Investigating protein-lipid interactions with implications for apoptosis using fluorescent cytochrome c

Abstract

In healthy cells, cytochrome c (cyt c) acts as an electron transport protein peripherally bound to the inner mitochondrial membrane lipid cardiolipin, but it has an important role in apoptosis - upon apoptotic stimuli, it is released from the mitochondria into the cytosol, where it sets off a cascade of activities leading to cell death. The signals and mechanisms which result in this release of cyt c are an intense topic of study, and the specific interactions of cyt c with cardiolipin are thought to be implicated in this process. In this work, we use fluorescent-labeled cyt c to observe the interactions of the protein with cardiolipin and related lipids, asking two questions:

1. How does the protein affect lipid membranes?

2. How do lipids affect the protein?

In chapter 2, we report that treatment of synthetic cardiolipin-containing phospholipid membranes with cyt c allows leakage across the membrane of not only the protein itself, but also of passive leakage markers. Using fluorescence confocal microscopy, we record images and videos of this leakage, demonstrating for the first time the formation of pores by cyt c in intact unilamellar membranes that are directly analogous to mitochondrial membranes. Our results suggest that if cardiolipin is mixed into the outer mitochondrial membrane near intermembrane contact sites, cyt c might contribute to its own escape from mitochondria during apoptosis.

In chapter 3, we describe a novel morphological transition undergone by cardiolipin-containing membranes treated with cyt c. We engineer vesicles with compositions such that they phase separate into coexisting liquid phases and cardiolipin preferentially localizes into one of the domains. The presence of cardiolipin-rich and cardiolipin-depleted domains within the same vesicle provides a built-in control experiment to simultaneously observe the behavior of two membrane compositions under identical conditions. We find that cyt c binds strongly to the cardiolipin-rich domains, and we observe that these domains start to form small buds and eventually fold up into a collapsed state.

Finally, in chapter 4, we find that cyt c responds to cis unsaturated fatty acids and phospholipids with a change in fluorescence of an added dye, which reflects a conformational change including a decrease in helical content and a detachment of the Met80 from the heme iron, and induction of peroxidase activity; these changes are thought to be associated with a pro-apoptotic conformation of the protein. These structural and functional changes are not observed when cyt c is treated with saturated fatty acids and phospholipids, while intermediate changes are in some cases observed for trans elaidic acid. While the more general question of the specificity of cyt c-lipid binding has been discussed in the literature for some time - with apparently contradictory results - to the best of our knowledge, this document is the first to demonstrate the preference of cyt c for cis unsaturated over trans unsaturated lipids.