Correlation Between Improved Mating Efficiency and Weakened Scaffold-Kinase Interaction in the Mating Pheromone Response Pathway Revealed by Interspecies Complementation

Abstract

Scaffold protein Ste5 and associated kinases, including Ste11, Ste7, and Fus3, are core components of the mating pheromone pathway, which is required to induce a mating response. Orthologs of these proteins are widely present in fungi, but to which extent one protein can be replaced by its ortholog is less well understood. Here, interspecies complementation was carried out to evaluate the functional homology of Ste5 and associated kinases in Kluyveromyces lactis, K. marxianus, and Saccharomyces cerevisiae. These three species occupy important positions in the evolution of hemiascomycetes. Results indicated that Ste5 and associated kinases in K. lactis and K. marxianus could be functionally replaced by their orthologs to different extents. However, the extent of sequence identity, either between full-length proteins or between domains, did not necessarily indicate the extent of functional replaceability. For example, Ste5, the most unconserved protein in sequence, achieved the highest average functional replaceability. Notably, swapping Ste5 between K. lactis and K. marxianus significantly promoted mating in both species and the weakened interaction between the Ste5 and Ste7 might contribute to this phenotype. Consistently, chimeric Ste5 displaying a higher affinity for Ste7 decreased the mating efficiency, while chimeric Ste5 displaying a lower affinity for Ste7 improved the mating efficiency. Furthermore, the length of a negatively charged segment in the Ste7-binding domain of Ste5 was negatively correlated with the mating efficiency in K. lactis and K. marxianus. Extending the length of the segment in KlSte5 improved its interaction with Ste7 and that might contribute to the reduced mating efficiency. Our study suggested a novel role of Ste5-Ste7 interaction in the negative regulation of the pheromone pathway. Meanwhile, Ste5 mutants displaying improved mating efficiency facilitated the breeding and selection of Kluyveromyces strains for industrial applications.

Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) pathway in Saccharomyces cerevisiae

“…The literature suggests a balance between Hsp70 promotion of degradation of misfolded proteins and Hsp90 promotion of folding to full maturation [ 60 , 64 , 104 , 108 ]. Current models suggest that Hsp90 uses both Ssa-dependent and Ssa-independent routes to fold proteins [ 40 , 41 , 44 , 103 – 105 , 109 ].…”

“…We also assessed mutations in 4 chaperones with dual functions in the Hsp70 and Hsp90 pathways: YDJ1 , STI1 , SSE1 and SSE2 . Ydj1 is the J domain/Hsp40 adapter that helps Hsp70/Hsp90 complexes be recognized by Rsp5 NEDD E3 ubiquitin ligase for ubiquitinylation and subsequent degradation of associated client protein by the proteasome [ 108 ]. Of all of the HSP proteins we investigated, Ydj1 is the only one predicted to regulate Gpa1 (Ga) which negatively regulates the mating signaling pathway ( Table 8 ).…”

Effects of HSP70 chaperones Ssa1 and Ssa2 on Ste5 scaffold and the mating mitogen-activated protein kinase (MAPK) Pathway in Saccharomyces cerevisiae

“…Fus3) or have fewer inhibitors. Additionally, genetic analysis suggests weakened binding of Ste5 to kinase may enhance mating (100). In addition, stress induced from changes in cell wall integrity from ssa1D ssa2D mutations may trigger activation of the invasive growth pathway to activate Kss1 which can partially substitute for Fus3 (Fig 1C).…”

“…In addition, counteractive effects of increased Fus3 abundance may relieve the reduction in the level of Ste5. In addition, genetic analysis of interspecies chimeras suggests that weaker binding of Ste5 to kinases may enhance mating (100).…”