Some assembly required: Contributions of Tom Stevens’ lab to the V‐ATPase field

The mechanisms by which a new subunit could be evolved within the V‐ATPase enzyme (or other multisubunit molecular machines) were not known. Therefore, in collaboration with Joe Thornton and colleagues, the Stevens lab computationally predicted and synthesized the most recent common ancestor to the Vma3 and Vma11 proteins (Anc.3‐11) as well as the ancestor to the more distantly related Vma16 subunit (Anc.16)—roughly estimated to be nearly a billion years ago. Following this “ancestral gene reconstruction,” the lab experimentally tested their function(s) in budding yeast.42 Incredibly, replacement of the entire yeast proteolipid system (deletion of all 3 native subunits) with the ancestral variants allowed for a functional V‐ATPase complex in vivo. Moreover, a mutational analysis of additional reconstructed variants revealed that inclusion of a small number of (loss‐of‐function) point mutations were sufficient to “ratchet” the newly evolved Vma11p ancestor into position as an obligate member of the structure.42 This work highlighted a possible general evolutionary mechanism for how biological complexity might arise by relatively “simple” molecular processes. A similar evolutionary approach was used to create an “ancestral a subunit,” a protein that had characteristics of both yeast subunit a isoforms Vph1p and Stv1p.