Adaptive Mutations in Yeast Amino Acid Transporters Expand Substrate Specificity

Kernekoncepter

Amino acid transporters can evolve novel substrate specificities through generalist intermediates, either by improving weak promiscuous activities or establishing new transport functions.

Resumé

The study investigates the evolution of substrate specificity in yeast amino acid transporters (YAT) from the APC superfamily. The authors first demonstrate that the substrate range of several wild-type YAT transporters is broader than previously reported, with some transporters exhibiting weak promiscuous activities towards additional substrates.

Using in vivo experimental evolution, the authors then evolve two YAT transporters, AGP1 and PUT4, towards new substrate specificities under selective pressure. They find that single mutations in the transmembrane regions of these transporters are sufficient to expand their substrate range, either by improving an existing weak activity or establishing transport of a new substrate.

Importantly, the adaptive mutations have distinct impacts on the fitness for the transporters' original substrates. Some mutations retain the ability to transport all original substrates, while others show decreased fitness for certain original substrates, indicating a trade-off between the ancestral and evolved functions.

The results showcase that membrane transporters can follow a similar evolutionary logic as soluble enzymes, evolving new functions through generalist intermediates. The authors propose that the observed promiscuous activities and standing genetic variation in transporters can prime organisms for rapid adaptation to new ecological niches.

Statistik

The growth rate of the Δ10AA strain expressing the wild-type AGP1 transporter on 2 mM citrulline (Cit) as the sole nitrogen source is 0.03 h-1.
The growth rate of the Δ10AA strain expressing the AGP1-G variant on 2 mM Cit is 0.09 h-1, a 3.2-fold increase compared to the wild-type.
The uptake rate of 1 mM 14C-Cit by the Δ10AA strain expressing the AGP1-G variant is 2.5-fold higher than the wild-type.
The growth rate of the Δ10AA strain expressing the wild-type PUT4 transporter on 2 mM aspartate (Asp) or glutamate (Glu) as the sole nitrogen source is 0.00 h-1.
The growth rate of the Δ10AA strain expressing the PUT4-S variant on 2 mM Asp or Glu is 0.10 h-1 and 0.12 h-1, respectively.
The uptake rate of 10 μM 14C-Ala or 14C-GABA by the Δ10AA strain expressing the PUT4-S variant is 2.5-fold and 3-fold lower, respectively, compared to the wild-type PUT4.

Citater

"Amino acid transporters can evolve novel functions through generalist intermediates, similar to classical enzymes."
"Each adaptive mutation comes with a distinct effect on the fitness for each of the original substrates, illustrating a trade-off between the ancestral and evolved functions."
"The evolved mutations in AGP1 are also positioned in the transmembrane helices that create the permeation pathway of LeuT-fold transporters."

Vigtigste indsigter udtrukket fra

Fitness landscape of substrate-adaptive mutations in evolved APC transporters

by Karapanagiot... kl. www.biorxiv.org 10-12-2023

https://www.biorxiv.org/content/10.1101/2023.10.12.562049v4

Dybere Forespørgsler

How do the evolutionary trajectories of transporters differ from those of soluble enzymes in terms of the emergence of new functions

The evolutionary trajectories of transporters differ from those of soluble enzymes in several key ways when it comes to the emergence of new functions. While soluble enzymes often evolve new functions by modifying existing genes or duplicating pre-existing ones, membrane transporters face unique challenges due to their role in facilitating the movement of molecules across cell membranes.
One significant difference is that membrane transporters do not break or make covalent bonds like enzymes do. This difference in function means that transporters experience fewer constraints in terms of the chemistry of their substrates. As a result, membrane transporters have the potential to transport a wide range of small molecules through lipid membranes, allowing for the evolution of transport systems for virtually every metabolite, ion, and xenobiotic.
Additionally, membrane transporters often belong to large gene families with multiple paralogs, each displaying distinct substrate specificity profiles. This gene family expansion through duplication and divergence can lead to the evolution of transporters with different substrate specificities, allowing organisms to adapt to changing environmental conditions and explore new ecological niches.
Overall, the evolutionary trajectories of transporters involve the acquisition of new functions through generalist intermediates, similar to soluble enzymes, but with unique considerations related to their role in membrane transport and the diversity of substrates they can interact with.

What are the potential mechanisms by which a single mutation can simultaneously improve transport of a new substrate while retaining the ability to transport original substrates

A single mutation can simultaneously improve transport of a new substrate while retaining the ability to transport original substrates through several potential mechanisms. One mechanism is through changes in the substrate-binding site of the transporter, which can alter the affinity and specificity of the transporter for different substrates. For example, in the case of the PUT4 transporter, the L207S mutation was found to be part of the substrate-binding site and led to an expansion of the substrate range to include Asp and Glu while still maintaining the ability to transport original substrates like Ala, GABA, Gly, Pro, Ser, and Val.
Another mechanism is through structural changes in the transmembrane helices of the transporter, which can impact the permeation pathway and the overall function of the protein. Mutations in key positions within the transmembrane domains can affect the transport properties of the transporter, allowing for the uptake of new substrates without compromising the ability to transport original substrates.
Furthermore, mutations that impact the surface expression or localization of the transporter can also influence its transport activity for different substrates. By altering the distribution of the transporter within the cell membrane, a mutation can modulate the interaction between the transporter and its substrates, leading to changes in substrate specificity and transport efficiency.
Overall, a single mutation can have multifaceted effects on the transport properties of a membrane transporter, enabling the protein to adapt to new environmental conditions while still fulfilling its original function.

What are the implications of the observed trade-offs between ancestral and evolved functions for the long-term evolution and specialization of membrane transporters in natural environments

The observed trade-offs between ancestral and evolved functions in membrane transporters have significant implications for their long-term evolution and specialization in natural environments. These trade-offs highlight the complexity of transporter evolution, where mutations that improve the transport of new substrates may come at the cost of reduced fitness for original substrates.
One implication is that the evolution of membrane transporters towards novel functions involves a delicate balance between maintaining ancestral functions and acquiring new capabilities. The trade-offs observed in this study demonstrate that mutations that enhance the transport of new substrates can have varying effects on the fitness of the organism for original substrates, illustrating the evolutionary constraints faced by transporters in adapting to changing environmental conditions.
Furthermore, the trade-offs between ancestral and evolved functions can influence the evolutionary trajectory of membrane transporters, shaping their specialization and adaptation to specific ecological niches. Mutations that confer a fitness advantage for new substrates may lead to the emergence of specialized transporters with distinct substrate specificities, allowing organisms to exploit new resources and thrive in diverse environments.
Overall, the trade-offs between ancestral and evolved functions in membrane transporters play a crucial role in the long-term evolution and specialization of these proteins, highlighting the dynamic nature of transporter evolution and the adaptive strategies employed by organisms to survive and thrive in their natural habitats.