Ancestral Protein Reconstruction of a membrane trafficking GTPase uncovers unanticipated properties of the ancestral protein and of modern Arf1 GTPases
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Abstract
The emergence of eukaryotes from their prokaryotic ancestors (eukaryogenesis) marked a fundamental shift in cellular organisation, with the appearance of intracellular compartments including the nucleus, the Golgi apparatus and endosomes. These organelles are part of the endomembrane system of eukaryotic cells, which mediates many processes, including secretion of proteins to the exterior of the cell, uptake of material by endocytosis, and compartmentalized degradation of cellular components. The period of eukaryogenesis after the merger of prokaryotic lineages but preceding the last eukaryotic common ancestor, is inferred to have involved a progressive increase in cellular complexity through expansion of organelle-specific protein machineries. However, the steps and stages of organelle emergence during this period are poorly understood as no extant organisms exist from this period, precluding the use of comparative genomics to determine the properties of ancestral proteins present. Membrane trafficking pathways linking organelles are regulated by Arf family GTPases, including Arf1 and Arf6, both present in the last eukaryotic common ancestor. Here we use ancestral sequence reconstruction and molecular cell biological characterization to explore the properties of the ancestor of the Arf1 and Arf6 GTPases. Arf1 has a major function at the Golgi apparatus in regulation of the secretory pathway, whereas Arf6 regulates endocytic pathways at the plasma membrane and endosomes. Our results indicate that the ancestral Arf1/6 protein localizes to both the Golgi and the plasma membrane. We find that localization to the plasma membrane is due to a C-terminal polybasic motif that unexpectedly is also found in a number of modern Arf1 proteins from a wide diversity of eukaryotes. Our data suggest that the ancestral Arf protein acted at both internal compartments and the cell periphery, a feature preserved in a number of modern Arf1 proteins.
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While ancient protein resurrection has been used to investigate deep nodes within the eukaryotic tree 31 and even aspects of LECA proteins 30, to our knowledge, this is the first attempt to reconstruct a pre-LECA protein.
Overall, the work carried out here is exciting for the reasons mentioned. The ancestral protein reconstruction was carried out rigorously, the predicted proteins were of very high confidence across sites, and the localization of the proteins clearly shows promiscuous functions that allow us to picture the evolution of essential endomembrane system functions. I think the questions being asked are very exciting, and this is a perfect use case for ASR. I look forward to seeing the functional characterization of these ancestral proteins taken further to include the other nodes of interest, validation in different …
While ancient protein resurrection has been used to investigate deep nodes within the eukaryotic tree 31 and even aspects of LECA proteins 30, to our knowledge, this is the first attempt to reconstruct a pre-LECA protein.
Overall, the work carried out here is exciting for the reasons mentioned. The ancestral protein reconstruction was carried out rigorously, the predicted proteins were of very high confidence across sites, and the localization of the proteins clearly shows promiscuous functions that allow us to picture the evolution of essential endomembrane system functions. I think the questions being asked are very exciting, and this is a perfect use case for ASR. I look forward to seeing the functional characterization of these ancestral proteins taken further to include the other nodes of interest, validation in different eukaryotic systems, and the characterization of more essential protein functions.
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he major localization of Arf1 is at the Golgi, where a highly conserved function is in vesicular traffic from the Golgi to either the ER or the endosomal system 27,42,43, whereas Arf6 localizes predominantly to the PM and endosomes, where it regulates the actin cytoskeleton and endocytic pathways
In coming papers, it would be great to see the functional characterization of these ancestral gene expanded much further. Localization is a great place to start, since much of the function of these genes is related to where they are being expressed. However, most of the functions of these extant proteins listed here were tested in the ancestral proteins. These test could be as simple as looking for morphological differences in organelles, or imaging trafficking inside of mutant cells. These are essential genes, so this could be done with …
he major localization of Arf1 is at the Golgi, where a highly conserved function is in vesicular traffic from the Golgi to either the ER or the endosomal system 27,42,43, whereas Arf6 localizes predominantly to the PM and endosomes, where it regulates the actin cytoskeleton and endocytic pathways
In coming papers, it would be great to see the functional characterization of these ancestral gene expanded much further. Localization is a great place to start, since much of the function of these genes is related to where they are being expressed. However, most of the functions of these extant proteins listed here were tested in the ancestral proteins. These test could be as simple as looking for morphological differences in organelles, or imaging trafficking inside of mutant cells. These are essential genes, so this could be done with inducible knockouts and gene rescues.
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We conclude that a property of the Arf1/6 predicted ancestral protein is that it had the capacity to localize to both the Golgi and the PM, and that PM localization is mediated by C-terminal basic residues.
really appreciate seeing not just the localization, but also the mechanisms of localization being tested in distantly-related eukaryotes. It really helps to confirm the functions of specific residues/domains and further supports that these traits were foundational to the early sub-functionalization of the endomembrane proteins.
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We mutated these lysine residues to glutamines, and expressed the mutants (Arf1/6PAML-3KQ and Arf1/6IQ-4KQ) in mammalian cells.
This is the first section of the text where '3KQ' and '4KQ' are explained, both in what they mean and why the mutations were carried out. However, these first come up in Figure 3. For that reason, I would move up the mention of these definitions in the text, or add it into the figure description.
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Thus we are confident that both the IQ-TREE and PAML reconstructions are robust and that together they cover the sequence space for possible ancestral reconstruction of the Arf1/6 ancestral protein
Looking at figure 2a, it appears that some sites that are of low confidence using one method are identical across both methods. For these low confidence sites, it is custom to test a secondary sequence that incudes the second most likely residues. I couldn't find supplement table 2, so it is possible you've already done that but i just couldn't look at the sequences to verify, and it wasn't clear from the text either.
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a constrained tree was constructed following the species relationships of the taxa sampled
It is common to find multiple gene duplication events inside of taxa, especially those representing multiple highly divergent species. Make sure you looked for signs of this in the unconstrained tree to see if there might be some unexpected taxa within your four main taxa. I do agree that it is very important to do species-relationship-guided constraints in creating these trees.
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