Cell wall-resident PIR proteins show an inverted architecture in Neurospora crassa , but keep their role as wall stabilizers

Proteins with internal repeats (PIRs) are the second most abundant class of fungal cell wall resident proteins. In yeasts, PIRs preserve the wall stability under stressful conditions. They are characterized by conserved N-terminal amino acid sequences repeated in tandem (PIR domains), and a Cys-rich C-terminal domain. Despite PIRs have been inferred in several filamentous fungi genomes, they have not been studied beyond yeasts. In this work, PIRs diversity, evolution and biological role, focused on a new PIRs class, were addressed. Bioinformatic inference of PIRs in fungi indicated they were an innovation in Ascomycota. Predicted PIRs clustered in two main groups: classical yeasts PIRs (N-terminal PIR domains; C-terminal Cys-rich domain), and PIRs from filamentous fungi with an inverted architecture (N-terminal Cys-rich domain; C-terminal PIR domains), which could harbor additional GPI-signals. As representatives of the second group, Neurospora crassa (Nc) PIR-1 (NCU04033) and PIR-2 (NCU07569) were studied. Confocal microscopy of eGFP-labeled PIR-1 and PIR-2 revealed they accumulate in apical plugs; additionally, PIR-1 requires the Kex2 processing site for correct maturation, and its predicted C-terminal GPI modification signal resulted functional. Moreover, Nc Δ pir-1 and Δ pir-2 single mutants showed a growth rate similar to that of Nc WT, but the double mutant Nc Δ pir-1 /Δ pir-2 grew significatively slower. Similarly, Nc Δ pir-1 and Nc Δ pir-2 were mildly sensitive to calcofluor white, although Nc Δ pir-1 /Δ pir-2 double mutant was severely impaired. Despite the inverted architecture of PIR-1 and PIR-2, they resulted in cell wall stabilizers as classical yeast PIRs.