Abstract
Phosphatidylcholine (PC) is a major phospholipid of all cellular membranes in eukaryotes, and it is also present in large quantities in both mitochondrial membranes. Since the final steps of PC biosynthesis take place in the endoplasmic reticulum, proper mitochondrial biogenesis necessitates PC transport to the mitochondria and intramitochondrial sorting of
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PC over both mitochondrial membranes. The mechanisms and regulatory aspects involved in these processes are unknown. The work described in this thesis was primarily aimed at elucidation of the mechanism of PC import and identification of the components involved. The yeast Saccharomyces cerevisiae was chosen as the model organism for these investigations, because it shares similar features of cellular organization and phospholipid composition and biosynthesis with higher eukaryotes. Its facultative requirement for mitochondria makes it a convenient model organism to study mitochondrial biogenesis. In addition, it offers the potential for classical genetics and genetic manipulation. This, in combination with its sequenced genome, allows for genetic as well as biochemical studies on candidate genes and gene products involved in PC import.
In chapter 2 of this thesis, the investigation of the transbilayer movement of PC across the outer mitochondrial membrane is described. An in vitro approach using isolated mitochondrial outer membrane vesicles (OMV) was applied, and the possible involvement of protein factors in this process was studied. Variations observed in mitochondrial PC and PE content were investigated by addressing the influence of growth phase and carbon source on the phospholipid composition and phospholipid biosynthetic activities, as described in chapter 3. In chapter 4, the possibility was examined, that the methyltransferases, catalyzing the conversion of phosphatidylethanolamine (PE) to PC, are able to act upon a substrate, localized in another membrane than the one in which the enzymes reside. Such a mechanism would preclude the need for actual transport of PC between the endoplasmic reticulum and the mitochondria. Subcellular fractions isolated from opi3 and cho2 knock-out strains were used. It is important to know that the integrity of mitochondria is preserved in in vitro studies, and the buffer requirements for maintaining mitochondrial intactness and membrane potential are reported in chapter 5. The possibility of developing a genetic approach for identifying yeast mutants in PC import depends greatly on knowledge on the role of PC in mitochondria, since such an approach requires an appropriate screening or selection method, preferably making use of PC dependent processes in mitochondria. Chapter 6 explores the interaction of PC with mitochondrial proteins, using a photoactivatable phospholipid analog, since this might shed light on the specific role of PC in mitochondria, or even allow the identification of proteins directly involved in the intramitochondrial transport of PC. Finally, in chapter 7, the results of these studies are summarized and discussed
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