Ancient Protein Resurrection of an ancestral Arf GTPase that regulates membrane trafficking uncovers dual cellular localization and unanticipated properties of modern Arf1 proteins

The evolution of eukaryotes, eukaryogenesis, marked a fundamental shift in cellular organisation for life on Earth, with the advent of the nucleus, mitochondria, and other compartmentalization. The organelles and machinery of the endomembrane system, being responsible for material synthesis, intracellular targeting, uptake and secretion, are key eukaryotic hallmark to be explained. The period of eukaryogenesis after the merger of prokaryotic lineages but preceding the Last Eukaryotic Common Ancestor, is inferred to have involved a rise in cellular complexity through expansion of organelle-specific protein machinery, but the steps and stages of organelle emergence is essentially a blackbox. This is particularly problematic since ancestral protein function is not necessarily inferable from modern components. Here we apply Ancient Protein Resurrection (Ancestral Sequence Reconstruction and molecular cell biological characterization) to explore the ancient evolution of the Arf GTPases. Arf1 is canonically understood to act in eukaryotic cells at the Golgi apparatus, while Arf6 acts at endosomes and the plasma membrane. We find that the inferred ancestral Arf1/6 protein acts at both Golgi apparatus and plasma membrane in both mammalian and yeast cells. Unexpectedly, we find that the mechanism of localization to the plasma membrane is due to a C-terminal motif found in Arf1 proteins from the wide diversity of modern eukaryotes, though not the best characterized models of yeast, plants, and humans. Our data suggests that the ancestral Arf protein acted at multiple locations in eukaryotic cells prior to the diversification of modern lineages and Arf1 proteins may do so to a greater extent than appreciated in organisms of medical and ecological importance today.