Abstract
F-box protein is a key protein of the SCF E3 ubiquitin ligase complex, responsible for substrate recognition and degradation through specific interactions. Previous studies have shown that F-box proteins play crucial roles in Cryptococcus sexual reproduction. However, the molecular mechanism by which F-box proteins regulate sexual reproduction in C. neoformans is unclear. In the study, we discovered the AICAR transformylase/IMP cyclohydrolase Ade16 as a substrate of Fbp1. Through protein interaction and stability experiments, we demonstrated that Ade16 is a substrate for Fbp1. To examine the role of ADE16 in C. neoformans, we constructed the iADE16 strains and ADE16(OE) strains to analyze the function of Ade16. Our results revealed that the iADE16 strains had a smaller capsule and showed growth defects under NaCl, while the ADE16(OE) strains were sensitive to SDS but not to Congo red, which is consistent with the stress phenotype of the fbp1Δ strains, indicating that the intracellular protein expression level after ADE16 overexpression was similar to that after FBP1 deletion. Interestingly, although iADE16 strains can produce basidiospores normally, ADE16(OE) strains can produce mating mycelia but not basidiospores after mating, which is consistent with the fbp1Δmutant strains, suggesting that Fbp1 is likely to regulate the sexual reproduction of C. neoformans through the modulation of Ade16. A fungal nuclei development assay showed that the nuclei of the ADE16(OE) strains failed to fuse in the bilateral mating, indicating that Ade16 plays a crucial role in the regulation of meiosis during mating. In summary, our findings have revealed a new determinant factor involved in fungal development related to the post-translational regulation of AICAR transformylase/IMP cyclohydrolase.