Distinct roles and actions of protein disulfide isomerase family enzymes in catalysis of nascent-chain disulfide bond formation
Chihiro Hirayama,
Kodai Machida,
Kentaro Noi,
Tadayoshi Murakawa,
Masaki Okumura,
Teru Ogura,
Hiroaki Imataka,
Kenji Inaba
Affiliations
Chihiro Hirayama
Institute of Multidisciplinary Research for Advanced Materials, Katahira 2-1-1, Aoba-ku, Tohoku University, Sendai, Miyagi 980-8577, Japan
Kodai Machida
Graduate School of Engineering, University of Hyogo, Himeji, Hyogo 671-2280, Japan
Kentaro Noi
Institute for NanoScience Design, Osaka University, Toyonaka, Osaka 560-8531, Japan
Tadayoshi Murakawa
Graduate School of Life Science and Technology, Tokyo Institute of Technology, Yokohama, Kanagawa, 226-8503, Japan
Masaki Okumura
Institute of Multidisciplinary Research for Advanced Materials, Katahira 2-1-1, Aoba-ku, Tohoku University, Sendai, Miyagi 980-8577, Japan; Frontier Research Institute for Interdisciplinary Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan
Teru Ogura
Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Kumamoto 860-0811, Japan; Faculty of Life Sciences, Kumamoto University, Kumamoto, Kumamoto 862-0973, Japan
Hiroaki Imataka
Graduate School of Engineering, University of Hyogo, Himeji, Hyogo 671-2280, Japan
Kenji Inaba
Institute of Multidisciplinary Research for Advanced Materials, Katahira 2-1-1, Aoba-ku, Tohoku University, Sendai, Miyagi 980-8577, Japan; Corresponding author
Summary: The mammalian endoplasmic reticulum (ER) harbors more than 20 members of the protein disulfide isomerase (PDI) family that act to maintain proteostasis. Herein, we developed an in vitro system for directly monitoring PDI- or ERp46-catalyzed disulfide bond formation in ribosome-associated nascent chains of human serum albumin. The results indicated that ERp46 more efficiently introduced disulfide bonds into nascent chains with a short segment exposed outside the ribosome exit site than PDI. Single-molecule analysis by high-speed atomic force microscopy further revealed that PDI binds nascent chains persistently, forming a stable face-to-face homodimer, whereas ERp46 binds for a shorter time in monomeric form, indicating their different mechanisms for substrate recognition and disulfide bond introduction. Thus, ERp46 serves as a more potent disulfide introducer especially during the early stages of translation, whereas PDI can catalyze disulfide formation when longer nascent chains emerge out from ribosome.
