Evolutionary Selection on Barrier Activity: Bar1 Is an Aspartyl Protease with Novel Substrate Specificity

Abstract

Peptide-based pheromones are used throughout the fungal kingdom for coordinating sexual responses between mating partners. Here, we address the properties and function of Bar1, an aspartyl protease that acts as a “barrier” and antagonist to pheromone signaling in multiple species. Candida albicans Bar1 was purified and shown to exhibit preferential cleavage of native α pheromone over pheromones from related fungal species. This result establishes that protease substrate specificity coevolved along with changes in its pheromone target. Pheromone cleavage by Bar1 occurred between residues Thr-5 and Asn-6 in the middle of the tridecapeptide sequence. Surprisingly, proteolytic activity was independent of the amino acid residues present at the scissile bond and instead relied on residues at the C terminus of α pheromone. Unlike most aspartyl proteases, Bar1 also exhibited a near-neutral pH optimum and was resistant to the class-wide inhibitor pepstatin A. In addition, genetic analysis was performed on C. albicans BAR1 and demonstrated that the protease not only regulates endogenous pheromone signaling but also can limit interspecies pheromone signaling. We discuss these findings and propose that the unusual substrate specificity of Bar1 is a consequence of its coevolution with the α pheromone receptor Ste2 for their shared peptide target.

Construction of multicellular yeast networks using the communication toolkit with variable specificity and attenuation

“…Notably, despite only limited number of identified functional proteases, they could be used to suppress almost all sender parts in our toolkit. Our analysis of promiscuity of barrier proteases did not support previously proposed co-evolution of Ste2 and Bar1 to recognize similar amino acid sequences of the α-factor 37 , since no apparent correlation between receptor and protease promiscuity could be observed (Fig. 6b, Supplementary Fig.…”

Asymmetric diversification of mating pheromones in fission yeast

“…This is because both Mam2 and Sxa2 are likely to depend on the same regions of P-factor activity. A recent study also suggests that coevolution of sterile 2 (Ste2) (a receptor for α-pheromone) and barrier 1 (Bar1) (a peptidase of α-pheromone) can occur, together with evolution of α-pheromone in Candida albicans , because Ste2 and Bar1 recognize the overlapping regions of α-pheromone [46]. In this study, although we did not obtain conclusive evidence that novel compatible combinations of the P-factor/Mam2 and P-factor/Sxa2 pairs can occur (S2C and S2D Fig), such coevolution might proceed little by little, even though reproductive isolation would not be prevented during this time.…”

Environmental Factors Can Influence Mitochondrial Inheritance in the Saccharomyces Yeast Hybrids

“…The strain of interest is patched (or stamped or replica plated) onto a sensitive tester lawn, and the formation of halos on the lawn (because of growth inhibition of the sensitive strain) indicates that the strain is the opposite mating type of the tester. Thus, the a/alpha diploids are not responsive to mating pheromone of either type [36, 37]. After the halo assay, the hybrid cells were also assessed on the plate containing glycerol as the only carbon source to determine whether these selected cells were respiration-competent cells.…”