Leishmania differentiation requires ubiquitin conjugation mediated by a UBC2-UEV1 E2 complex

Post-translational modifications such as ubiquitination are important for orchestrating the cellular transformations that occur as the Leishmania parasite differentiates between its main morphological forms, the promastigote and amastigote. 2 E1 ubiquitin-activating (E1), 13 E2 ubiquitin-conjugating (E2), 79 E3 ubiquitin ligase (E3) and 20 deubiquitinating cysteine peptidase (DUB) genes can be identified in the Leishmania mexicana genome but, currently, little is known about the role of E1, E2 and E3 enzymes in this parasite. Bar-seq analysis of 23 E1, E2 and HECT/RBR E3 null mutants generated in promastigotes using CRISPR-Cas9 revealed numerous loss-of-fitness phenotypes in promastigote to amastigote differentiation and mammalian infection. The E2s UBC1/CDC34, UBC2 and UEV1 and the HECT E3 ligase HECT2 are required for the successful transformation from promastigote to amastigote and UBA1b, UBC9, UBC14, HECT7 and HECT11 are required for normal proliferation during mouse infection. Of all ubiquitination enzyme null mutants examined in the screen, Delta ubc2 and Delta uev1 exhibited the most extreme loss-of-fitness during differentiation. Null mutants could not be generated for the E1 UBA1a or the E2s UBC3, UBC7, UBC12 and UBC13, suggesting these genes are essential in promastigotes. X-ray crystal structure analysis of UBC2 and UEV1, orthologues of human UBE2N and UBE2V1/UBE2V2 respectively, reveal a heterodimer with a highly conserved structure and interface. Furthermore, recombinant L. mexicana UBA1a can load ubiquitin onto UBC2, allowing UBC2-UEV1 to form K63-linked di-ubiquitin chains in vitro. Notably, UBC2 can cooperate in vitro with human E3s RNF8 and BIRC2 to form non-K63-linked polyubiquitin chains, showing that UBC2 can facilitate ubiquitination independent of UEV1, but association of UBC2 with UEV1 inhibits this ability. Our study demonstrates the dual essentiality of UBC2 and UEV1 in the differentiation and intracellular survival of L. mexicana and shows that the interaction between these two proteins is crucial for regulation of their ubiquitination activity and function. Author summary The post-translational modification of proteins is key for allowing Leishmania parasites to transition between the different life cycle stages that exist in its insect vector and mammalian host. In particular, components of the ubiquitin system are important for the transformation of Leishmania from its insect (promastigote) to mammalian (amastigote) stage and normal infection in mice. However, little is known about the role of the enzymes that generate ubiquitin modifications in Leishmania. Here we characterise 28 enzymes of the ubiquitination pathway and show that many are required for life cycle progression or mouse infection by this parasite. Two proteins, UBC2 and UEV1, were selected for further study based on their importance in the promastigote to amastigote transition. We demonstrate that UBC2 and UEV1 form a heterodimer capable of carrying out ubiquitination and that the structural basis for this activity is conserved between Leishmania, Saccharomyces cerevisiae and humans. We also show that the interaction of UBC2 with UEV1 alters the nature of the ubiquitination activity performed by UBC2. Overall, we demonstrate the important role that ubiquitination enzymes play in the life cycle and infection process of Leishmania and explore the biochemistry underlying UBC2 and UEV1 function.