Unraveling the principles of peroxisome homeostasis in yeast: the need for confocal methods.

Hallmark of eukaryotic cells is the presence of several specific metabolic compartments, the organelles. The advantage of this compartmentalization is apparently that segregation of sets of enzymes from the cytosol or the creation of unique micro-environments with specific (bio)chemical properties can increase the efficiency of certain metabolic pathways. As a consequence, the cell had to evolve highly complex regulatory mechanisms for the generation and maintenance of these compartments. Also, it is of vital importance that the function of the various organelles is continuously adapting to ensure that the cell directs its resources optimally towards specialized functions (as in differentiated cells) and survival, a process called homeostasis. One of the important modes to achieve this is to precisely control the abundance and function of the organelles. The unraveling of the mechanisms involved in these processes is one of the main challenges of modem cell biology. In the research group Eukaryotic Microbiology the principles of peroxisome function and homeostasis are studied for over two decades now. Peroxisomes are ubiquitous organelles that were discovered rather late (1954). The organelles contain as basic constituents hydrogen peroxide-producing oxidases as well as enzymes involved in the breakdown of hydrogen peroxide and free radicals. The functional diversity of the organelles is unprecidented. So far, more than 50 biochemical pathways have been characterized in peroxisomes. These range from catabolic processes like b-oxidation of very long chain fatty acids in mammals, photorespiration in plants to the oxidative primary metabolism of various carbon and nitrogen sources in fungi5962 to anabolic processes like amino acid synthesis in yeast16 and cholesterol and plasmalogen biosynthesis in man59. This indicates that the peroxisome is not only the site of reactive oxygen metabolism, but also crucially involved in membrane lipid composition and function. The importance of peroxisomes is probably best illustrated by the existence of peroxisome diseases like Zellweger syndrome, which are associated with major neurological impairments that may result in an early death. The recent achievements on peroxisome biogenesis are mainly based on genetic and ultrastructural approaches and have advanced to a level that several of the proteins, essential for peroxisome assembly and maintenance, are now known. However, the knowledge on the principles of these processes is still largely descriptive. Additional non-invasive techniques are required to deepen our insights e.g. to understand the in vivo sequence and nature of protein-protein interactions of the matrix protein import cascade and the initial events in organelle biogenesis, multiplication and turnover. We aim to use novel revolutionary confocal laser scanning microscopy (CLSM) methods that have become available now. The scientific background, relevance and innovation of this work are detailed below.