Mechanisms of LAM-mediated intracellular sterol traffic and its regulation by conserved kinases

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Understanding how cells control cholesterol trafficThe purpose of this project is to improve understanding of traffic of cholesterol inside cells, a poorly understood process of vital importance to the health of all cells, from humans to fungi and plants. Cells are separated from the outside world by the plasma membrane, which consists of a bilayer (two apposed layers) of fat molecules (lipids) that prevent the escape of molecules inside the cell. The key lipid for the plasma membrane is cholesterol, which strengthens the membrane and prevents it from dividing up into different regions, which would be toxic and may contribute to a wide variety of societally important diseases from atherosclerosis to Alzheimer's. When the physical properties of the plasma membrane have to change, for example when body temperature increases, levels of cholesterol in the plasma membrane must change so that its physical properties match the new conditions. Cholesterol flow into and out of the plasma membrane is carried out by specialised sterol transfer proteins, which fold into the overall shape of a box with a hinged lid. They accommodate a single cholesterol molecule inside, and they can repeatedly shuttle sterol from one site inside cells to another. We recently discovered a large family of sterol transfer proteins called LAMs in all cells in humans, fungi and plants. LAMs transfer excess cholesterol away from the plasma membrane to an internal organelle called the endoplasmic reticulum, where is converted into an oil form that is stored in fat bodies for later use. Cells have several pathways that respond to new conditions, each pathway containing multiple proteins. Most of these pathways are regulated by the activity of proteins called kinases, which physically modify downstream pathway components to switch them on. Among the major kinases found in all cells in all complex life forms is TORC2. Like a thermostat counteracting temperature changes, TORC2 responds to changes in the plasma membrane by modifying components in several pathways that all counteract the original changes. TORC2 is known to affect many different lipids of the plasma membrane, but so far it has never been linked directly to cholesterol.This three year project is based on our new evidence that cells use TORC2 to control the activity of LAMs. We will find out if the important sterol trafficking function of LAMs is controlled by TORC2 as our data suggests. We will also test the paradigm that master regulators like TORC2 have multiple outputs that combine to create synergy. For this we will study how cells combine changes in cholesterol with other changes in lipids of the plasma membrane that TORC2 enacts. We have also found out that cells sometimes rapidly destroy LAMs. Targeted protein destruction is a very common way in which pathways are controlled; indeed a major way TORC2 works is by altering the activity of the machinery that destroys specific proteins. Therefore, we will find out how LAMs are destroyed and whether destruction of LAM proteins is a second layer of control imposed by TORC2 on cholesterol traffic.Our work will be carried out in budding yeast where the LAM and TORC2 pathways are better understood than in human cells. However, all the participating proteins and lipids involved in yeast have counterparts in humans, and we will examine if the effects we discover in yeast apply in human cells. The practical applications following on from our basic research will be to help devise ways to manipulate many processes where cholesterol movement is important. Because LAM proteins and TORC2 are found in all complex life forms, our research will produce knowledge that is applicable to plants, fungi, and animals. Our results will add to the understanding not only of healthy ageing (atherosclerosis, Alzheimer's, type II diabetes), but also for food security since cholesterol traffic is vital both to crop plants and to fungi that destroy crops.