UC Berkeley

AAA+ ATPases hydrolyze ATP to perform diverse cellular functions. While these complex machines have been the subject of biochemical studies for years, the details on what these motors contribute to cellular processes, how these motors perform work on their substrates, and the identify of their substrates remains unknown for many AAA+ ATPases. Specifically, it is unclear how the activity of two such enzymes, Pex1/Pex6 and Msp1, brings about their cellular functions. It is known that Pex1/Pex6 is essential for peroxisome biogenesis, but the identity of Pex1/Pex6’s substrate is unknown. Furthermore, an understanding of how Pex1/Pex6 processes this substrate, and how this processing contributes to peroxisome biogenesis, is lacking. Msp1 is a poorly studied AAA+ ATPase embedded in the outer-mitochondrial membrane. It is responsible for extracting mislocalized tail-anchored proteins from this surface, but the mechanism of action it uses for extraction and how it recognizes mislocalized proteins remain unclear. Furthermore, although Msp1 has been shown to be present at peroxisomes, details regarding how its function may differ from its mitochondrial duties are unexplored. For my dissertation, I designed and established in vitro assays to study the biochemical mechanisms of both Pex1/Pex6 and Msp1.

To better understand Pex1/Pex6’s role at the peroxisomal membrane, I focused on characterizing its primarily peroxisomal binding partner, Pex15. I solved the crystal structure of the soluble core domain of Pex15, and exploited its biochemical features to develop an assay that measures its mechanical unfolding. Furthermore, I demonstrated that the Pex1/Pex6 ATPase is capable of unfolding the soluble Pex15 domain, constituting the first direct evidence that Pex1/Pex6 unfolds substrates. I utilized these assays to define both, motor mechanism and substrate requirements to establish that the intrinsically disordered regions of Pex15 play crucial roles in Pex1/Pex6 interaction and engagement.

Although Pex15 functions as a peroxisomal tether for Pex1/Pex6, it occasionally mislocalizes to the outer-mitochondrial membrane, where it becomes a substrate for Msp1. Msp1 is believed to function as a hexamer; however, it fails to oligomerize when the N-terminal transmembrane domain is removed, presenting a challenge to in vitro characterization of this motor. I developed a system to study Msp1 activity in vitro by fusing it to a hexamerizing scaffold to encourage oligomerization. I used this system to characterize Msp1’s mechanism of action, provided direct evidence that it is capable of unfolding proteins by processive threading, and showed that it is promiscuous in substrate selection. Lastly, I demonstrated that Pex3 directly inhibits Msp1 unfoldase activity. Overall, my work helped elucidate AAA+ motor function and further our understanding of peroxisome biogenesis and proteome quality control at the peroxisome and mitochondria.