RESUMEN
Phosphoribosyl pyrophosphate synthetase, which is encoded by the Prs gene, catalyses the reaction of ribose-5-phosphate and adenine ribonucleotide triphosphate (ATP) and has central importance in cellular metabolism. However, knowledge about how Prs family members function and contribute to total 5-phosphoribosyl-α-1-pyrophosphate (PRPP) synthetase activity is limited. In this study, we identified that the filamentous fungus Aspergillus nidulans genome contains three PRPP synthase-homologous genes (AnprsA, AnprsB and AnprsC), among which AnprsB and AnprsC but not AnprsA are auxotrophic genes. Transcriptional expression profiles revealed that the mRNA levels of AnprsA, AnprsB and AnprsC are dynamic during germination, hyphal growth and sporulation and that they all showed abundant expression during the vigorous hyphal growth time point. Inhibiting the expression of AnprsB or AnprsC in conditional strains produced more effects on the total PRPP synthetase activity than did inhibiting AnprsA, thus indicating that different AnPrs proteins are unequal in their contributions to Prs enzyme activity. In addition, the constitutive overexpression of AnprsA or AnprsC could significantly rescue the defective phenotype of the AnprsB-absent strain, suggesting that the function of AnprsB is not a specific consequence of this auxotrophic gene but instead comes from the contribution of Prs proteins to PRPP synthetase activity.
Asunto(s)
Aspergillus nidulans/genética , Aspergillus nidulans/metabolismo , Hifa/crecimiento & desarrollo , Ribosa-Fosfato Pirofosfoquinasa/genética , Esporas Fúngicas/crecimiento & desarrollo , Adenosina Trifosfato/química , Aspergillus nidulans/crecimiento & desarrollo , Eliminación de Gen , Técnicas de Inactivación de Genes , Hifa/genética , Fosforribosil Pirofosfato/biosíntesis , ARN Mensajero/genética , Ribosamonofosfatos/química , Esporas Fúngicas/genéticaRESUMEN
CpcB (cross pathway control B) encodes a yeast Cpc2 and mammalian RACK1 (receptor for activated protein kinase C) ortholog, which is a WD repeat protein with functional homology to the ß subunit of heterotrimeric G proteins in Aspergillus fumigatus. Previous study has reported that CpcB governs growth and development in both A. fumigatus and Aspergillus nidulans. However, little is known about the functional identities of CpcB orthologs and their relationships with G protein complexes. In this study, we verified that cytoplasmic AfCpcB acts as a Gß-like protein ortholog and plays important roles in hyphal growth, conidiophore morphology, cell wall integrity, and virulence in A. fumigatus. Furthermore, double deletion of AfcpcB and AfgpaB (Gα) causes a similar phenotype to AfgpaB mutant with abnormal multiple septa conidiophores but exhibits sparse conidiation with white and fluffy colonies. Thus, the exacerbated conidiation defect suggests that AfcpcB has its own specific function compared to the Gα subunit of AfgpaB or the G-protein complex. In addition, complementation assays using AfcpcB orthologs of A. nidulans and yeasts (Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans) suggest that all tested fungal AfcpcB orthologs under the A. fumigatus native promoter can largely restore hyphal growth defects in AfcpcB deletion mutant, but only the A. nidulans cpcB ortholog completely rescues the ΔAfcpcB conidiation defect, suggesting that CpcB acts as a Gß-like protein ortholog in the Aspergilli, but may have unique and important unexplored functions that required for conidiation, which is absent in yeast.
Asunto(s)
Aspergillus fumigatus/crecimiento & desarrollo , Aspergillus fumigatus/patogenicidad , Proteínas de Unión al GTP/metabolismo , Regulación Fúngica de la Expresión Génica , Hifa/crecimiento & desarrollo , Esporas Fúngicas/crecimiento & desarrollo , Aspergillus fumigatus/genética , Eliminación de Gen , Prueba de Complementación GenéticaRESUMEN
Protein phosphatase 2A (PP2A) is a major intracellular protein phosphatase that regulates multiple aspects of cell growth and metabolism. Different activities of PP2A and subcellular localization are determined by its regulatory subunits. Here we identified and characterized the functions of two protein phosphatase regulatory subunit homologs, ParA and PabA, in Aspergillus nidulans. Our results demonstrate that ParA localizes to the septum site and that deletion of parA causes hyperseptation, while overexpression of parA abolishes septum formation; this suggests that ParA may function as a negative regulator of septation. In comparison, PabA displays a clear colocalization pattern with 4',6-diamidino-2-phenylindole (DAPI)-stained nuclei, and deletion of pabA induces a remarkable delayed-septation phenotype. Both parA and pabA are required for hyphal growth, conidiation, and self-fertilization, likely to maintain normal levels of PP2A activity. Most interestingly, parA deletion is capable of suppressing septation defects in pabA mutants, suggesting that ParA counteracts PabA during the septation process. In contrast, double mutants of parA and pabA led to synthetic defects in colony growth, indicating that ParA functions synthetically with PabA during hyphal growth. Moreover, unlike the case for PP2A-Par1 and PP2A-Pab1 in yeast (which are negative regulators that inactivate the septation initiation network [SIN]), loss of ParA or PabA fails to suppress defects of temperature-sensitive mutants of the SEPH kinase of the SIN. Thus, our findings support the previously unrealized evidence that the B-family subunits of PP2A have comprehensive functions as partners of heterotrimeric enzyme complexes of PP2A, both spatially and temporally, in A. nidulans.