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

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.