RBX1

RBX1 (PF3D7_0319100) is the shared RING-domain catalytic subunit of Cullin-RING ligase (CRL) complexes that drive protein ubiquitination in Plasmodium (PMID:38416790). In P. falciparum it assembles into at least two distinct enzymatically active CRLs: a PfSCF complex comprising PfCullin-1, PfRbx1, PfSkp1, PfFBXO1 and calcyclin-binding protein (PMID:38416790), and a CRL4-like complex built on a cullin-4/cullin-2 scaffold with the DDB1 adaptor and WD40-repeat substrate receptors including WIG1 (PMID:38416790, PMID:39207167). Both complexes are competent for in vitro ubiquitination, with RBX1 supplying the RING activity common to each (PMID:38416790). RBX1 also binds directly to a novel parasite-specific E2 ubiquitin-conjugating enzyme, PF3D7_0811400, an interaction dependent on the E2's N-terminus (PMID:40249735). Functionally, the PfCullin-1-linked CRL is required for inner-membrane biogenesis and DNA replication, while the PfCullin-4-linked CRL supports DNA replication (PMID:40592953). In P. berghei, the orthologous RBX1 is confirmed as a component of the SCF-FBXO1 complex alongside SKP1, CUL1, FBXL2, polyubiquitin and Nedd8 (PMID:36898988). Beyond complex membership and these replication/membrane phenotypes, the specific substrates ubiquitinated by RBX1-containing CRLs have not been characterized in the available corpus.

1. 2023 Medium

   Establishing whether a Plasmodium RBX1 participates in a canonical SCF architecture was needed to place parasite CRL machinery in the ubiquitin system; showing RBX1 co-purifies with SCF components in P. berghei anchored it as a bona fide SCF subunit.

   Evidence Immunoprecipitation followed by mass spectrometry of FBXO1-HA/FBXL2-HA in P. berghei

   PMID:36898988

   Open questions at the time

   • Single-lab co-IP without RBX1 loss-of-function to test its individual requirement
   • Does not identify substrates of the P. berghei SCF-FBXO1 complex
   • P. falciparum orthology inferred, not directly tested here
2. 2024 High

   It was unknown whether P. falciparum RBX1 forms functional multi-complex CRLs; demonstrating its incorporation into both an enzymatically active PfSCF and a CRL4-like complex established RBX1 as a shared catalytic RING subunit of distinct parasite ligases.

   Evidence Reciprocal immunoprecipitation, in vitro protein-protein interaction and in vitro ubiquitination assays in P. falciparum

   PMID:38416790

   Open questions at the time

   • Substrates ubiquitinated by either complex not defined
   • Contribution of RBX1 alone versus the assembled complex to activity not dissected
   • Structural basis of RBX1 partitioning between cullin scaffolds unresolved
3. 2024 Medium

   Defining the receptor architecture of the P. falciparum CRL4-like complex clarified how substrate specificity might be achieved; identifying RBX1 with a cullin-4 scaffold, DDB1, and WD40-repeat receptors including WIG1 mapped the adaptor/receptor layer around the RING subunit.

   Evidence Co-immunoprecipitation with mass spectrometry/proteomics in P. falciparum asexual blood stages

   PMID:39207167

   Open questions at the time

   • Direct substrates recruited by the WD40 receptors not identified
   • Which receptors act via RBX1-dependent ubiquitination not established
4. 2025 Medium

   Connecting RBX1 to an E2 enzyme was needed to define the catalytic partner delivering ubiquitin; showing direct interaction with the parasite-specific E2 PF3D7_0811400 via its N-terminus identified the conjugating enzyme working with RBX1.

   Evidence Co-immunoprecipitation of myc-PfRbx1 with FLAG-tagged wild-type versus N-terminal truncation PF3D7_0811400, immunoblotting

   PMID:40249735

   Open questions at the time

   • Single-lab co-IP without reciprocal in vitro reconstitution of E2-RBX1 ubiquitin transfer
   • Whether this E2 serves all RBX1 CRLs or a specific subset not addressed
5. 2025 Medium

   The cellular processes requiring RBX1-containing CRLs were unknown; conditional deletion linked the PfCullin-1 CRL to inner-membrane biogenesis and DNA replication and the PfCullin-4 CRL to DNA replication, assigning biological roles to the RBX1 ligases.

   Evidence Endogenous HA-tagging, DiCre/LoxP conditional deletion, immunoprecipitation and proteomics in P. falciparum 3D7

   PMID:40592953

   Open questions at the time

   • Substrates whose ubiquitination underlies the membrane and replication phenotypes not identified
   • Direct requirement of the RBX1 subunit itself versus the cullin scaffolds not separated

• The physiological substrates targeted by RBX1-containing CRLs, and how RBX1 is partitioned among distinct cullin scaffolds, remain unresolved.
• No substrate of any RBX1 CRL identified in the corpus
• No structural model of RBX1 within the parasite complexes
• Mechanism selecting cullin-1 versus cullin-4/cullin-2 scaffolds unknown