Project Summary/Abstract Cells have developed complex stress responses to identify and eliminate misfolded proteins. An exciting development in recent years has been the recognition that lipid homeostasis is critical for protein quality control. The Unfolded Protein Response (UPR) senses misfolded proteins in the endoplasmic reticulum and orchestrates a broad program of cellular remodeling to address this threat. Various defects in lipid metabolism trigger the UPR, and the UPR in turn controls the expression of some lipid metabolic genes. Furthermore, in contrast to the canonical luminal signaling mechanism for misfolded proteins, recent work indicates the presence of a second sensor pathway that detects defects in the ER membrane ("bilayer stress"). The functional properties of membranes (e.g. thickness, fluidity, curvature) are largely determined by their compositions. Far from early conceptions of membranes as static or inert structures, we now understand that membranes are highly dynamic and capable of altering their compositions in response to changing cellular conditions/needs. This proposal focuses on a poorly understood group of lipids known as very long chain fatty acids (VLCFAs) which are relatively unabundant but perform critical functions. We hypothesize that VLCFAs play key roles in protein quality control and membrane homeostasis. To inhibit VLCFA utilization, we have studied a mutant of the major VLCFA CoA synthetase, Fat1. Our preliminary data indicate that Fat1 plays an important role in ER homeostasis, and its loss triggers compensatory induction of the UPR. To understand the basis for this effect, we carried out a mass spectrometry-based lipidomic analysis. Remarkably, the fat1Δ mutant showed a dramatic increase in membrane saturation which is a known inducer of the UPR. This effect is mediated, at least in part, via partial loss of function of Ole1, the sole fatty acyl desaturase in yeast. In Aim 1, we will determine the mechanism by which VLCFAs regulate membrane homeostasis and the UPR. Recent data implicate membrane saturation as a key determinant of alpha-synuclein toxicity, which is responsible for Parkinson's disease. Our data indicate that Fat1 is an important regulator of synuclein toxicity. In Aim 2, we will determine mechanism by which VLCFAs regulate synuclein toxicity in yeast and Drosophila. Completion of this proposal is expected to provide both basic and disease-oriented mechanistic insight into this emerging but fundamental area of cell biology.