Mutant/mutation
The mutant expresses a 3xHA-tagged version of DDB1.
The mutant was published in bioRxiv preprint doi: https://doi.org/10.1101/2023.07.19.549332

Protein (function)
From the paper: 'Plasmodium encodes a second cullin annotated as cullin-2 (CUL4 - PF3D7_0629800) that, compared to human cullins, shares maximum sequence identity with Cullin-4B. To keep in line with the nomenclature, we here propose to rename this protein CUL4.
Here we confirm that CUL4 forms a CRL4-related complex in P. falciparum asexual blood stages that consists of RBX1, the adaptor protein DDB1 and a distinct set of putative receptor proteins containing WD40 repeat domains.

Affinity purification of PbCUL4-HA from 4 min activated P. berghei gametocytes also identified  RBX1 (PBANKA_0806200), DDB1 (PBANKA_0807500), Nedd8 (PBANKA_1411400), and polyubiquitin 140 (PolyUb – PBANKA_0610300) highlighting the conservation of a CRL4-based complex in multiple stages of  the Plasmodium lifecycle. The orthologues of the WD containing protein WIG1 (PBANKA_1216700) and the ubiquitin carboxyl-terminal hydrolase (PbUCH7 -143 PBANKA_0210600) were also enriched. To confirm these interactions, we generated P. berghei lines expressing HA-tagged alleles of PbDDB1 (RMgm-5395) and PbWIG1(RMgm-5396).

We further show that this CRL4-related complex is expressed in P. berghei gametocytes with at least one putative receptor WD protein expressed at both stages. While this WD protein is not required for the proliferation of asexual blood stages, its disruption leads to a complete block in microgamete formation (see RMgm-5394). We thus decided to name this protein WD repeat protein Important for Gametogenesis 1 (WIG1). Proteomic analyses indicate that WIG1 disruption alters proteostasis of ciliary proteins and components of the DNA replication machinery during gametocytogenesis. Further analysis by ultrastructure expansion microscopy (U-ExM) indicates that WIG1-dependent depletion of ciliary proteins is associated with altered formation of the microgametocyte MTOCs and defects in DNA replication.

Phenotype
Immunoblotting confirmed expression of DDB1-HA in gametocytes.
Immunofluorescence assays indicated a cytoplasmic distribution of PbDDB1-HA in schizonts and gametocytes

Additional information
Affinity purification of PbCUL4-HA from 4 min activated P. berghei gametocytes also identified  RBX1 (PBANKA_0806200), DDB1 (PBANKA_0807500), Nedd8 (PBANKA_1411400), and polyubiquitin 140 (PolyUb – PBANKA_0610300) highlighting the conservation of a CRL4-based complex in multiple stages of  the Plasmodium lifecycle. The orthologues of the WD containing protein WIG1 (PBANKA_1216700) and the ubiquitin carboxyl-terminal hydrolase (PbUCH7 -143 PBANKA_0210600) were also enriched. To confirm these interactions, we generated P. berghei lines expressing HA-tagged alleles of PbDDB1 (RMgm-5395) and PbWIG1(RMgm-5396).
We took advantage of the highly efficient PlasmoGEM vectors to attempt to delete the last 217 codons of the WIG1 gene (PBANKA_1216700) in P. berghei. A WIG1-GD line was readily obtained (RMgm-5394) and cloned suggesting no defect in the proliferation of asexual blood stages. Similarly, the WIG1-GD line produced microscopically normal female and male gametocytes, as assessed by Giemsa staining. However, upon activation by XA, no exflagellation events could be observed. 

Affinity purification of PbDDB1-HA and PbWIG1-HA from 4 min activated P. berghei gametocytes followed by label-free semiquantitative mass spectrometry confirmed enrichment of PbCUL4, PbNedd8, PbRBX1, PbDDB1 and PbWIG1 compared with previous immunoprecipitations of the FBXO1 substrate receptor of the SCF1/CRL1 complex. In addition, another conserved. Plasmodium protein of unknown function, PBANKA_1225100, was also enriched in PbCUL4-HA and PbDDB1-HA immunoprecipitates from P. berghei gametocytes. Altogether these results highlight the conserved expression of a CUL4/RBX1/DDB1/WIG1 complex in both P. falciparum schizonts and P. berghei gametocytes

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