RHOBTB3

RHOBTB3 is an atypical Rho family GTPase that functions as a Golgi-associated ATPase and scaffolding protein, coordinating membrane trafficking, ubiquitin-dependent protein degradation, and cell cycle control. It hydrolyzes ATP rather than GTP, with maximal ATPase activity achieved upon Rab9 binding that relieves autoinhibition, and cooperates with Rab9 and TIP47 in endosome-to-trans-Golgi network transport (PMID:19490898); at Golgi–endosome contacts it additionally assembles a complex with SHIP164 and Vps26B to drive Rab14-positive early endosome bud formation (PMID:38565878). RHOBTB3 serves as a substrate adaptor for CUL3/RBX1 E3 ubiquitin ligase complexes on the Golgi, targeting cyclin E for ubiquitylation to regulate S/G2 cell cycle progression (PMID:24145166), and mediating K27-linked polyubiquitination of AMBRA1 to suppress autophagy (PMID:39404422). RHOBTB3 also organizes a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes HIFα hydroxylation and subsequent ubiquitination under normoxia; hypoxia disrupts this scaffold, stabilizing HIFα (PMID:26215701).

1. 2009 High

   Establishing that RHOBTB3 is an ATPase rather than a GTPase, and that Rab9-dependent relief of autoinhibition couples its enzymatic activity to endosome-to-TGN trafficking, provided the first mechanistic framework for this atypical Rho family member.

   Evidence In vitro ATPase assays, direct binding reconstitution with Rab9, domain-swap rescue experiments, and co-immunoprecipitation in mammalian cells

   PMID:19490898

   Open questions at the time

   • Structural basis for autoinhibition and its relief by Rab9 is unknown
   • Role of ATP hydrolysis versus ATP binding in trafficking has not been dissected
   • TIP47 release model lacks direct reconstitution of vesicle docking/fusion
2. 2012 Medium

   Discovery that RHOBTB3 binds the 5-HT7a serotonin receptor and inhibits its proteasomal degradation — independent of CUL3 recruitment — revealed a non-ubiquitin-ligase protective function for RHOBTB3 at the ER/plasma membrane.

   Evidence Yeast two-hybrid screen, co-IP with domain mapping, immunofluorescence co-localization, proteasome inhibitor experiments in HEK293T cells

   PMID:22245496

   Open questions at the time

   • Mechanism by which RHOBTB3 inhibits receptor degradation without mediating ubiquitination is unresolved
   • Interaction has not been confirmed by an independent lab
   • Physiological significance in serotonergic signaling is untested
3. 2013 High

   Identification of RHOBTB3 as a Golgi-localized CUL3/RBX1 E3 ligase adaptor that ubiquitylates cyclin E established its role as a cell cycle regulator linking Golgi integrity to S/G2 transition.

   Evidence Reciprocal co-IP, RNAi depletion with cell cycle arrest and Golgi fragmentation phenotypes, in vivo ubiquitylation assays, Golgi-targeting experiments

   PMID:24145166

   Open questions at the time

   • How Golgi localization is required for ligase activity is mechanistically unclear
   • Whether cyclin E is ubiquitylated directly on the Golgi membrane or after release is unresolved
   • Relationship between Golgi fragmentation and cyclin E accumulation is correlative
4. 2015 High

   Assembly of a RHOBTB3/LIMD1–PHD2–VHL scaffold that promotes both HIFα hydroxylation and ubiquitination demonstrated that RHOBTB3 coordinates an oxygen-sensing degradation complex disrupted by hypoxia.

   Evidence Co-IP, in vitro hydroxylation assay, ubiquitination assay, RNAi with HIFα readouts, xenograft model

   PMID:26215701

   Open questions at the time

   • Whether scaffold assembly is constitutive or regulated by signals other than hypoxia is unknown
   • Subcellular site of RHOBTB3–PHD2–VHL complex assembly has not been determined
   • Stoichiometry and structural organization of the pentameric complex are uncharacterized
5. 2019 Medium

   Genetic knockout in mice revealed that RHOBTB3 restrains platelet alpha-granule secretion and supports adhesion under flow, extending its functional repertoire to hemostasis.

   Evidence RhoBTB3-knockout mouse, platelet aggregation/secretion assays, flow adhesion assay, flow cytometry

   PMID:30754723

   Open questions at the time

   • Molecular mechanism linking RHOBTB3 to granule secretion machinery is unknown
   • Whether the phenotype depends on CUL3-dependent ubiquitination or ATPase activity is untested
   • In vivo thrombosis or bleeding phenotypes were not reported
6. 2020 Medium

   High-content screening confirmed that RHOBTB3 depletion alters Golgi morphology and carrier dynamics at the Golgi–ER interface, broadening its trafficking role beyond the endosome–TGN axis.

   Evidence RNAi-based imaging screen, live-cell carrier tracking, immunofluorescence

   PMID:32054068

   Open questions at the time

   • Whether Golgi–ER carrier phenotype is a direct consequence of RHOBTB3 ATPase or ubiquitin ligase activity is unknown
   • Molecular cargo affected in this route is not identified
   • Single-lab observation awaiting independent replication
7. 2024 High

   Identification of a RHOBTB3–SHIP164–Vps26B complex at Golgi–endosome contacts that drives Rab14-positive endosome bud formation established a new contact-site-based membrane remodeling function.

   Evidence Co-IP, RNAi depletion with rescue using wild-type and lipid-transfer-defective SHIP164 mutants, live-cell imaging, Rab14 effector pulldown

   PMID:38565878

   Open questions at the time

   • Whether RHOBTB3 ATPase activity is required for bud formation is untested
   • Lipid species transferred by SHIP164 at these contacts are not identified
   • Structural basis for RHOBTB3–SHIP164–Vps26B tricomplex is unknown
8. 2024 Medium

   Demonstrating that RHOBTB3 mediates K27-linked polyubiquitination of AMBRA1 at K45 to suppress autophagy added autophagy regulation to its CUL3-dependent substrates.

   Evidence IP/mass spectrometry substrate identification, site-specific K45 mutagenesis, ubiquitination assay, epistasis by double knockdown, autophagy flux assays

   PMID:39404422

   Open questions at the time

   • Single-lab finding; independent validation of K27-linked ubiquitination specificity is needed
   • Whether this regulation occurs at the Golgi like cyclin E ubiquitylation is unknown
   • Physiological contexts triggering RHOBTB3-dependent AMBRA1 degradation are uncharacterized

• How RHOBTB3's dual enzymatic activities — ATP hydrolysis and CUL3-dependent E3 ligase adaptor function — are coordinated on the Golgi membrane, and whether they serve the same or independent trafficking and signaling pathways, remains unresolved.
• No structural model of full-length RHOBTB3 or its CUL3-bound form exists
• Whether ATPase and ubiquitin ligase activities are mutually exclusive or cooperative is unknown
• In vivo phenotyping beyond platelets (e.g., conditional tissue-specific knockouts) has not been reported