Correlation Between Improved Mating Efficiency and Weakened Scaffold-Kinase Interaction in the Mating Pheromone...

Area of Science:

• Molecular and Cellular Biology
• Yeast Genetics
• Evolutionary Biology

Background:

• The mating pheromone pathway, involving scaffold protein Ste5 and kinases (Ste11, Ste7, Fus3), is crucial for yeast mating.
• Orthologs of these proteins are common in fungi, but their functional interchangeability remains poorly understood.

Purpose of the Study:

• To evaluate the functional homology of Ste5 and associated kinases in *Kluyveromyces lactis*, *K. marxianus*, and *Saccharomyces cerevisiae* through interspecies complementation.
• To investigate the relationship between sequence identity and functional replaceability of these proteins.
• To explore the role of Ste5-Ste7 interaction in regulating the pheromone pathway and its impact on mating efficiency.

Main Methods:

• Interspecies complementation assays were performed using Ste5 and kinase orthologs from *K. lactis*, *K. marxianus*, and *S. cerevisiae*.
• Sequence identity analysis of full-length proteins and specific domains was conducted.
• Chimeric Ste5 proteins with altered Ste7-binding affinities and modifications to a negatively charged segment were engineered and tested.

Main Results:

• Ste5 and associated kinases from *K. lactis* and *K. marxianus* showed varying degrees of functional replaceability by orthologs.
• Sequence identity did not directly correlate with functional replaceability; Ste5, despite low sequence conservation, exhibited high functional interchangeability.
• Swapping Ste5 between *K. lactis* and *K. marxianus* enhanced mating, linked to weakened Ste5-Ste7 interaction. Altering Ste5-Ste7 affinity modulated mating efficiency, with reduced affinity improving it.
• A negatively charged segment in the Ste7-binding domain of Ste5 negatively correlated with mating efficiency; extending this segment in KlSte5 reduced mating.

Conclusions:

• Ste5-Ste7 interaction plays a novel role in the negative regulation of the yeast pheromone pathway.
• Functional homology of mating pathway components can be significant even with low sequence conservation.
• Engineered Ste5 mutants with improved mating efficiency hold potential for breeding and selecting *Kluyveromyces* strains for industrial applications.