Mutant/mutation
The mutant expresses a C-terminal 3XHA–mCherry-tagged version of ATG8.
To check if Plasmodium Atg4 retains Atg8 processing activity despite its exposed glycine, we investigated the importance of cysteine protease for Atg8 conjugation. Atg8 was endogenously tagged with 3XHA–mCherry at the C-terminus to mask its exposed glycine.

Protein (function)
Autophagy (macroautophagy) is a catabolic process conserved from yeast to mammals. During macroautophagy, the Atg8 protein is conjugated to phosphatidylethanolamine (PE) in autophagic membranes. Atg8 localizes on isolated membranes and autophagosomes and has been considered a marker of the induction and progression of autophagy. The presence of Atg8 on the autophagosome is related to its many roles, such as cargo selection, fusion, and membrane tethering and expansion. In yeast, Atg8 is synthesized with a terminal peptide, which is immediately cleaved by the cysteine protease autophagy-related protein 4 (Atg4), revealing a glycine at the C-terminus, which then acts as a substrate in the ubiquitin-like conjugation reaction mediated by Atg7 (E1-activating enzyme) and Atg3 (E2 conjugating enzyme), which mediates its conjugation to PE on the autophagosome membrane. The conjugation of Atg8 to the membrane is reversible, and Atg8 is deconjugated from the outer surface upon autophagosome closure. Deconjugation is important for replenishing Atg8 for further autophagosome formation Two different classes of cysteine proteases have been found to play a role in deconjugatio: one is Atg4 of the C4 cysteine protease family, which cleaves at the C-terminus of the Atg8–PE bond, and the other is ovarian tumor unit (Otu) of the DUB family, which irreversibly inactivates Atg8 by cleaving at the N-terminus of the Atg8–PE bond. In Apicomplexan parasites, two cysteine proteases, Atg4 and ovarian tumor unit (Otu), have been identified to delipidate Atg8 to release this protein from membranes. Here, we investigated the role of cysteine proteases in Atg8 conjugation and deconjugation and found that the Plasmodium parasite consists of both activities.

From the paper:
'We successfully disrupted the genes individually; however, simultaneously, they were refractory to deletion and essential for parasite survival. Mutants lacking Atg4 and Otu showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast; however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling' .
Attempts to generate and select Atg4/Otu double knockout parasites were unsuccessful.

Phenotype
To check if Plasmodium Atg4 retains Atg8 processing activity despite its exposed glycine, we investigated the importance of cysteine protease for Atg8 conjugation. Atg8 was endogenously tagged with 3XHA–mCherry at the C-terminus to mask its exposed glycine.
Atg8-3XHA-mCherry schizont cultures were treated with the cysteine protease inhibitor E64 to evaluate cysteine protease activity. The treated and untreated schizonts were fixed and analyzed by IFA using anti-Atg8 and anti-mCherry antibodies. Without an inhibitor, mCherry was cleaved, as confirmed by independent signals of Atg8 and mCherry that did not colocalize. However, E64-treated Atg8-3XHA-mCherry schizonts showed overlapping staining patterns. The treated Atg8-3XHAmCherry and WT schizonts also failed to properly segregate their nuclei and were found to be arrested during schizogony, which was due to the general inhibition of parasites by E64. Atg8 conjugation was not affected in the arrested WT parasites, suggesting that parasites could conjugate Atg8 with exposed glycine.
Furthermore, the cleavage of ATG8 was ascertained by western blot analysis of Atg8-3XHA-mCherry schizonts using anti-ATG8 and anti-HA antibodies. We found processing of 3XHA-mCherry in untreated samples, while no processing was observed in E64-treated parasites. These results indicate that in the presence of an E64 inhibitor, cysteine proteases failed to process the 3XHA-mCherry tag from the C-terminus of Atg8 and subsequently failed to conjugate on the apicoplast membrane.

See also RMgm-1475 and RMgm-1953 for unsuccessful attempts to knock-out the Atg8 gene, indicating an essential function of ATG8 for growth/multiplication of asexual blood stages.

Additional information
To functionally characterize the role of Atg4 (PBANKA_1025400) and Otu (PBANKA_0515350) in the Plasmodium berghei life cycle, these genes were disrupted using double-crossover homologous recombination (see RMgm-5457 and RMgm-5458). In addition, attempts were preformed to generate and select Atg4/Otu double knockout parasites. These attempts were unsuccessful (see RMgm-5459).
The following was found:
Mutants lacking Atg4 (RMgm-5457) and Otu (RMgm-5458) showed normal blood and mosquito stage development. All mice infected with Otu KO sporozoites became patent; however, Atg4 KO sporozoites either failed to establish blood infection or showed delayed patency. Through in vitro and in vivo analysis, we found that Atg4 KO sporozoites invade and normally develop into early liver stages. However, nuclear and organelle differentiation was severely hampered during late stages and failed to mature into hepatic merozoites. We found a higher level of Atg8 in Atg4 KO parasites, and the deconjugation of Atg8 was hampered. We confirmed Otu localization on the apicoplast (see mutant RMgm-5460); however, parasites lacking Otu showed no visible developmental defects. Our data suggest that Atg4 is the primary deconjugating enzyme and that Otu cannot replace its function completely because it cleaves the peptide bond at the N-terminal side of glycine, thereby irreversibly inactivating Atg8 during its recycling' .
Evidence is presented that:
- Cysteine proteases are essential for Atg8 processing
- Atg4 is a deconjugating enzyme, and Atg4 KO parasites failed to deconjugate Atg8
- Atg4 is critical for late liver stage development
- Atg4 KO parasites show impaired (liver-stage) merozoite formation
- Atg4 is dispensable for exocytosis of micronemes
- Atg8‑PE deconjugation is essential for organelle biogenesis during liver stage development
- Atg4/Otu double gene deletion is lethal for blood‑stage propagation

Other mutants