PUBL

PUBL (plastid ubiquitin-like protein) is a divergent, ubiquitin-derived protein that drives the import of nucleus-encoded proteins across the periplastid membrane of the apicoplast in Toxoplasma gondii, an activity essential for parasite viability (PMID:28655825). It operates within an apicoplast-specific ERAD-like machinery together with the AAA ATPase CDC48AP, with which it acts epistatically to translocate apicoplast proteins across the periplastid membrane (PMID:28655825). Mechanistically, the residues of PUBL homologous to those required for ubiquitin conjugation onto target proteins are essential for import, whereas residues homologous to polyubiquitination and preprotein-processing determinants are dispensable, indicating that a conjugation-like chemistry rather than canonical polyubiquitin signaling underlies its translocation function (PMID:28655825). Beyond these findings, no further mechanistic detail has been characterized in the available corpus.

1. 2017 High

   Whether a ubiquitin-derived protein contributes to apicoplast biogenesis was unknown; identifying PUBL as an apicoplast-localized, ubiquitin-ancestor-derived protein essential for protein import across the periplastid membrane established a dedicated import factor whose loss is lethal.

   Evidence Conditional null mutants and genetic complementation with apicoplast protein import assays in Toxoplasma gondii

   PMID:28655825

   Open questions at the time

   • Direct biochemical demonstration of PUBL conjugation to a substrate not shown
   • Substrate identity of imported proteins not defined
2. 2017 High

   How PUBL couples to the translocation machinery was unresolved; epistasis with the AAA ATPase CDC48AP placed PUBL within an apicoplast-specific ERAD-like complex that drives translocation across the periplastid membrane.

   Evidence Genetic epistasis using double conditional null mutants of PUBL and CDC48AP with a periplastid-membrane translocation readout in Toxoplasma gondii

   PMID:28655825

   Open questions at the time

   • Physical composition and stoichiometry of the ERAD-like complex not resolved
   • Mechanism by which CDC48AP extracts PUBL-marked substrates not shown
3. 2017 High

   Which biochemical features of PUBL matter for import was unknown; mutagenesis showed that conjugation-homologous residues are essential while polyubiquitination- and preprotein-processing-homologous residues are dispensable, defining a conjugation-like rather than canonical ubiquitin-signaling mechanism.

   Evidence Site-directed mutagenesis of ubiquitin-homologous residues with genetic complementation and import assays in Toxoplasma gondii

   PMID:28655825

   Open questions at the time

   • The specific conjugation target of PUBL not identified
   • Enzymes activating/conjugating PUBL not characterized

• The direct molecular substrate(s) of PUBL conjugation and the full enzymatic cascade (activating/conjugating machinery) that couple PUBL modification to CDC48AP-driven translocation remain undefined.
• No conjugation substrate identified
• Activating/conjugating enzymes for PUBL unknown
• Structural basis of PUBL function not determined