A Chemical Genetic Approach To The Study Of Cellular Transport

Abstract

The focus of this thesis is the use of chemical genetics to study two different aspects of membrane biology, (a) the mechanisms underlying cellular lipid transport and (b) the intersection between endocytic and exocytic traffic. The broad goals of chemical genetics are to find novel chemical tools to study biological systems and to identify novel molecular players therein. A key advantage of small molecules is their ability to rapidly and reversibly interfere with biology which qualifies them as ideal reagents to study the dynamics of complex processes (1, 2). Cellular cholesterol homeostasis is maintained at the level of synthesis and catabolism of cholesterol, and through regulation of cellular lipid import and -export. Lipids can be acquired from the extracellular environment by receptor-mediated endocytosis of specialized carriers, such as low-density lipoproteins (LDL), or by dedicated surface membrane transport proteins (3). LDL-receptor mediated endocytosis moves LDL particles into endosomal/lysosomal compartments where cholesterol and other lipids are transferred into the cell proper, concomitantly with the degradation of the lipoprotein (4). In contrast, surface membrane lipid transporters directly transfer lipids between extracellular ligands and the cell surface bilayer independent of cellular endocytic activity. The transport mechanisms of two lipid transport proteins, Scavenger Receptor, Class B, type I (SR-BI), and ABCA1 (ATP Binding Cassette transporter A1), are investigated in the first part of this thesis. Both transporters play critical roles in lipoprotein metabolism, atherosclerosis and cardiovascular disease (5, 6). The second part of this thesis describes a study of the mechanisms of intracellular trafficking. For a multicellular organism to function properly, it is imperative that cells coordinate their activities. Careful modulation of exocytic- and endocytic traffic allows cells to communicate with each other via controlled surface expression of membrane proteins, and via the secretion and internalization of soluble molecules. The information that these soluble molecules carry can be interpreted in autocrine, paracrine or endocrine fashion. Because many molecules need to be directed (and restricted) to their appropriate intracellular location following their cellular synthesis or acquisition from the outside world, controlling cellular trafficking also ensures the preservation of the identity of subcellular compartments. Finally, to fulfill their metabolic needs, cells use intracellular trafficking pathways to take up nutrients, and other molecules they cannot synthesize, from the outside world (3). Experiments described in the second part of this thesis aspire to characterize molecular elements that regulate both endocytosis and exocytosis. This line of research experiments is motivated by a desire to deepen our understanding of the basic machinery of cellular trafficPloegh, H.L. [Promotor]Kirchhausen, T. [Copromotor