Assistant Professor, Department of Biology
Title: The Role of Trafficking from the trans-Golgi Network and Endosomes in Cell Survival
Research Objectives
Membrane traffic, the movement of proteins between subcellular compartments, is vital to cell survival and is often dysregulated in human diseases such as cancer, cardiovascular disease, and diabetes. Many of these diseases are also characterized by fundamental changes in metabolism; however, it unclear how these changes regulate and alter membrane traffic. In this proposal, we will use the small budding yeast Saccharomyces cerevisiae to examine how membrane traffic from the trans-Golgi network and endosomes (TGN-E) helps to ensure cell survival during metabolic stress induced by nitrogen and aminoacid starvation. Using a combination of genetic and biochemical methods, we will (1) test if ablating traffic from the TGN-E affects cell survival, (2) determine if TGN-E trafficking is required to deliver dispensable proteins, and (3) examine how amino acid sensing signaling pathways alter TGN-E traffic during nitrogen and amino acid starvation. Because the metabolism of yeast can mimic both normal and cancerous cells, our studies will provide important insight into mechanisms that ensure normal cell survival and that cancer cells may manipulate to outcompete other cells.
Background and Significance
A distinguishing feature of eukaryotic organisms is the compartmentalization of the cell into smaller membrane-bound organelles. These organelles sequester specific and fundamental cellular functions such as protein synthesis and degradation, detoxification, and lipid production. The ability to move and transport proteins and other cargo between organelles, collectively called membrane traffic, is essential for organelle function. Trafficking at the TGN-E is important for delivering and regulating the localization of proteins that function at the cell surface. These include nutrient transporters, such as the insulin regulated glucose transporter GLUT4 implicated in Type II diabetes and signaling receptors implicated in cancers, such as the epidermal growth factor receptor. TGN-E trafficking also regulates the delivery of proteins to the lysosome, a primarily a degradative compartment, that prevents tumorigenic signaling and is vital for survival following acute cellular starvation (Leto and Saltiel, 2012; Shachar et al., 2011; Woodman, 2009).
Cell survival is dependent on homeostasis, the ability to maintain a constant internal environment despite changes in the surrounding environment. This includes maintaining a steady supply of nitrogen and amino acids, which are required for DNA, protein, and vitamin synthesis. Cells generally maintain low intracellular reserves of nitrogen and amino acids; therefore, the cell imports a large amount of nitrogen and amino acids from the extracellular environment. Acute changes to the environment, such as amino acid starvation, lead to immediate cellular stress. An immediate survival response to replenish the cell’s supply of free amino acids is mounted by altering the traffic of dispensable proteins to lysosome/vacuole for degradation. The internalization and degradation of plasma membrane proteins has been shown to be a source of dispensable proteins (Jones et al., 2012). However, the TGN-E is a major hub of newly synthesized proteins and diverting traffic from TGN-E to the vacuole may significantly contribute to this supply. The focus of this proposal will be to examine the role of the TGN-E in delivering dispensable proteins following nitrogen and amino acid starvation.
The various steps in trafficking cargo, from sorting and packaging cargo into transport vesicles and then directing vesicles to their target destination involves a number of proteins and protein complexes. Dysfunctions in or the inability to regulate trafficking proteins at the TGN-E is associated with many types of diseases and disorders including cancer, diabetes, and neurodegeneration (Bryant et al., 2002; Liang et al., 2008; Tian and Abel, 2001; Woodman, 2009). In yeast, a number of proteins, called adaptors, are responsible for sorting and packaging cargo into transport vesicles at the TGN-E. These include the clathrin adaptors (Gga2, Ent5, Ent3, and AP-1), the AP-3 complex, and Ent4 (Aoh et al., 2011; Costaguta et al., 2006; Deng et al., 2009). Ablation of these adaptors often leads to a growth defect, suggesting they are important for cell survival. We will directly test if ablation of these adaptors affects survival during acute nitrogen and amino acid starvation.
I will utilize the yeast Saccharomyces cerevisiae to examine TGN-E trafficking. Many trafficking pathways are similar in yeast and metazoans, such that human proteins can frequently complement for the loss of yeast proteins and vice versa. Indeed, studies in yeast laid the foundation for our understanding of trafficking. Yeast offer many technical advantages that will be essential for dissecting the role of TGN-E trafficking in cell survival. In addition to standard biochemical methodologies, the ease of genetic manipulation makes mutant construction simple. The ability to examine genetic interactions using high- throughput genetic and biochemical screening assays using commercially available mutant libraries will be a powerful tool to understand how signaling can affect TGN-E traffic.