Studying Chaperones in Oxidative Environments Using Fluorescence Microscopy Tools and Approaches in Live Cells
Aronson, Deborah E.
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The secretory pathway, beginning with the endoplasmic reticulum (ER) followed by the Golgi complex (GC), function consecutively to facilitate the sorting, processing and quality control steps necessary to conclude with a properly folded secreted protein. Many neurodegenerative, metabolic and genetic diseases are caused by pathology instigated by a misfolded or misprocessed protein. Determining how the folding and ER quality control (ERQC) machinery produces correctly folded secretory proteins and regulates the degradation of misfolded protein is essential to help elucidate their role in the pathology of an ever-expanding list of diseases. To address these questions, we believe it is crucial to characterize the molecular availability of ER and Golgi complex chaperones under normal and stressed conditions. ER resident proteins, and a vast and varied substrate pool of secretory proteins, enter the ER where ER chaperones facilitate folding and posttranslationally modify substrates. One such modification, disulfide bond formation, is essential for many cysteine-containing secretory proteins to achieve their native conformation. The oxidizing environment of the ER and the ER chaperone, protein disulfide isomerase (PDI), facilitate this key modification. To assess the molecular availability of PDI and ERp44 and investigate their roles in chaperone folding and ER stress, we created fluorescently tagged fusion proteins for quantitative live cell imaging analyses including fluorescent recovery after photobleaching (FRAP). During our studies, we discovered that because of its rapid and efficient folding capabilities, Superfolder GFP (sfGFP) is the optimal fluorescent protein (FP) for creating chaperone-FP in oxidizing environments including the ER. Using these tools, we determined ERp44 is targeted to the GC via a structural targeting motif, which is necessary and sufficient for GC localization, despite its KDEL sequence. Using microscopy photobleaching techniques, we also found that the dynamics and molecular availability of PDI-mGFP is affected by substrate binding in live cells. Together, we have created and optimized a variety of live cell and biochemical chaperone assays, determined the best employment of various FPs for the creation of chaperone fusion proteins and characterized aspects of the molecular availability of PDI and ERp44 at steady state.