UC San Diego

Protein transport through the secretory pathway is an essential process for all living organisms. While studies over the last three decades have enormously increased our understanding of this event, many of the components involved in the process of protein secretion have yet to be identified, and we are still lacking mechanistic insight into many steps of the secretory pathway. The main objective of this work is to identify new components involved in the secretory pathway. This will be achieved through the establishment of a functional genomics screen in drosophila cells and successive characterization of candidate genes as well as through a pharmacological approach in mammalian cells. Chapter I will describe the functional genomics approach that led to the identification of new components of the secretory pathway. Genome wide RNA interference (RNAi) knockdown was performed on Drosophila cell line stably expressing a secreted form of horseradish peroxidase (HRP). In this cell line, the systematic depletion of the product (mRNA) of genes involved in secretion results in a defect in the release of HRP into the medium. The amount of HRP measured by chemiluminescence provides an indication of the level of secretion upon the loss of a certain gene product. This functional genomics screen approach has led to the identification of approximately 100 genes. The initial characterization of one of these genes is reported. The following chapters will describe the pharmacological approach, which relies on the use of a natural product (norrisolide), a compound able to cause Golgi complex fragmentation and to block secretion in mammalian cells (Brady et al., 2004; Guizzunti et al., 2006; Guizzunti et al., 2007). In chapter II it is introduced norrisolide, a natural product able to induce irreversible fragmentation of the Golgi complex. The Golgi apparatus, a central organelle of the secretory pathway is a dynamic structure whose organization is maintained by a balance of membrane input and output. The fragmentation of the Golgi complex by norrisolide provides a mean to identify the mechanisms by which Golgi organization is maintained and regulated. Norrisolide activity is analyzed through the use of norrisolide-derived probes. We will show how norrisolide's core is necessary and sufficient to induce Golgi fragmentation. A fluorescent analogue of norrisolide will be used to visualize the intracellular localization of norrisolide's target. Chapter III shows the design of trifunctional probes based on norrisolide's structure that can be used to identify norrisolide's target. These probes are designed to contain norrisolide's core, a crosslinking agent and a tag. Different crosslinkers and tags are discussed