VPS36

VPS36 (EAP45) is the ubiquitin-binding subunit of the mammalian ESCRT-II complex that couples recognition of ubiquitinated cargo to endosomal membrane lipids during multivesicular body sorting (PMID:15755741, PMID:17057716). Cargo recognition is mediated by its GLUE domain, a split pleckstrin-homology fold that binds ubiquitin along one edge of its beta-sandwich while simultaneously engaging a subset of 3-phosphoinositides, structurally integrating ubiquitin and phosphoinositide sensing on late endosomes (PMID:15755741, PMID:17057716). Membrane recruitment of ESCRT-II is reinforced by direct interaction of VPS36 (and VPS22) with RILP, which couples the early and late endocytic machinery and influences EGF cargo sorting toward degradation (PMID:17010938). VPS36 sets the kinetics of receptor downregulation: Arl4A binding to VPS36 stabilizes the VPS36–ESCRT-III association and attenuates CHMP2A-dependent recruitment of the deubiquitinase USP8, prolonging EGFR ubiquitination and deterring its transport from endosomes to lysosomes (PMID:38030597). Through ubiquitin-dependent cargo sorting VPS36 also negatively regulates Hedgehog signaling by directing Smoothened away from the cell surface in the absence of Hh signal (PMID:23843610). Beyond endosomal sorting, VPS36 participates in cytokinesis and is co-opted at the plasma membrane during HIV-1 budding through domain-specific contributions, with the N-terminal H0 domain mediating association with budding Gag and the GLUE domain being more critical for cytokinesis (PMID:34580994).

1. 2005 High

   Establishing how ESCRT-II reads cargo, the discovery that VPS36 harbors the GLUE domain showed a single module can recognize both ubiquitin and endosomal lipids, explaining how ubiquitinated cargo is captured at the membrane.

   Evidence Biochemical ubiquitin- and phosphoinositide-binding assays with immunofluorescence colocalization on late endosomes

   PMID:15755741

   Open questions at the time

   • Does not resolve the structural basis of simultaneous ubiquitin/lipid binding
   • Selectivity among 3-phosphoinositides not fully defined
2. 2006 High

   The crystal structure of the GLUE domain settled how dual recognition is achieved, revealing a split-PH fold that binds ubiquitin along one edge of its beta-sandwich while accommodating phospholipid recognition.

   Evidence X-ray crystallography with biochemical binding validation of the human EAP45 GLUE domain

   PMID:17057716

   Open questions at the time

   • Structure of GLUE within the assembled ESCRT-II complex not determined
   • Membrane-bound conformation not captured
3. 2006 Medium

   Identifying RILP as a direct VPS36/VPS22 partner answered how ESCRT-II is recruited to membranes, linking early sorting endosome machinery to ESCRT-II-dependent cargo degradation.

   Evidence Co-immunoprecipitation, domain mapping, endosome marker analysis and EGF degradation assay (single lab)

   PMID:17010938

   Open questions at the time

   • Recruitment mechanism shown largely via overexpression
   • Direct contribution to physiological ESCRT-II assembly not isolated
4. 2013 Medium

   Genetic work in Drosophila connected VPS36 cargo sorting to signaling control, showing Vps36 recognizes ubiquitinated Smoothened to restrain Hedgehog pathway activity.

   Evidence Drosophila loss-of-function genetics with ubiquitination, surface trafficking, and Hh epistasis assays (single lab)

   PMID:23843610

   Open questions at the time

   • Conservation of Smo regulation in mammalian VPS36 not established
   • Ubiquitin ligase directing Smo not identified here
5. 2019 Medium

   A virus interaction screen revealed a non-canonical, GLUE-independent VPS36 binding and antiviral role, with VPS36 suppressing PEDV replication by lowering ORF3 levels through a lysosome-independent route.

   Evidence IP/mass spectrometry, co-IP domain mapping, overexpression/knockdown viral replication assays with lysosomal inhibitors (single lab)

   PMID:31022991

   Open questions at the time

   • Mechanism of ORF3 reduction not defined
   • Domain mediating the VPS36-ORF3 interaction unmapped
6. 2021 Medium

   Imaging during HIV-1 budding assigned domain-specific roles to VPS36, separating an H0-dependent association with budding Gag from a GLUE-dependent role in cytokinesis.

   Evidence SNAP-tag fixed and live-cell imaging with domain truncation/mutation (single lab)

   PMID:34580994

   Open questions at the time

   • Functional necessity of EAP45 for HIV-1 release not quantified
   • Mechanism of virion packaging unresolved
7. 2023 High

   Defining a regulatory input on receptor downregulation, Arl4A binding to VPS36 was shown to stabilize ESCRT-III association and block USP8 recruitment, thereby slowing EGFR deubiquitination and lysosomal delivery.

   Evidence Co-IP, pulldown, mutagenesis, endosomal fractionation, EGFR degradation and USP8 recruitment assays, live-cell imaging

   PMID:38030597

   Open questions at the time

   • Generality of Arl4A control beyond EGFR cargo not established
   • Structural basis of Arl4A-VPS36 contact not resolved

• How VPS36-dependent cargo selection and ESCRT-II/III dynamics are integrated across diverse cargoes and signaling contexts in mammalian cells remains incompletely defined.
• Mammalian Smoothened regulation by VPS36 untested
• Cargo-specific deubiquitination control beyond EGFR unknown
• In situ ESCRT-II assembly structure lacking