Identification and characterisation of the Pex3-Inp1 complex as the first peroxisome-plasma membrane tether

Eukaryotic cells have evolved molecular mechanisms that control organelle size, number and position. Molecular tethers are required for organelle positioning, multiplication and establishment of interorganellar contact sites. The balance between organelle tethering and motility determines the intracellular distribution of organelles and their segregation during cell division. In Saccharomyces cerevisiae, correct peroxisome distribution is achieved by the opposing processes of cortical anchoring in the mother cell and Myosin-dependent transport towards the bud. The Inp1-Pex3 tethering complex is required for peroxisome retention during cell division and for peroxisome positioning along the mother cortex. As has been postulated for other organelles, yeast peroxisomes interact with many cellular structures including the plasma membrane, ER, vacuole, mitochondria and lipid bodies. Components of some interorganellar peroxisomal contact sites have recently been identified whereas others are still completely uncharacterised including the plasma membrane- peroxisome (PM-PER) contact site. The work presented in this thesis identifies Inp1 as the first known plasma membrane-peroxisome (PM-PER) tether by demonstrating that Inp1 meets the predefined criteria which a contact site tether protein must adhere to. This thesis first describes a conserved Pex3 binding motif in the C-terminal region of Inp1. This motif bears a striking resemblance to the Pex3 binding site present on Pex19 and both in vitro and in vivo evidence is presented which illustrates that Pex19 and Inp1 can compete for binding to Pex3. In addition, the N-terminal 100 amino acids of Inp1 are shown to localise to the plasma membrane, bind to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) and, when artificially attached to the peroxisomal membrane, restore retention by relocating peroxisomes to the cell periphery in inp1Δ cells. In this study, Inp1 is shown to be present in the correct sub-cellular location to interact with both the plasma membrane and peroxisomal membrane and the data illustrates the structural and functional capacity of Inp1 to be a PM-PER tether. Through detailed analysis of the molecular function of Inp1, the work in this thesis identifies a novel role for Inp1 as a PM- PER tether and concludes that tethering of peroxisomes to the plasma membrane is required for peroxisome retention. This is the first molecular characterisation of the PM-PER tether and has allowed for the proposal of a new model for peroxisome retention.