The role of defined lipid molecular species in yeast subcellular membranes

Eukaryotic cells are compartmentalized into organelles of distinct chemical composition and biological function. These subcellular organelles are not created de novo, but ate inherited from the mother to the daughter cell upon cell division. Organelles thus may contain inheritable structural information that is independent of the DNA- encoded information of the genome. The nature of this structural information is poorly understood, but the fact that each of the organellar membrane has a distinct and often characteristic lipid composition raises the possibility that membranes contain inheritable structural information, encoded in their membrane-specific protein/lipid composition. How this lipid composition is determined and maintained, however, is only poorly understood. Addressing this question is the subject of the proposed research project. Our approach to study this complex question is to follow the fate of selected and precisely defined lipid molecular species in different subcellular membranes. This is a new approach to study membrane structure and function, that is only made possible by more recent technical advances in rapidly and sensitively analyzing the lipid molecular species composition of biological membranes by nano-electrospray tandem mass spectroscopy (ESI-MS/MS). A lipid molecular species is a lipid of defined chemical structure, i.e. of known acyl chain- and head group substituents. Cellular membranes are typically composed of dozens of different lipid molecular species whose detailed characterization was not practical before the advent of ESI-MS/MS. In our previous work, we performed a comprehensive characterization of the lipid molecular species composition of nine different subcellular membranes from yeast by ESI-MS/MS. This analysis allowed us to identify certain lipid molecular species that appear to be characteristic for a particular subcellular membrane. The fate of these lipid species will now be analyzed in more detail. The synthesis of an unusual very-long-chain fatty acid (C26) substituted phosphatidylinositol will be characterized by biochemical and genetic methods. Furthermore, the synthesis, transport, and turn-over of one lipid molecular species that is typically found at the plasma membrane will be followed in a number of different yeast mutants. The results of these studies are expected to significantly improve our understanding of cellular membranes at a number of different levels, ranging from the compartmentalization of biosynthetic pathways to the turn-over and remodeling of lipids in defined subcellular membranes.

For a cell to grow and divide proteins have to be transported to and retained in their appropriate subcellular membrane. Aim of this project was to analyze the role of lipids in this process. Through two independent studies, we could show that the acyl chain composition of membrane lipids critically affects targeting and stabilization of integral membrane proteins. First, inhibiting acyl chain desaturation in the endoplasmic reticulum results in a rapid and reversible subcellular relocalization of the single essential delta-9 acyl chain desaturase of yeast. These results indicate that the desaturase, which is itself an integral membrane protein, has an intrinsic affinity for more ordered saturated membrane domains. The second study revealed an important role of very-long chain fatty acid containing sphingolipids in the subcellular transport and stabilization of a major plasma membrane enzyme, the proton pumping ATPase. Mutant cells containing C22 rather than the normal C26 fatty acids fail to stabilize the ATPase at the plasma membrane and transport the enzyme to the vacuole where it is degraded. This defect in retention of the ATPase at the plasma membrane correlates with a lack of raft association of the protein, as assessed by its solubility in mild detergents. These observations suggest that more fluid membrane domains may be preferentially internalized and endocytosed by the cell.