Phosphatidylcholine-protein interactions and remodeling of cardiolipin in yeast mitochondria

Abstract

The aim of the research described in the thesis was i) to gain insight into the significance of phosphatidylcholine (PC) for the functioning of the glycerol-3-phosphate dehydrogenase Gut2 and the proline oxidase Put1 in yeast mitochondria, and ii) to advance the understanding of acyl chain remodeling of the specific mitochondrial lipid cardiolipin (CL). Based on the prominent labeling of Gut2 by the photoactivatable PC analogue TID-PC, it was hypothesized that Gut2 activity depends on PC. Such a dependence might serve to align carbon/energy and lipid metabolic pathways. From in vivo and in vitro experiments with PC-biosynthetic mutants, it was concluded that PC is most likely involved in binding Gut2 to the membrane, rather than in stimulating its activity. A role of PC in modulating Gut2 activity to regulate glycerol-3-phosphate availability for lipid synthesis was deemed unlikely. Since it could not be excluded that residual PC remaining after PC depletion was sufficient for Gut2 activity, the use of PC-free systems is suggested to clarify the significance of the Gut2-PC interaction further. Subsequently, the focus was on the possible role of PC in the utilization of proline as nitrogen source, involving the mitochondrial proline oxidase Put1. The investigation was mainly based on the finding in an mRNA profiling study that the transcript levels of the PUT1 and GAP1 genes (encoding Put1 and the general amino acid permease Gap1, respectively) were increased in PC-depleted cells. In vivo and in vitro experiments demonstrated that PC indeed plays a role in proline utilization. Although a reduced proline uptake appeared to be the bottleneck for growth of opi3 cells in the absence of choline, PC depletion was also found to impact proline metabolism. Intriguingly, the presumed precursor of Put1 gradually accumulated in cells subjected to PC depletion unlike precursors of other mitochondrial proteins. In the second part of the thesis, acb1 mutants (lacking the acylCoA-binding protein Acb1) were exploited to gain insight into CL remodeling, triggered by the finding that CL from Acb1-depleted cells contains elevated levels of acyl chains shorter than C16. The experimental data demonstrated that the CL remodeling system in S. cerevisiae does not strive for the removal of those shorter chains, but rather, that it specifically focuses on the replacement of C16:0 chains in CL molecules. The CL remodeling mechanism proposed (selective outflow of acyl chains via the CL-specific lipase Cld1 followed by non-selective inflow via the transacylase Taz1) is able to account for the CL profile observed in S. cerevisiae including those in taz1 and acb1 cells, and for the accumulation of MLCL in the former and not in the latter mutant strain. In spite of years of research, not much is known about the biological significance of CL remodeling. It is speculated that remodeling aims to increase the non-bilayer propensity of CL in order to enhance mitochondrial functioning and/or efficiency.