FBXO1

TgFBXO1 is a bona fide F-box protein of Toxoplasma gondii that binds SKP1 to serve as a substrate receptor within an SCF (SKP1-Cullin-F-box) E3 ubiquitin ligase complex, and it functions as an effector of daughter cell scaffold assembly during parasite replication (PMID:31348812). In interphase parasites it localizes to the Inner Membrane Complex, and early in replication it translocates to the developing daughter cell scaffold in a cell-cycle-coupled manner tied to centrosome duplication (PMID:31348812). This scaffold recruitment requires N-myristoylation of TgFBXO1 and depends on the small GTPase TgRab11b, placing TgFBXO1 downstream of TgRab11b in the pathway governing daughter cell scaffold formation (PMID:31348812). Loss of TgFBXO1 impairs scaffold function and blocks parasite growth (PMID:31348812). Unlike the F-box proteins FBXO13 and FBXO14, TgFBXO1 abundance is independent of oxygen-dependent SKP1 glycosylation controlled by the O2-sensing hydroxylase PHYa (PMID:39307307). The ubiquitylation substrates of the TgFBXO1-containing SCF complex have not been identified in the available corpus.

1. 2019 Medium

   Whether TgFBXO1 acts as a canonical SCF substrate receptor was unknown; demonstrating direct SKP1 binding established it as a bona fide F-box protein and component of an E3 ubiquitin ligase complex.

   Evidence Interactome screen with reciprocal Co-IP/pulldown confirming SKP1 binding in T. gondii

   PMID:31348812

   Open questions at the time

   • No ubiquitylation substrate of the TgFBXO1-SCF complex identified
   • Cullin and full complex composition not defined for this receptor
2. 2019 Medium

   The cellular role of TgFBXO1 was unknown; cell-cycle-resolved imaging established that it moves from the interphase IMC to the developing daughter cell scaffold in coordination with centrosome duplication.

   Evidence Fluorescence/live imaging and cell-cycle-stage-specific localization with centrosome and kinetochore markers

   PMID:31348812

   Open questions at the time

   • Molecular trigger coupling relocalization to centrosome duplication not defined
   • Does not establish whether ubiquitin ligase activity is required at the scaffold
3. 2019 Medium

   How TgFBXO1 is targeted to the daughter scaffold was unknown; mutagenesis and epistasis showed recruitment requires N-myristoylation and the small GTPase TgRab11b, defining an ordered targeting pathway.

   Evidence N-myristoylation-site mutagenesis and TgRab11b loss-of-function, each with localization readout

   PMID:31348812

   Open questions at the time

   • Mechanism linking TgRab11b to TgFBXO1 delivery not defined
   • Whether myristoylation and TgRab11b act in the same or parallel steps not resolved
4. 2019 Medium

   Whether TgFBXO1 is functionally important was unknown; loss-of-function showed it is required for daughter cell scaffold function and parasite growth, establishing it as a scaffold effector.

   Evidence Knockdown/knockout with parasite growth assay and scaffold phenotype analysis

   PMID:31348812

   Open questions at the time

   • Molecular basis of the scaffold defect not defined
   • Substrate(s) whose regulation underlies the phenotype unknown
5. 2024 Medium

   Whether TgFBXO1 stability is under oxygen-dependent control via SKP1 glycosylation was unknown; PHYa deletion analysis showed FBXO1 abundance is unaffected, distinguishing it from oxygen-regulated F-box proteins FBXO13 and FBXO14.

   Evidence PHYa deletion mutant with quantitative interactome and protein abundance measurements

   PMID:39307307

   Open questions at the time

   • What does regulate TgFBXO1 abundance/turnover is unknown
   • Negative finding from a single interactome comparison

• The ubiquitylation substrates of the TgFBXO1-SCF complex and the biochemical basis of its role in daughter cell scaffold assembly remain unidentified.
• No substrate identified for the TgFBXO1 substrate receptor
• Link between E3 ligase activity and scaffold function not established