The plasma membrane houses an array of cell surface membrane proteins that perform diverse and critical cellular functions. Cells internalise surface proteins, ligands, lipids, and extracellular material to endosomal compartments inside the cell. Internalised proteins are subsequently trafficked to different intracellular locations or are recycled back to the surface. We are interested in the routes surface proteins use to repopulate the plasma membrane and the machinery that controls these trafficking pathways.
To study surface recycling mechanisms we primarily use budding yeast as a model organism, which has many powerful genetic and biochemical tools to study conserved intracellular trafficking pathways. Two example assays used to measure yeast recycling are described in Figure 1.
Figure 1: The red fluorescent dye FM4-64 (left) binds to lipids on the plasma membrane and is internalised to endosomes (after ~ 5 minutes) and eventually progresses to the yeast lysosome / vacuole. However, most internalised dye is recycled from endosomes out of the cell. As FM4-64 fluorescence is quenched upon secretion, a decrease in fluorescence can be used as a measure of recycling kinetics. We have also engineered a recycling reporter based on a GFP labelled surface receptor (right). The receptor is fused to the catalytic domain of a deubiquitinating enzyme, thereby blocking its entry into the degradation pathway. The reporter is instead restricted to early endosomes, where it efficiently recycles to the surface in wild-type cells but remains trapped in endosomes in recycling defective mutants.
Using the GFP-tagged recycling reporter described above a comprehensive genetic screen was conducted, where the reporter was expressed in a collection of haploid deletion mutants. Of the ~5000 non-essential genes in yeast a cohort of mutants that were defective in recycling were identified and verified. These proteins define the contours of a mechanistic map of the recycling pathway, encompassing a variety of molecular functions, including metabolic control, lipid synthesis and organization, membrane fission, ion flux and transcription (Figure 2).
Figure 2: The genetic screen identified 89 mutants that are defective for recycling. The proteins encoded by these mutants were clustered based on their known protein-protein interactions and functional associations using STRING pathway analysis software.
The lab is currently funded through a Sir Henry Dale Fellowship supported by Wellcome and The Royal Society, in addition to support from the biology department at The University of York. The lab is extremely grateful for this generous funding.