VPS36

VPS36 (EAP45) is the ubiquitin- and phosphoinositide-binding subunit of the ESCRT-II complex that couples recognition of ubiquitinated transmembrane cargo to endosomal membranes during multivesicular body (MVB) sorting. Its GLUE domain adopts a split pleckstrin-homology fold that simultaneously engages ubiquitin and 3-phosphoinositides, providing the molecular basis for ESCRT-II cargo selection (PMID:15755741, PMID:17057716); in Drosophila, Vps36-mediated ubiquitin recognition on Smoothened controls Hedgehog pathway output by directing receptor internalization (PMID:23843610). VPS36 is recruited to endosomes in part through RILP interaction (PMID:17010938), and its association with the small GTPase Arl4A stabilizes ESCRT-II/III coupling, modulates CHMP2A-dependent USP8 recruitment, and thereby regulates the kinetics of EGFR ubiquitination and lysosomal delivery (PMID:38030597). Beyond canonical MVB sorting, VPS36 is co-opted during HIV-1 budding through its N-terminal H0 domain, which is functionally separable from the GLUE domain used in cytokinetic abscission (PMID:34580994).

1. 2005 High

   Identifying how ESCRT-II contacts ubiquitinated cargo: discovery that the GLUE domain of VPS36 binds both ubiquitin and 3-phosphoinositides established VPS36 as the dual-specificity adaptor linking ESCRT-II to endosomal membranes bearing ubiquitinated cargo.

   Evidence Biochemical binding assays (ubiquitin and phosphoinositide pull-downs) and fluorescence colocalization on late endosomes

   PMID:15755741

   Open questions at the time

   • Structural basis of simultaneous ubiquitin and lipid binding was unknown
   • Functional consequence of disrupting GLUE-ubiquitin binding in vivo was not tested
2. 2006 High

   Structural resolution of the GLUE domain as a split PH fold explained how a single domain can bind both ubiquitin and phospholipid, providing the first atomic model for ESCRT-II cargo–membrane coupling.

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

   PMID:17057716

   Open questions at the time

   • No structure of GLUE bound simultaneously to ubiquitin and lipid
   • Relative contribution of ubiquitin- vs. lipid-binding surfaces in intact ESCRT-II not dissected
3. 2006 Medium

   The question of how ESCRT-II reaches endosomal membranes was partially answered by showing that the Rab7/Rab34 effector RILP directly binds VPS36 and VPS22, linking Rab-dependent membrane identity to ESCRT-II recruitment.

   Evidence Co-immunoprecipitation, domain mapping, and EGF sorting assays in mammalian cells

   PMID:17010938

   Open questions at the time

   • Interaction shown by overexpression Co-IP; endogenous reciprocal pull-down not performed
   • Whether RILP is essential for ESCRT-II recruitment in physiological trafficking is unclear
   • Relationship between RILP-mediated and PI3P-mediated ESCRT-II recruitment not resolved
4. 2013 High

   Demonstrating physiological specificity of VPS36 ubiquitin recognition, Drosophila Vps36 was shown to sort ubiquitinated Smoothened for internalization, with its loss causing membrane Smo accumulation and aberrant Hedgehog signaling — establishing ESCRT-II as a signaling regulator.

   Evidence Drosophila genetic loss-of-function, ubiquitination assays, trafficking and Hedgehog pathway epistasis

   PMID:23843610

   Open questions at the time

   • Whether VPS36 directly contacts Smo ubiquitin moieties via its GLUE domain was not shown biochemically
   • Mammalian relevance of VPS36-Hedgehog connection not tested
5. 2019 Medium

   Expanding VPS36 function beyond endogenous cargo, interaction with PEDV ORF3 independent of the GLUE domain indicated that VPS36 can restrict viral replication through a non-canonical binding interface.

   Evidence Co-IP/mass spectrometry identification of VPS36–ORF3 interaction; overexpression/knockdown of VPS36 with viral replication assays

   PMID:31022991

   Open questions at the time

   • Single-laboratory finding; no reciprocal validation by alternative binding method
   • Mechanism by which VPS36 reduces ORF3 levels (not lysosomal) is unresolved
   • Relevance to other coronaviruses not examined
6. 2021 Medium

   Domain-separation experiments revealed that VPS36's N-terminal H0 domain — distinct from GLUE — mediates association with HIV-1 Gag at the plasma membrane, while the GLUE domain is preferentially used during cytokinetic abscission, establishing that ESCRT-II exploits different VPS36 surfaces for viral vs. cellular functions.

   Evidence SNAP-tag live/fixed imaging with domain deletion constructs in HIV-1-producing and dividing cells

   PMID:34580994

   Open questions at the time

   • Direct binding between H0 domain and Gag not demonstrated biochemically
   • Whether H0-mediated recruitment is required (rather than redundant) for HIV-1 budding efficiency is unknown
7. 2023 High

   The small GTPase Arl4A was identified as a direct endosomal regulator of VPS36 that stabilizes ESCRT-II/III coupling and modulates downstream USP8 deubiquitinase access, explaining how EGFR ubiquitination dynamics and lysosomal delivery are tuned at the ESCRT-II–III transition.

   Evidence Reciprocal Co-IP, direct binding assays, siRNA epistasis, EGFR ubiquitination and degradation kinetics

   PMID:38030597

   Open questions at the time

   • Structural basis of Arl4A–VPS36 interaction and whether it competes with ubiquitin or lipid binding is unknown
   • Generality of Arl4A regulation beyond EGFR cargo not tested
   • Whether Arl4A-GTP cycling controls the duration of ESCRT-II–III association in vivo is unresolved

• A unified structural model of full-length mammalian VPS36 in complex with ESCRT-II, simultaneously engaging ubiquitin, PI3P, and Arl4A on an endosomal membrane, is still lacking; the relative hierarchy among RILP, PI3P, and Arl4A in controlling ESCRT-II membrane recruitment remains undefined.
• No cryo-EM or crystal structure of mammalian ESCRT-II holo-complex with VPS36 GLUE domain in membrane-bound state
• Quantitative dissection of RILP vs. Arl4A vs. PI3P contributions to endosomal ESCRT-II recruitment is missing
• Role of VPS36 in non-endosomal ESCRT functions (e.g., nuclear envelope sealing, autophagy) is unexplored in primary literature