9752962 Robinson This is a POWRE Research Enhancement Award. During cellular remodeling, a process known as autophagy plays a role in transporting superfluous cytoplasmic proteins and organelles to the lysosome for degradation. Such cellular remodeling occurs during differentiation, nutrient deprivation, metamorphosis, aging and transformation; autophagy also removes abnormal organelles that may accumulate for various reasons. Thus, autophagy is a vitally important process. During autophagy, a double membrane structure surrounds or engulfs a region of cytosol containing the material to be degraded, creating a double membrane bound organelle, the autophagosome. Autophagosomes deliver their contents for degradation by fusing the outer membrane with the lysosomal or vacuolar membrane. The objective of this project is to unravel the molecular and cellular mechanisms involved in organelle autophagy. The experiments will be performed in the yeast, Saccharomyces cerevisiae. This organism is commonly used as a model for cell biological studies because of the advantages it offers for genetic, cell biological, and molecular analyses. The experimental approach will be to label the peroxisomes of the yeast cells with green fluorescent protein (GFP) and use digital time-lapse fluorescent microscopy as well as quantitative fluorimetry to observe and measure peroxisome autophagy. Some of the planned experiments will test whether autophagy can be a selective process where specific superfluous structures are selectively targeted to the autophagic pathway. To do this, rates of peroxisome and mitochondrial degradation will be simultaneously measured under conditions where one organelle, but not the other, is required for cell viability. A second hypothesis that will be tested is that general membrane trafficking components as well as key components of the cell's cytoskeleton play a role in the formation, transport or function of the autophagosomes in yeast. These experiments will combine assa ys for organelle autophagy with methods available in yeast to specifically inhibit specific protein functions. The experiments will specifically assess potential roles in autophagy of two proteins involved in membrane fusion, NSF and Kar2p, and two components of the yeast cytoskeletal system, microtubules and actin. Finally, genetic screens will be carried out to identify new molecules involved in autophagy. Fluorescence-activated cell sorting will be used to enrich for mutant cells that retain GFP-labeled peroxisomes due to delayed or abolished autophagy. The characterization of mutants collected by this screen is part of the longer term goal of understanding the molecular mechanisms of autophagy but is beyond the scope of the present award. This project represents a return to active cell biological research for the Principal Investigator, after a major hiatus in her career. The support from a POWRE award will allow the Principal Investigator to develop the necessary preliminary results for a novel and promising avenue of research. ***
