Functional diversification of Yeast Amino acid Transporters
Understanding how new protein functions emerge over evolutionary time is a central question in biology. This thesis addresses it using membrane transporters as a model system, focusing on the Yeast Amino Acid Transporter (YAT) family in Saccharomyces cerevisiae. These proteins mediate the amino acid uptake across the plasma membrane and represent a diverse group regarding substrate specificity and regulation; yet their functional range, structure and evolutionary flexibility remain incompletely understood.
By integrating evolutionary and biochemical approaches, I show that YATs are more functionally plastic than previously recognized. Using growth-based assays and directed evolution, I demonstrate that several transporters display unexpected substrate promiscuity and that single point mutations can significantly broaden substrate specificity without abolishing original functions. These changes occur not only in the substrate-binding core but also in distal regions, highlighting the importance of long-range structural effects.I further characterize the lysine transporter Lyp1, revealing that it can transport multiple additional amino acids across a wide affinity range, from micromolar to millimolar.
This suggests that weak, latent interactions with non-canonical substrates may provide a starting point for evolving new functions.Given the strong link between structure and function, this thesis also addresses challenges in structural characterization. I present strategies for improving cryo-EM analysis of small transporters like Lyp1 and evaluate safer alternatives to uranyl acetate for negative-stain electron microscopy, identifying effective lanthanide-based stains.
Overall, this work provides a holistic view of how genetic variation, protein structure, and biochemical activity interact to shape the evolution and diversification of membrane transporters.