The development and characterization of Cardiolipin‐specific fluorescent protein markers to assess and understand the localization and behavior of Cardiolipin within mitochondrial membranes of yeast cells

Abstract

Mitochondria are double‐membrane organelles that generate most of the energy used by cells and play key roles in cellular growth, death and differentiation. Continuous remodeling and redistribution of the mitochondrial membrane structure are critical for integrating organelle physiology with cellular needs. Cardiolipin (CL) is a diphosphotidyl lipid found almost exclusively in bacteria and in the mitochondrial inner membrane of eukaryotes. CL distribution within mitochondrial membranes is important in determining membrane structure, regulating bioenergetic processes, and recruiting factors involved in cell death and mitochondrial dynamics to the outer mitochondrial membrane. However, CL's exact distribution and functional localization within mitochondria is poorly understood because there exists no means for detecting and visualizing CL in live cells. We have developed a CL sensor based on the CL‐binding properties of the “InsertB” domain of Mgm‐1, a yeast mitochondrial inner membrane fusion protein. In in vitro assays, Insert B specifically binds CL‐containing liposomes. In live yeast and mammalian cells, a fluorescent‐tagged Insert B protein forms distinct foci within mitochondria depending on the presence of CL. By making a series of site‐directed mutations in highly conserved regions of Insert B, we are further characterizing the marker's affinity and specificity for CL. We are now using this tool to better understand the localization and behavior of CL‐enriched regions within mitochondrial membranes and its association with mitochondrial function.Support or Funding InformationUC Davis Molecular and Cellular Biology Summer Fellowship for Undergraduate Research. Special thanks to Dr. Katherine Labbé and Dr. Jodi Nunnari.