Jessica Cope L&S Biological Sciences
Purification and Analysis of Pex2/10/12 Ligase Complex and Ubc4 Enzyme
Peroxisomes are membrane bound organelles found in nearly all eukaryotic cells and are indispensable for human health. The peroxisomal matrix contains many enzymes which are synthesized in the cytosol and then imported into the peroxisomes via the protein, Pex5. Pex5 binds to target proteins in the cytosol then chaperones the client protein into the peroxisomes. An E3 ubiquitin ligase complex made of three proteins: Pex2, Pex10, and Pex12 then mediates the transfer of a ubiquitin from the E2 ubiquitin conjugating enzyme Ubc4 to Pex5. Once ubiquitinated, Pex5 returns to the cytosol through the Pex2/10/12 complex to be either recycled or degraded. A current model of the Pex2/10/12 complex results in steric clashes during this interaction, leading me to hypothesize that a conformational change is necessary. In this project I will be expressing, purifying, and visualizing these components using cryo-EM. Because the Pex2/10/12 complex facilitates the ubiquitination of Pex 5 by Ubc4, ascertaining how Ubc4 interacts with Pex2/10/12 may be imperative to determining the mechanism of Pex5 retrotranslocation.