Tiivistelmä – Referat – Abstract
OCRL1 is a phosphatase that cleaves phosphatidylinositol 4,5-bisphosphate (PI4,5P) to phosphatidylinositol-4-phosphate (PI4P). ORP4 is a lipid binding/transport protein implicated in G-protein coupled signaling, cellular calcium homeostasis, proliferation and viability. OCRL1 and ORP4 are found in the endoplasmic reticulum and membrane contact sites throughout the endosomal system and the Golgi complex. OCRL1 is also present in the plasma membrane and vesicular structures. ORP4 has high affinity for binding sterols through the OSBP related domain (ORD). ORP4 also interacts with vimentin intermediate filaments via the ORD and influences the localization and organization of vimentin. The membrane lipid phosphatidylinositol (PI) can be phosphorylated either singly or in combination at three different positions on its inositol ring (D-3, D-4, and D-5)—yielding 8 possible phosphoinositides; the interconversion between the species is regulated by kinases and phosphatases that either add or remove phosphate groups from the various positions on the ring. Phosphoinositide metabolism is heavily involved in signal transduction pathways and cytoskeletal organization. Interestingly, it has also been found to be spatially regulated with distinct phosphoinositides being enriched in particular membrane compartments. BiFC assays can provide an important tool for visualizing protein-protein interactions. Not only is BiFC able to determine protein-protein proximity but it is also able to localize the interaction with regard to compartment membranes and structures. This study examined the interaction of ORP4 with OCRL1 by using BiFC analysis. We were able to determine that the protein pair seems to be in close proximity in the endoplasmic reticulum near the Golgi. Interaction only took place when the OCRL1 was tagged with VB at the C-terminal
