Protein folding coupled with disulfide bond formation, that is oxidative protein folding, proceeds mainly in the endoplasmic reticulum (ER). Greater than 20 Protein Disulfide Isomerase family members (PDIs) are conserved in the mammalian ER to catalyze this reaction. However, it remains an important open question how PDIs recognize various substrates and guide their proper folding through disulfide bond formation and isomerization. The goal of this study is to understand how protein homeostasis is maintained in the mammalian ER. To this end, I employ multiple approaches including single-molecule observation by high-speed AFM, NMR/SAXS analyses in solution, X-ray crystal structure analysis, and several biochemical assays. Diabetes and neurodegenerative diseases are caused by impairment of the protein quality control systems in cells, and hence this study will provide molecular insights into the mechanism underlying these diseases.