UCSF

Maintaining protein homeostasis is essential for a proper functioning cell. From the time a polypeptide chain begins to be synthesized to the time the final product arrives at its destination there is an incredible amount of regulation taking place. The dysregulation of these processes can have disastrous effects for the cell. In this thesis I will be exploring two important areas of regulation: translation initiation and protein targeting. In Chapter 1, I will show structural, biochemical, and cell-based data characterizing the AAA ATPase ATAD1. ATAD1 is tasked with removing mistargeted tail-anchored proteins from the mitochondrial outer membrane. Through cryo-electron microscopy, we solved the structure of this protein with a peptide trapped in the central pore and designed a cell-based assay to validate structure-based predictions. We identified conserved structural elements that are essential for the function of ATAD1, in particular, the unusually aromatic nature of the pore loops that enable ATAD1 to extract it’s hydrophobic substrates.

In Chapter 2, I will be describing our characterization of an alternative translation initiation factor eukaryotic initiation factor 2A (eIF2A). From the initial discovery of this protein in the 1970’s, eIF2A has been implicated in non-canonical translation initiation processes and has been shown to be important for responding to stress. Using available genome-wide genetic interaction datasets for Saccharomyces cerevisiae, we identified and characterized the genetic interaction between eIF2A and TIF1 (the DEAD-box helicase component of the eIF4F complex, eIF4A). We found that strains with both proteins deleted exhibited translational reprogramming that resulted in a sensitivity to metabolic stressors. As an exciting entry into detailed in vitro characterization, we also overcame a hurtle in the recombinant expression of the human version of eIF2A and have begun re-examining the previously held assumptions about the mechanism of eIF2A-dependent translation.