Fitness landscape of substrate-adaptive mutations in evolved APC transporters

Reviewer #1 (Public Review):

Summary:
The evolution of transporter specificity is currently unclear. Did solute carrier systems evolve independently in response to a cellular need to transport a specific metabolite in combination with a specific ion or counter metabolite, or did they evolve specificity from an ancestral protein that could transport and counter-transport most metabolites? The present study addresses this question by applying selective pressure to Saccharomyces cerevisiae and studying the mutational landscape of two well-characterised amino acid transporters. The data suggest that AA transporters likely evolved from an ancestral transporter and then specific sub-families evolved specificity depending on specific evolutionary pressure.

Strengths:
The work is based on sound logic and the experimental methodology is well thought through. The data appear accurate, and where ambiguity is observed (as in the case of citruline uptake by AGP1), in vitro transport assays are carried out to verify transport function.

Weaknesses:
Although the data and findings are well described, the study lacked additional contextual information that would support a clear take-home message.

Reviewer #2 (Public Review):

Summary:
This paper describes evolution experiments performed on yeast amino acid transporters aiming at the enlargement of the substrate range of these proteins. Yeast cells lacking 10 endogenous amino acid transporters and thus being strongly impaired to feed on amino acids were again complemented with amino acid transporters from yeast and grown on media with amino acids as the sole nitrogen source.

In the first set of experiments, complementation was done with seven different yeast amino acid transporters, followed by measuring growth rates. Despite most of them have been described before in other experimental contexts, the authors could show that many of them have a broader substrate range than initially thought.

Moving to the evolution experiments, the authors used the OrthoRep system to perform random mutagenesis of the transporter gene while it is actively expressed in yeast. The evolution experiments were conducted such that the medium would allow for poor/slow growth of cells expressing the wt transporters, but much better/faster growth if the amino acid transporter would mutate to efficiently take up a poorly transported (as in the case of citrulline and AGP1) or non-transported (as in case of Asp/Glu and PUT4) amino acid.

This way and using Sanger sequencing of plasmids isolated from faster-growing clones, the authors identified a number of mutations that were repeatedly present in biological replicates. When these mutations were re-introduced into the transporter using site-directed mutagenesis, faster growth on the said amino acids was confirmed. Growth phenotype data were attempted to be confirmed by uptake experiments using radioactive amino acids; however, the radioactive uptake data and growth-dependent analyses do not fully match, hinting at the existence of further parameters than only amino acid uptake alone to impact the growth rates.

When mapped to Alphafold prediction models on the transporters, the mutations mapped to the substrate permeation site, which suggests that the changes allow for more favourable molecular interactions with the newly transported amino acids.

Finally, the authors compared the growth rates of the evolved transporter variants with those of the wt transporter and found that some variants exhibit a somewhat diminished capacity to transport its original range of amino acids, while other variants were as fit as the wt transporter in terms of uptake of its original range of amino acids.

Based on these findings, the authors conclude that transporters can evolve novel substrates through generalist intermediates, either by increasing a weak activity or by establishing a new one.

Strengths:
The study provides evidence in favour of an evolutionary model, wherein a transporter can "learn" to translocate novel substrates without "forgetting" what it used to transport before. This evolutionary concept has been proposed for enzymes before, and this study shows that it also can be applied to transporters. The concept behind the study is easy to understand, i.e. improving growth by uptake of more amino acids as nitrogen source. In addition, the study contains a large and extensive characterization of the transporter variants, including growth assays and radioactive uptake measurements.

Weaknesses:
The authors took a genetic gain-of-function approach based on random mutagenesis of the transporter. While this has worked out for two transporters/substrate combinations, I wonder how comprehensive and general the insights are. In such approaches, it is difficult to know which mutation space is finally covered/tested. And information that can be gained from loss-of-function analyses is missed. The entire conclusions are grounded on a handful of variants analyzed. Accordingly, the outcome is somewhat anecdotal; in some cases, the fitness of the variants was changed and in others not. Highlighting the amino acid changes in the context of the structural models is interesting, but does not fully explain why the variants exhibit changed substrate ranges. Two important technical elements have not been studied in detail by the authors, but may well play a certain role in the interpretation of the results. Firstly, the authors did not quantify the amount of transporter being present on the cell surface; altered surface expression can impact uptake rates and thus growth rates. Secondly, the authors have not assessed whether overexpressing wt versus variant transporters has an impact on the growth rate per se. Overexpressing transporters from plasmids is quite a burden for the cells and often impacts growth rates. Variants may be more or less of a burden, an effect that may (or may also not) go hand in hand with increased/decreased surface production levels.

And finally, I was somewhat missing an evolutionary analysis of these transporters to gain insights into whether the identified substitutions also occurred during natural evolution under real-life conditions.

Reviewer #3 (Public Review):

The goal of the current manuscript is to investigate how changes in transporter substrate specificity emerge through experimental evolution. The authors investigate the APC family of amino acid transporters, a large family with many related transporters that together cover the spectrum of amino acid uptake in yeast.

The authors use a clever approach for their experimental evolutions. By deleting 10 amino acid uptake transporters in yeast, they develop a strain that relies on amino acid import by introducing APC transporters under nitrogen-limiting conditions. They can thus evolve transporters towards the transport of new substrates if no other nitrogen source is available. The main takeaway from the paper is that it is relatively easy for the spectrum of substrates in a particular transporter of this family to shift, as a number of single mutants are identified that modulate substrate specificity. In general, transporters evolved towards gain-of-function mutations (better or new activities) and also confer transport promiscuity, expanding the range of amino acids transported.

The data in the paper support the conclusions, in general, and the outcomes (evolution towards promiscuity) agree with the literature available for soluble enzymes. However, it is also a possibility that the design of these experiments selects for promiscuity among amino acids. The selections were designed such that yeast had access to amino acids that were already transported, with a greater abundance of the amino acid that was the target of selection. Under these conditions, it seems probable that the fittest variants will provide the yeast access to all amino acid substrates in the media, and unlikely that a specificity swap would occur, limiting the yeast to only the new amino acid.

The authors also examine the fitness costs of mutants, but only in the narrow context of growth on a single (original) amino acid under conditions of nitrogen limitation. Amino acid uptake is typically tightly controlled because some amino acids (or their carbon degradation products) are toxic in excess. This paper does not address or discuss whether there might be a fitness cost to promiscuous mutants in conditions where nitrogen is not limiting.

Author Response

Reviewer #1 (Public Review):

Summary:

The evolution of transporter specificity is currently unclear. Did solute carrier systems evolve independently in response to a cellular need to transport a specific metabolite in combination with a specific ion or counter metabolite, or did they evolve specificity from an ancestral protein that could transport and counter-transport most metabolites? The present study addresses this question by applying selective pressure to Saccharomyces cerevisiae and studying the mutational landscape of two well-characterised amino acid transporters. The data suggest that AA transporters likely evolved from an ancestral transporter and then specific sub-families evolved specificity depending on specific evolutionary pressure.

Strengths:

The work is based on sound logic and the experimental methodology is well thought through. The data appear accurate, and where ambiguity is observed (as in the case of citruline uptake by AGP1), in vitro transport assays are carried out to verify transport function.

Weaknesses:

Although the data and findings are well described, the study lacked additional contextual information that would support a clear take-home message.

We appreciate the reviewer’s positive assessment of the work, and the helpful comment to summarize the findings into a short take-home message. We chose not to discuss protein evolution theories in detail to keep the text as concise as possible. However, we do acknowledge the fact that the reader might want to see our results embedded in more context. In a revised version, we will integrate our findings more with the pertinent literature, which will show how our results align with theoretical models for protein evolution towards novel functions. We will also discuss in more detail how our laboratory results could be translated into a “natural” setting of evolution.

Reviewer #2 (Public Review):

Summary:

This paper describes evolution experiments performed on yeast amino acid transporters aiming at the enlargement of the substrate range of these proteins. Yeast cells lacking 10 endogenous amino acid transporters and thus being strongly impaired to feed on amino acids were again complemented with amino acid transporters from yeast and grown on media with amino acids as the sole nitrogen source.

In the first set of experiments, complementation was done with seven different yeast amino acid transporters, followed by measuring growth rates. Despite most of them have been described before in other experimental contexts, the authors could show that many of them have a broader substrate range than initially thought.

Moving to the evolution experiments, the authors used the OrthoRep system to perform random mutagenesis of the transporter gene while it is actively expressed in yeast. The evolution experiments were conducted such that the medium would allow for poor/slow growth of cells expressing the wt transporters, but much better/faster growth if the amino acid transporter would mutate to efficiently take up a poorly transported (as in the case of citrulline and AGP1) or non-transported (as in case of Asp/Glu and PUT4) amino acid.

This way and using Sanger sequencing of plasmids isolated from faster-growing clones, the authors identified a number of mutations that were repeatedly present in biological replicates. When these mutations were re-introduced into the transporter using site-directed mutagenesis, faster growth on the said amino acids was confirmed. Growth phenotype data were attempted to be confirmed by uptake experiments using radioactive amino acids; however, the radioactive uptake data and growth-dependent analyses do not fully match, hinting at the existence of further parameters than only amino acid uptake alone to impact the growth rates.

When mapped to Alphafold prediction models on the transporters, the mutations mapped to the substrate permeation site, which suggests that the changes allow for more favourable molecular interactions with the newly transported amino acids.

Finally, the authors compared the growth rates of the evolved transporter variants with those of the wt transporter and found that some variants exhibit a somewhat diminished capacity to transport its original range of amino acids, while other variants were as fit as the wt transporter in terms of uptake of its original range of amino acids.

Based on these findings, the authors conclude that transporters can evolve novel substrates through generalist intermediates, either by increasing a weak activity or by establishing a new one.

Strengths:

The study provides evidence in favour of an evolutionary model, wherein a transporter can "learn" to translocate novel substrates without "forgetting" what it used to transport before. This evolutionary concept has been proposed for enzymes before, and this study shows that it also can be applied to transporters. The concept behind the study is easy to understand, i.e. improving growth by uptake of more amino acids as nitrogen source. In addition, the study contains a large and extensive characterization of the transporter variants, including growth assays and radioactive uptake measurements.

Weaknesses:

The authors took a genetic gain-of-function approach based on random mutagenesis of the transporter. While this has worked out for two transporters/substrate combinations, I wonder how comprehensive and general the insights are. In such approaches, it is difficult to know which mutation space is finally covered/tested. And information that can be gained from loss-of-function analyses is missed. The entire conclusions are grounded on a handful of variants analyzed. Accordingly, the outcome is somewhat anecdotal; in some cases, the fitness of the variants was changed and in others not. Highlighting the amino acid changes in the context of the structural models is interesting, but does not fully explain why the variants exhibit changed substrate ranges. Two important technical elements have not been studied in detail by the authors, but may well play a certain role in the interpretation of the results. Firstly, the authors did not quantify the amount of transporter being present on the cell surface; altered surface expression can impact uptake rates and thus growth rates. Secondly, the authors have not assessed whether overexpressing wt versus variant transporters has an impact on the growth rate per se. Overexpressing transporters from plasmids is quite a burden for the cells and often impacts growth rates. Variants may be more or less of a burden, an effect that may (or may also not) go hand in hand with increased/decreased surface production levels.

And finally, I was somewhat missing an evolutionary analysis of these transporters to gain insights into whether the identified substitutions also occurred during natural evolution under real-life conditions.

First of all, we thank the reviewer for the attention to detail with which they have read the manuscript, and the very helpful comments on how to improve it. We will indeed take on some of the suggestions in a revised version of the text:

Regarding the match of growth rate and uptake rate measurements, we plan to plot their correlation in a graph.

Regarding the amount of transporter on the plasma membrane, we acknowledge that the visual representation of the fluorescence micrographs already in the text might not be enough. We therefore will quantify expression levels from said micrographs and include the information in the manuscript.

On a similar note, we had already measured the growth rates of all transporter variant cultures in the absence of selection for amino acid uptake (i.e., in medium with ammonium as the nitrogen source; Figure 4 - Supplement figure 1). We will include the measured growth rates in the text to give an indication of what the impact of transporter overexpression is on the growth rate per se.

Regarding the proposed analysis of natural transporter sequences, we do see the possible value in such an analysis. However, it is currently out of scope for the present study. The reasons are 1) that preliminary analyses show that the sequence similarity of functionally verified/annotated transporters is too low to reliably pinpoint a phenotype to a single residue, and 2) that we do not envision that the variants that we discovered are necessarily beneficial in a natural setting, where fine-grained regulation of amino acid transport may be more important than a broad substrate range. Regarding the generality of the insights, we do agree on the reviewer’s comment that we “only” analyzed a relatively small number of variants. However, the target of the study was not to generate high-throughput data on a large set of variants (e.g., by NGS of the whole culture) but to provide in-depth data for characterized and verified variants in a clean genetic background (i.e., verified phenotype and fitness measurements on all native and novel substrates).

As to the mutation space, we will include an estimate in a revised version of the text. We estimate that a majority of all possible single mutants is covered in the first and second passages of the selection experiment, which is corroborated by the fact that we repeatedly find the same mutants in biological replicates.

Regarding the mentioned loss-of-function analyses, we are unsure about what the reviewer intends with this statement at this point. To briefly summarize, we feel that our results are a good indication that transporters can evolve new functions analogously to enzymes. We explicitly do not imply that this is the only way to evolve novelty.

Reviewer #3 (Public Review):

The goal of the current manuscript is to investigate how changes in transporter substrate specificity emerge through experimental evolution. The authors investigate the APC family of amino acid transporters, a large family with many related transporters that together cover the spectrum of amino acid uptake in yeast.

The authors use a clever approach for their experimental evolutions. By deleting 10 amino acid uptake transporters in yeast, they develop a strain that relies on amino acid import by introducing APC transporters under nitrogen-limiting conditions. They can thus evolve transporters towards the transport of new substrates if no other nitrogen source is available. The main takeaway from the paper is that it is relatively easy for the spectrum of substrates in a particular transporter of this family to shift, as a number of single mutants are identified that modulate substrate specificity. In general, transporters evolved towards gain-of-function mutations (better or new activities) and also confer transport promiscuity, expanding the range of amino acids transported.

The data in the paper support the conclusions, in general, and the outcomes (evolution towards promiscuity) agree with the literature available for soluble enzymes. However, it is also a possibility that the design of these experiments selects for promiscuity among amino acids. The selections were designed such that yeast had access to amino acids that were already transported, with a greater abundance of the amino acid that was the target of selection. Under these conditions, it seems probable that the fittest variants will provide the yeast access to all amino acid substrates in the media, and unlikely that a specificity swap would occur, limiting the yeast to only the new amino acid.

The authors also examine the fitness costs of mutants, but only in the narrow context of growth on a single (original) amino acid under conditions of nitrogen limitation. Amino acid uptake is typically tightly controlled because some amino acids (or their carbon degradation products) are toxic in excess. This paper does not address or discuss whether there might be a fitness cost to promiscuous mutants in conditions where nitrogen is not limiting.

We are grateful for the reviewer’s insightful comments on the paper.

Regarding the design of our experiments, we followed the concept of directed evolution as described by pioneers of the field, in which the starting point for evolving a protein is to have a basic level of that activity. In the case of AGP1, the promiscuous activity is Cit uptake. We recognize that elimination of all the already transported amino acids from the evolution media could also yield very insightful results. However, we aimed to simulate the effect of the evolutionary pressure acting in a “natural” environment, where the uptake of the specific amino acid is not initially crucial for its survival. In the case of PUT4, the experimental design was chosen to ensure the initial survival of the culture (since neither Glu nor Asp support the growth of the strain) by providing a low level of already transported amino acids. In the revised manuscript, we will state this more clearly.

Regarding the second point, we agree that a short discussion about the potentially detrimental effects of promiscuous transporters would be beneficial for the reader. We will touch on this aspect in the revised version of the text. Indeed, our system is intentionally simplified, as we try to take regulation of transport out of the equation (e.g., by using the constitutive ADH1 promoter as opposed to a nitrogen-regulated one). In a natural setting, microorganisms encounter fluctuations of nutrient availability, necessitating tight control of nutrient transport. This is probably a major reason why microorganisms typically encode transporters with redundant specificities (i.e., promiscuous and specific ones). Otherwise, one very broad-range nutrient transporter would suffice. In our system, we artificially select for broad-range transport, which is reflected in the observed phenotypes of the evolved transporters. We expect that in a natural setting, a broad-range transporter would be a stepping stone to evolve a narrow-range transporter with a new specificity (which is actually what we see in the double-mutant AGP1-NV, with lowered fitness in original substrates and increased fitness in Cit).