Mutant/mutation
The mutant expresses a C-terminal 3xHA-tagged version of SKP1

Protein (function)
Ubiquitin is a 76-residue protein that is usually added through a covalent bond to lysine residues in substrate proteins. Protein ubiquitination is a three-step process during which ubiquitin is first activated by an E1 ubiquitin-activating enzyme, transferred to an E2 ubiquitin-conjugating enzyme and then transferred to a substrate selected by an E3  ubiquitin ligase. E3 ligases are generally multiprotein complexes and are classified into four families: HECT, RING-finger, Ubox, and PHD-finger..Among the RING-finger ligases, the cullin-RINGE3 ligases (CRLs) represent a diverse group that includes ligases such as the Skp1/Cullin/F-box (SCF) protein complex and the anaphase promoting complex/cyclosome (APC/C). These CRLs have important roles in the ubiquitination of proteins involved in the cell cycle. Four APC/C components have been identified in the Plasmodium genome: APC3, APC10, APC11, as well as the adaptor protein CDC20. APC3 and CDC20 were shown to be important for Plasmodium microgametogenesis17,18. SCF complexes contain three invariant proteins, RBX1, Cullin and SKP1, in addition to a variable F-box protein. Cullin serves as a scaffold protein which N-terminally binds to SKP1 and C-terminally binds to a RING- finger protein (RING-box protein 1 - RBX1) and an E2 ubiquitin-conjugating enzyme. F-box proteins generally contain a ~50 amino acid F-box domain which functions as a receptor by binding to the SCF complex. F-box domains are commonly found in concert with other protein–protein interaction motifs, such as leucine-rich, WD repeat and ankyrin repeats, which mediate interactions with their substrate-specific interaction motif.
Genome mining in Plasmodium identified SKP1, Cullin1, and RBX1 together with two F-box domain-containing proteins. 

Phenotype
Immunoblotting confirmed the expression of fusion proteins in gametocytes with the expected mobility for RBX1-HA and SKP1-HA, but no signal above the background could be observed for CUL1-HA. Immunofluorescence assays (IFA) revealed a cell-wide distribution of both RBX1-HA and SKP1-HA in both activated macro- and microgametocytes, while no signal above the detection limit was detected for CUL1-HA gametocytes.

While a faint cytoplasmic signal could be observed for RBX1-HA in blood-stage schizonts, no specific enrichment was observed. SKP1-HA, FBXO1-HA, and FBXL2-HA showed a pattern that was reminiscent of the dynamic localisation of the glideosome-associated protein 45 (GAP45).

A signal above the detection limit was also observed for SKP1-HA, FBXO1-HA and FBXL2-HA in ookinetes.

These results show that components of the SCF(FBXO1) complex identified in gametocytes are also expressed in blood-stage schizonts and ookinetes suggesting multistage requirements for these proteins

See further below.

Additional information
From the paper:
'Plasmodium genome mining previously identified orthologues of four proteins possibly belonging to Skp1/Cullin/F-box (SCF) complexes: Cullin1 (CUL1 - PBANKA_1426500), RBX1 (PBANKA_0806200), SKP1 (PBANKA_1142900), Nedd8 (PBANKA_1411400), and a second cullin domain-containing protein (CUL2 - PBANKA_1128600).

We first set out to identify whether these proteins form a complex in Plasmodium using affinity purification of triple HA-tagged proteins. Transgenic P. berghei lines expressing triple HA-tagged RBX1 (RMgm-5335), SKP1 (RMgm-5333) and CUL1 (RMgm-5334). To identify interacting proteins for RBX1-HA and SKP1-HA, we used affinity purification of triple HA-tagged proteins from synchronised mitotic gametocytes 4–6min post-activation followed by label-free semiquantitative mass spectrometry. Supporting the notion of an SKP1/CUL1 complex, CUL1, Nedd8 and polyubiquitin (PolyUb – PBANKA_ 0610300) were enriched proteins co-purifying with RBX1-HA and SKP1-HA. Among the proteins most enriched in both immunoprecipitates were also two proteins containing either F-box (FBXO1 – PBANKA_1118900) or leucine-rich repeats (FBXL2 or LRR11,  PBANKA_0925100) that could represent possible adaptors of the SKP1/CUL1 complex. We also identified the IMC sub-compartment protein 1 (ISP1 - PBANKA_1209400), suggesting that this protein, which is important to define the polarity of ookinetes, may be regulated by SCF-dependent ubiquitination.
A protein of unknown function, PBANKA_1358700 was also enriched in all immunoprecipitates.'
 
'To confirm FBXO1 and FBXL2 are components of SKP1/CUL1 complexes, we generated transgenic P. berghei lines expressing endogenously triple HA-tagged alleles of FBXO1 (RMgm-5336) and FBXL2 (RMgm-5337). 
Immunoblotting confirmed the expression of fusion proteins in gametocytes with the expected mobility. IFA showed a similar cytoplasmic distribution of FBXO1-HA and FBXL2-HA in both macro- and microgametes with slightly brighter staining at structures that likely correspond to mitotic spindles and forming axonemes in the microgametes. Immunoprecipitations of FBXO1-HA or FBXL2-HA enriched peptides from SKP1, CUL1, RBX1, PolyUb and Nedd8, while the most enriched protei nwas ISP1. CDPK1, a regulator of merozoite formation, gamete egress and ookinete development, was also  slightly enriched in FBXO1-HA immuno-precipitates.'

'These results indicate that gametocytes express a conserved SKP1/RBX1/CUL1 complex with at least two possible adaptor proteins, FBXO1 and FBXL2. The enrichment of CDPK1 in FBXO1-HA and SKP1-HA immuno-precipitates  suggests a possible interplay between phosphorylation and ubiquitination to regulate sexual development.'

'To study the function of SCFF(BXO1) complex, we first attempted to knock-out skp1 (RMgm-5330), cul1 (RMgm-5331), and rbx1 (RMgm-5332). These attempts were unsuccessful indicating an essential function during asexual blood stage growth/multiplication'.

'The endogenous  skp1, cul1 and rbx1 genes were taged with an auxin-inducible degron (AID) coupled to an HA epitope tag that allows degradation of the fusion protein in the presence of auxin in a strain expressing the Tir1 protein (RMgm-1305; Philip and Waters, 2015). However, no significant degradation of the targeted proteins nor defects in exflagellation could be observed upon auxin addition in non-clonal populations This prevented  to further use this system to interrogate the functions of these proteins across multiple stages.'

'To infer potential function in gametocytes, we then opted for stage-specific knockdowns by placing the endogenous cul1 or rbx1 genes under the control of the promoter of the blood-stage schizont-specific promoter ama1 (PBANKA_0915000), which is active in schizonts but silent in gametocytes. Cloned parasite lines expressing cul1 (Pama1CUL1; RMgm-5338) and rbx (Pama1RBX; RMgm-5339) under control of the ama-1 promoter were  obtained, but none of these showed quantifiable defects in exflagellation (possibly due to sufficient expression levels?). However, the Pama1CUL1 clone mainly formed retort ookinetes, a phenotype that was partially rescued by fertilisation with competent Nek4-KO microgametes, suggestive of a role for cul1 in either sexual lineage.'

We then interrogated the requirement for the putative adaptor proteins FBXO1, FBXL2 and the protein of unknown function PBANKA_1358700 that was enriched in SCFFBXO1 immuno-precipitates. We were able to obtain a KO clonal line for PBANKA_1358700 (RMgm-5340), but no defects in asexual blood stages nor in exflagellation were detected.
No FBXL2-KO transient populations could be observed (RMgm-5341) and no major protein degradation was observed in an FBXL2-AID/HA line upon treatment with auxin. However, no exflagellation defect could be detected upon auxin treatment preventing us from further functional analysis of this protein.
We obtained a clonal FBXO1-AID/HA line as well as a clonal line, FBXO1-GD (for Gene Disruption; RMgm-5342), in which 207 fbxo1 bases were deleted, disrupting the coding sequence at the 353rd amino acid. Phenotypic analysis of FBXO1-GD showed that FBXO1 is required for normal growth of asexual blood stages, microgametogenesis and ookinete development. Slight growth/multiplication delay of FBXO1-GD asexual blood stages and strongly impaired microgamete formation as determined by the percentage of microgametocytes leading to exflagellating microgametes. Flagellar movement of the forming microgametes commenced while the parasite was still inside the host cell and delayed egress of male gametes from the host erythrocyte. 
FBXO1-GD parasites did not form banana-shaped ookinetes and only retort stages were observed.
Evidence is presented that that: 
- FBXO1 expression during gametocytogenesis is required for macro- and microgamete egress and centrosome partitioning during microgametogenesis.
- FBXO1-GD gametocytes show significantly reduced ubiquitination levels upon activation
- FBXO1 playse a role in maintaining the integrity of the pellicle and the subpellicular microtubule network during ookinete development.
- FBXO1 and CDPK1 share a molecular environment and show related cellular requirements during sexual development
- CDPK1 negatively regulates FBXO1 function by downregulating its expression level in gametocytes

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