Grant: Discovery Grants Program - Individual

Cells contain numerous compartments that are separated from each other by membranes, allowing different compartments to perform distinct functions. For example, mitochondria generate energy for cell metabolism, the nucleus controls gene expression, the endoplasmic reticulum (ER) synthesizes membrane proteins, lysosomes function as the digestive system of the cell, and endosomes can take up extracellular material and direct it to lysosomes or recycle it back to the plasma membrane that surrounds the cell. All of these membrane compartments communicate with each other and work in conjunction to maintain normal cell function. Of the ~10,000 different proteins in any given cell, about one-third of these proteins are membrane proteins. Each of these membrane proteins must be sorted and transported to their correct sub-cellular membrane compartment. Such membrane protein trafficking is an essential, fundamental process in all cells, and must be tightly regulated and controlled. Most protein trafficking occurs in small membrane vesicles that can shuttle between one membrane compartment and another, delivering cargo between compartments. For membrane proteins destined for the plasma membrane, their journey starts in the ER from where they are transported to the Golgi complex, which serves as major sorting hub to direct proteins to lysosomes, endosomes or the plasma membrane. Deciding which protein goes where is based on the recognition of specific signals present in each cargo protein by different proteins that make up the transport vesicle. While sorting signals and carrier proteins that regulate protein shuttling between the ER and Golgi have been well characterized in recent years, we have a very poor understanding of what regulates membrane protein trafficking from the Golgi to the plasma membrane. The goal of my NSERC-funded research program is to more clearly define how cells regulate this essential process.Using a simple membrane protein (named p14) as a model system, we began four years ago to explore how this protein is transported to the plasma membrane. We discovered a short sequence of amino acids in p14 that was required for plasma membrane localization; when this sequence was mutated, p14 failed to exit from the Golgi. We further discovered that the function of this Golgi export signal was based on the presence of three basic (meaning positively charged) amino acids contained within a polybasic motif (PBM). This was the first example of a Golgi export signal based on a PBM. We also discovered that p14 interacted with three different cellular proteins previously identified as components of the recycling endosome pathway. Based on these results, we hypothesize the p14 PBM functions as Golgi-plasma membrane trafficking signal by sorting p14 from the Golgi into recycling endosomes that then deliver p14 to the plasma membrane.The goal of this NSERC proposal is to more determine how this underappreciated, yet essential, trafficking pathway functions by determining the role of the p14 PBM in this process. To achieve this goal, we propose to characterize p14 interactions with these endocytic recycling proteins, to identify additional proteins involved in this process, and to directly examine how this process occurs using video microscopy on live cells. The p14 protein provides a simple, tractable system to define pathways involved in Golgi-plasma membrane trafficking, a poorly understood stage of cellular trafficking pathways. Our proposed studies have the potential to more clearly discern the nature of these essential protein trafficking pathways and to provide mechanistic insights into this process.