Project Summary Understanding how cells adapt to stress and repair damage is one of the highest priorities in cell biology and health research. The endoplasmic reticulum (ER) is a key source of lipid and proteins for building and maintaining several organelles in cells, and in response to stimuli it is capable of directing resources into an assortment of transport pathways. The signaling protein Ypt1/Rab1 is a Rab GTPase (Rab) that plays essential roles in how the ER directs resources and executes quality control of damaged organelles including mitochondria and peroxisomes. Our long-term goal is to understand the mechanisms for how lipid and protein cargos are routed and re-routed to specific itineraries in response to cellular stimuli and stresses, which will inform development of targeted therapeutic interventions to address diseases. We have found that the GTPase accelerating protein Gyp8, an evolutionarily conserved but poorly understood negative regulator of Ypt1/Rab1 signaling, localizes to the ER, peroxisomes and mitochondria and impinges on Ypt1 signaling. Our central hypotheses are that Gyp8 functions to modulate Ypt1/Rab1 signaling in the early secretory pathway (ER) and at non-secretory membranes (peroxisomes and mitochondria) that are subject to frequent damage and quality control via Ypt1/Rab1-dependent selective autophagy. Also, since Gyp8 localizes to organelles that commonly tether/dock to exchange materials in essential metabolic pathways, we predict that Gyp8 regulates Rab-dependent tethering interactions, particularly among organelles specialized for lipid storage and metabolism. To test our central hypotheses and advance understanding of several membrane biogenesis pathways that originate at the ER, we will pursue these specific aims: 1) Identify intra- and extra-genic factors that control the subcellular itinerary and activity of Gyp8; 2) Determine target Rab GTPase(s) regulated by Gyp8 in the secretory pathway; and 3) Define the role of Gyp8 in regulating peroxisomal and mitochondrial dynamics. The proposed research is innovative both for area of focus and technical approach. While Ypt1/Rab1 signaling controls multiple transport pathways essential to cellular health, understanding of where and when signal must be terminated to accomplish each of its roles is particularly incomplete. The experimental plan combines gold standard biochemical and imaging techniques in organelle and vesicular transport (enzyme-coupled kinetic transport assays and 3D electron tomography) with systems biology approaches (synthetic gene array and affinity capture mass spectrometry proteomics). The proposed research is significant because defining how Ypt1/Rab1 is regulated to support and exercise quality control of ER, mitochondria and peroxisomes is foundational to understanding aspects of cardiovascular, neurodegenerative and metabolic disorders.