CAREER: Investigating the mechanisms of peroxisomal membrane protein biogenesis

Animal, plant, and fungal cells are divided by internal membranes that define unique compartments termed organelles. These organelles allow the cell to create different chemical environments. For example, an organelle called the peroxisome sequesters metabolic enzymes. These enzymes may produce toxic byproducts, such as reactive oxygen species, alongside enzymes that degrade those toxic byproducts to protect the cell. Peroxisome function is essential to human health, agricultural success, and offers rich potential for the discovery of novel biochemical pathways in newly sequenced eukaryotes. Furthermore, there is growing interest in synthetic biology in using peroxisomal sequestration to improve production titers of small molecules of commercial interest. However, our ability to predict and control peroxisome function is hampered by our lack of understanding of its membrane proteins, which control the availability of metabolites inside the peroxisome. The goals of this proposal are to understand how cells make, target, and maintain the proteins in the peroxisome membrane. This project sheds light on the evolution of peroxisomes, enabling their use as synthetic organelles, and improving the understanding of their role in disease and biotechnology. In addition, the educational component to this grant improves outcomes in biology education through increasing access to research experiences for transfer students. Membrane proteins control the transport of small molecules across membranes, regulate signaling cascades, and define the unique identity of each organelle. These proteins can reach the peroxisome through two routes: direct insertion from the cytosol, or indirect trafficking from the endoplasmic reticulum (ER). The latter mechanism involves well-defined insertases at the ER but utilizes poorly defined ER-to-peroxisome trafficking machinery. Two proteins, PEX3 and PEX19, are essential to both routes, though their mechanisms of action in each route are poorly understood. The scientific goals of this project are to elucidate how PEX3 and PEX19 interact with each other and peroxisomal membrane proteins to define the minimal requirements for direct insertion. The project discovers the mechanisms used for ER-to-peroxisome trafficking of peroxisome membrane proteins and determines factors that mediate membrane protein stability at the peroxisome membrane. The approaches feature peptide arrays for assessing the binding preferences of Pex3 and Pex19, Laurdan anisotropy for assessing membrane fluidity and fluorescence imaging for localization studies. Improved understanding of peroxisome membrane protein biogenesis has the potential to reveal a novel mechanism of membrane protein insertion, improve predictions of peroxisome proteomes and metabolic function, and elucidate the contributions of peroxisome membrane quality control to disease outcomes. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria. NSF Award ID: 2543258 | Program: 01003031DB NSF RESEARCH & RELATED ACTIVIT,01002627DB NSF RESEARCH & RELATED ACTIVIT | Principal Investigator: Brooke Gardner | Institution: University of California-Santa Barbara, SANTA BARBARA, CA | Award Amount: $1,201,130 View on NSF Award Search: https://www.nsf.gov/awardsearch/show-award/?AWD_ID=2543258 View on Research.gov: https://www.research.gov/awardapi-service/v1/awards/2543258.html