID:11916981), for neuronal extracellular vesicle secretion that supports brain angiogenesis (PMID:35330682), and for phagophore closure during autophagy (PMID:36907049). Loss of VPS28 also causes lysosome-dependent transcriptional reprogramming toward aerobic glycolysis via NF-κB and JNK pathway activation (PMID:12663786, PMID:8817003).

Mechanistic history

Synthesis pass · year-by-year structured walk · 8 steps
  1. 1996 High

    Identification of Vps28p as a class E VPS protein resolved a key gap in understanding how transport intermediates form at the prevacuolar endosome, establishing that loss of this cytosolic factor produces the characteristic multilamellar class E compartment and CPY missorting.

    Evidence Yeast null mutant with fluorescence/EM immunolocalization and pulse-chase sorting assays

    PMID:8817003

    Open questions at the time
    • Molecular partners and complex membership unknown
    • Mechanism of membrane sorting activity undefined
    • No mammalian homolog characterized
  2. 2000 High

    Demonstration that human VPS28 directly binds TSG101 and co-localizes on endosomal membranes upon VPS4 inactivation defined VPS28 as a bona fide ESCRT-I subunit in mammals and established the TSG101–VPS28 interaction as the complex's organizational axis.

    Evidence Reciprocal co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 expression with confocal microscopy

    PMID:11134028

    Open questions at the time
    • Stoichiometry and additional subunits of ESCRT-I not determined
    • How ESCRT-I connects to downstream ESCRT machinery unknown
  3. 2002 High

    Functional antibody microinjection showed that VPS28 is required for endosomal clearance of ubiquitinated cargo and EGFR degradation, moving VPS28 from a structural component to an active participant in receptor downregulation.

    Evidence Anti-VPS28 antibody microinjection with EGF trafficking assay, ubiquitin-agarose pulldown, immunofluorescence colocalization

    PMID:11916981

    Open questions at the time
    • How VPS28/ESCRT-I communicates with ESCRT-III not resolved
    • Whether VPS28 directly contacts ubiquitin or acts through TSG101 unclear
  4. 2003 High

    Mapping of the ESCRT network revealed that VPS28's binding site on TSG101 is essential for HIV-1 budding, and that the entire class E VPS machinery including VPS28 participates in both MVB biogenesis and viral egress.

    Evidence TSG101 mutagenesis with HIV-1 budding complementation assays; systematic interaction mapping and dominant-negative validation

    PMID:12663786 PMID:14505570

    Open questions at the time
    • Structural basis of VPS28–TSG101 interaction unresolved
    • Whether VPS28 has autonomous functions beyond scaffolding unknown
  5. 2006 High

    Crystal structure of the VPS28 C-terminal domain revealed a four-helical bundle that recruits ESCRT-III via direct binding to Vps20, solving the mechanistic question of how ESCRT-I hands off to ESCRT-III; mutations on the conserved Vps20-binding surface abolished both the interaction and viral budding rescue.

    Evidence X-ray crystallography at 3.05 Å, site-directed mutagenesis, EIAV Gag late-domain rescue assay

    PMID:16749904

    Open questions at the time
    • Full-length ESCRT-I structure with VPS28 not yet available
    • Whether VPS28-CTD has additional binding partners beyond Vps20 not explored
  6. 2005 Medium

    Genetic epistasis in Candida albicans placed Vps28 upstream of both Rim101-dependent and Rim101-independent pH-responsive pathways, revealing that ESCRT-I controls transcriptional signaling beyond simple endosomal sorting, with consequences for pathogen virulence.

    Evidence Gene deletion, epistasis with constitutively active Rim101, mouse virulence model

    PMID:16299290

    Open questions at the time
    • Mechanism of Rim101-independent signaling through Vps28 not defined
    • Relevance to mammalian signaling pathways not established
  7. 2022 Medium

    VPS28 was shown to be essential for neuronal MVB formation and extracellular vesicle secretion, with neuronal EVs carrying VEGF-A to promote brain angiogenesis; EV rescue experiments established a causal neurovascular communication axis dependent on VPS28.

    Evidence Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue, mouse cortical neuron EV assay

    PMID:35330682

    Open questions at the time
    • Whether VPS28 has cell-type-specific roles beyond neurons not tested
    • Direct VEGF-A sorting into EVs by VPS28 not demonstrated biochemically
  8. 2023 Medium

    Chemoproteomic identification of VPS28 as the direct target of arctigenin, whose binding triggers VPS28 proteasomal degradation and blocks phagophore closure, established VPS28 as functionally required for autophagosome maturation.

    Evidence Photo-crosslinkable chemoproteomic probes, proteasome inhibition rescue, phagophore closure phenotyping in PANC-1 cells

    PMID:36907049

    Open questions at the time
    • Whether VPS28 acts in phagophore closure through ESCRT-III recruitment or a distinct mechanism is untested
    • Generalizability beyond PANC-1 cells not shown

Open questions

Synthesis pass · forward-looking unresolved questions
  • It remains unknown how VPS28-dependent lysosomal flux restrains NF-κB/JNK-driven glycolytic reprogramming, whether VPS28 has ESCRT-I-independent functions, and what regulates VPS28 protein turnover beyond LRSAM1/TSG101 co-dependency.
  • Mechanism linking ESCRT-I loss to NF-κB/JNK activation not molecularly defined
  • Post-translational regulation of VPS28 poorly characterized
  • Full-length ESCRT-I structural model including VPS28 lacking

Mechanism profile

Synthesis pass · controlled-vocabulary classification · explore literature graph →
Molecular activity
GO:0005198 structural molecule activity 3 GO:0060090 molecular adaptor activity 2
Localization
GO:0005768 endosome 2 GO:0005829 cytosol 2 GO:0031410 cytoplasmic vesicle 2
Pathway
R-HSA-5653656 Vesicle-mediated transport 4 R-HSA-1643685 Disease 2 R-HSA-9612973 Autophagy 1
Partners
ALIXTSG101VPS20VPS37
Complex memberships
ESCRT-I

Evidence

Reading pass · 14 per-paper findings extracted from the source corpus
Year Finding Method Journal Conf PMIDs
1996 Yeast Vps28p is a 28 kDa cytoplasmic hydrophilic protein required for efficient anterograde and retrograde transport out of the prevacuolar/endosomal compartment. Loss of VPS28 causes accumulation of vacuolar, endocytic, and late Golgi markers in an aberrant multilamellar endosome-like 'class E' compartment, with ~40-50% of carboxypeptidase Y missorted, placing Vps28p in the class E VPS pathway that facilitates formation of transport intermediates at the prevacuolar endosome. Genetic disruption (null mutant), fluorescence microscopy with FM 4-64 and marker proteins, immunolocalization by electron microscopy, pulse-chase 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 to form part of a multiprotein ESCRT-I complex. Direct binding requires structural information within the conserved C-terminal portion of TSG101. Upon expression of dominant-negative VPS4, a portion of both TSG101 and hVPS28 translocates from the cytosol to the surface of enlarged endosomal vacuoles, implicating them directly in endosomal sorting. Co-immunoprecipitation, chemical cross-linking, dominant-negative VPS4 overexpression, subcellular fractionation/confocal microscopy The Journal of biological chemistry High 11134028
2002 The TSG101/hVPS28 cytosolic complex binds ubiquitin-agarose, and hVPS28 localizes to endosomes containing internalized EGF receptor and ubiquitinated proteins. Microinjection of anti-hVPS28 antibody retards EGF degradation and causes endosomal accumulation of ubiquitin-protein conjugates, establishing that hVPS28 (as part of ESCRT-I) acts in the removal of ubiquitinated cargo from endosomes. Ubiquitin-agarose pulldown, immunofluorescence colocalization, antibody microinjection with EGF trafficking assay The Journal of cell biology High 11916981
2003 VPS28 is a component of ESCRT-I that binds to a sequence near the TSG101 C-terminus and is recruited to the plasma membrane by HIV-1 Gag. The integrity of the VPS28-binding site within TSG101 is required for HIV-1 particle budding. TSG101 also exhibits multimerization activity, and mutations disrupting VPS28 binding or multimerization impair TSG101's ability to support HIV-1 budding. Co-immunoprecipitation, complementation assay with artificially recruited TSG101 mutants, HIV-1 budding assay (particle release quantification) Journal of virology High 12663786
2003 VPS28 is part of the ESCRT-I complex, which is connected to ESCRT-III via AIP1/ALIX. The entire class E VPS protein network (including VPS28) participates in HIV-1 release and MVB biogenesis, as dominant-negative mutants of late-acting class E proteins arrest HIV-1 budding through both plasma and endosomal membranes. Protein-protein interaction mapping, dominant-negative expression, HIV budding assay, identification of 22 human class E proteins Cell High 14505570
2006 The crystal structure of the conserved C-terminal domain of yeast Vps28 (Vps28-CTD) was solved at 3.05 Å resolution, revealing a four-helical bundle that folds independently. Co-expression experiments showed Vps28-CTD does not directly participate in ESCRT-I assembly (with Vps23/Vps37) but instead acts as an adaptor module. Mutagenesis of a strictly conserved surface on Vps28-CTD abolished interaction with the ESCRT-III factor Vps20. Vps28-CTD is sufficient to rescue an EIAV Gag late-domain deletion, and mutations abolishing Vps20 interaction also prevent this rescue, demonstrating that Vps28-CTD recruits ESCRT-III via Vps20. X-ray crystallography (3.05 Å), 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 transcriptional regulation downstream of the Rim pathway, controlling pH-responsive target genes PHR1 and PHR2. Deletion of VPS28 has a more severe effect on alkaline growth than RIM101 deletion, and this effect is only partially suppressed by a constitutively active Rim101p, indicating VPS28 acts both through RIM101-dependent and RIM101-independent pathways. VPS28 deletion also significantly reduces virulence in a mouse model. Gene deletion, genetic epistasis (rim101 suppression assay), reporter gene analysis, mouse virulence model Infection and immunity Medium 16299290
2009 The YRKL sequence of influenza A virus M1 functions as an L-domain motif that interacts with VPS28 (a component of ESCRT-I) and Cdc42. Co-immunoprecipitation showed M1 binds VPS28 via the YRKL motif. siRNA depletion of VPS28 reduced influenza virus production, indicating VPS28 participates in the influenza virus life cycle via M1 interaction. Co-immunoprecipitation, Western blotting, siRNA knockdown with virus titer measurement, position-independent L-domain insertion assay Journal of virology Medium 16474136
2009 Influenza A virus M1 binds VPS28 (confirmed by pulldown), but confocal microscopy showed no colocalization between M1 and VPS28 or VPS4 in infected cells, and siRNA depletion of endogenous VPS28 had no significant effect on influenza virus replication or filamentous virion production, indicating VPS28/ESCRT-I is not required for influenza budding. siRNA knockdown, confocal microscopy, virus replication assay (filamentous and non-filamentous strains), dominant-negative VPS4 overexpression, VPS28 overexpression Virology Medium 19524996
2019 In Drosophila larval adipocytes, the Vps28 component of ESCRT-I is required for maintenance of normal intracellular levels of Awd (the fly homolog of NME1/2). Loss of Vps28 disrupts normal intracellular trafficking of Awd, and Awd partly colocalizes with the ESCRT accessory component ALiX in fat body cells, suggesting ESCRT-I-dependent endosomal routing controls NME1/2 protein levels. Genetic loss-of-function in Drosophila, fluorescence microscopy with endosomal markers (CD63), colocalization with ALiX Frontiers in physiology Medium 31427986
2021 LRSAM1 deregulation significantly decreases VPS28 protein levels in CMT2P patient lymphoblastoid cell lines and in LRSAM1-knockdown SH-SY5Y cells. TSG101 downregulation also reduces VPS28 expression, suggesting VPS28 protein stability depends on TSG101 (its ESCRT-I partner) and is indirectly regulated by the LRSAM1 ubiquitin ligase. RNAi knockdown in cell lines, expression analysis in patient-derived lymphoblastoid cells PloS one Low 30726272
2022 VPS28 is essential for the sprouting of brain central arteries and blood-brain barrier integrity in zebrafish. Neuron-enriched Vps28 regulates the formation of intracellular multivesicular bodies (MVBs) to control extracellular vesicle (EV) secretion by neurons. Neuronal EVs containing VEGF-A act as key regulators in neurovascular communication, and EVs from zebrafish embryos or mouse cortical neurons partially rescued brain vasculature defects and BBB leakage caused by Vps28 disruption. Zebrafish vps28 morpholino knockdown, live imaging, EV isolation and rescue experiments, mouse cortical neuron EV assay, VEGF-A trafficking analysis iScience Medium 35330682
2023 VPS28, a key subunit of ESCRT-I implicated in phagophore closure, is a direct target of the natural compound arctigenin. Chemoproteomics using photo-crosslinkable probes identified VPS28 as the direct cellular binding partner of arctigenin, which triggers VPS28 degradation via the ubiquitin-proteasome pathway and induces a phagophore closure-blockade phenotype in PANC-1 cells, establishing VPS28 as required for autophagosome (phagophore) closure. Chemoproteomic profiling with photo-crosslinkable probes in living cells, ubiquitin-proteasome pathway inhibition assay, phagophore closure phenotype analysis Bioorganic chemistry Medium 36907049
2024 ESCRT-I deficiency (loss of TSG101 or VPS28) reduces expression of genes encoding fatty acid and amino acid oxidation enzymes, increases glycolytic gene expression, causes intracellular lipid accumulation and increased lactate production. Mechanistically, this transcriptional reprogramming toward aerobic glycolysis is driven by activation of canonical NFκB and JNK signaling pathways. Inhibiting lysosomal activity phenocopies these effects, indicating ESCRT-I restricts glycolysis by mediating lysosomal degradation. Transcriptome analysis of TSG101/VPS28 knockout cells, metabolic assays (lactate, lipid staining, respiration), NFκB/JNK pathway inhibition, lysosomal inhibitor phenocopy bioRxivpreprint Medium

Source papers

Stage 0 corpus · 54 papers · ranked by NIH iCite citations
Year Title Journal Citations PMID
2005 Towards a proteome-scale map of the human protein-protein interaction network. Nature 2090 16189514
2005 A human protein-protein interaction network: a resource for annotating the proteome. Cell 1704 16169070
2002 Generation and initial analysis of more than 15,000 full-length human and mouse cDNA sequences. Proceedings of the National Academy of Sciences of the United States of America 1479 12477932
2015 The BioPlex Network: A Systematic Exploration of the Human Interactome. Cell 1118 26186194
2017 Architecture of the human interactome defines protein communities and disease networks. Nature 1085 28514442
2015 A human interactome in three quantitative dimensions organized by stoichiometries and abundances. Cell 1015 26496610
2014 A proteome-scale map of the human interactome network. Cell 977 25416956
2020 A reference map of the human binary protein interactome. Nature 849 32296183
2003 Complete sequencing and characterization of 21,243 full-length human cDNAs. Nature genetics 754 14702039
2021 Dual proteome-scale networks reveal cell-specific remodeling of the human interactome. Cell 705 33961781
2003 The protein network of HIV budding. Cell 695 14505570
2011 Phylogenetic-based propagation of functional annotations within the Gene Ontology consortium. Briefings in bioinformatics 656 21873635
2008 An empirical framework for binary interactome mapping. Nature methods 652 19060904
2010 Membrane budding and scission by the ESCRT machinery: it's all in the neck. Nature reviews. Molecular cell biology 612 20588296
2008 Large-scale proteomics and phosphoproteomics of urinary exosomes. Journal of the American Society of Nephrology : JASN 607 19056867
2007 Human ESCRT and ALIX proteins interact with proteins of the midbody and function in cytokinesis. The EMBO journal 595 17853893
2004 The status, quality, and expansion of the NIH full-length cDNA project: the Mammalian Gene Collection (MGC). Genome research 438 15489334
2022 OpenCell: Endogenous tagging for the cartography of human cellular organization. Science (New York, N.Y.) 432 35271311
2016 Widespread Expansion of Protein Interaction Capabilities by Alternative Splicing. Cell 423 26871637
2010 Systematic analysis of human protein complexes identifies chromosome segregation proteins. Science (New York, N.Y.) 421 20360068
2003 Protein sorting into multivesicular endosomes. Current opinion in cell biology 409 12892785
2015 Panorama of ancient metazoan macromolecular complexes. Nature 407 26344197
2006 Proteomic and biochemical analysis of purified human immunodeficiency virus type 1 produced from infected monocyte-derived macrophages. Journal of virology 382 16940516
2000 Secretory protein trafficking and organelle dynamics in living cells. Annual review of cell and developmental biology 380 11031247
2003 Divergent retroviral late-budding domains recruit vacuolar protein sorting factors by using alternative adaptor proteins. Proceedings of the National Academy of Sciences of the United States of America 337 14519844
1996 Multilamellar endosome-like compartment accumulates in the yeast vps28 vacuolar protein sorting mutant. Molecular biology of the cell 251 8817003
2009 Proteomic analysis of human parotid gland exosomes by multidimensional protein identification technology (MudPIT). Journal of proteome research 237 19199708
2002 Mammalian class E vps proteins recognize ubiquitin and act in the removal of endosomal protein-ubiquitin conjugates. The Journal of cell biology 234 11916981
2010 MHC class II-associated proteins in B-cell exosomes and potential functional implications for exosome biogenesis. Immunology and cell biology 221 20458337
2003 Role of ESCRT-I in retroviral budding. Journal of virology 218 12663786
2009 Proteomic analysis of integrin-associated complexes identifies RCC2 as a dual regulator of Rac1 and Arf6. Science signaling 207 19738201
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 VPS28 regulates triglyceride synthesis via ubiquitination in bovine mammary epithelial cells. Scientific reports 1 39732879
2024 Mechanism to disrupt ESCRT-mediated intracellular trafficking through Vps28-small molecules interaction: an in silico approach. Journal of biomolecular structure & dynamics 0 39668793