Identification of the redox partners of ERdj5/JPDI, a PDI family member, from an animal tissue.
ERdj5 (also known as JPDI) is a member of PDI family conserved in higher eukaryotes. This protein possesses an N-terminal J domain and C-terminal four thioredoxin domains each having a redox active site motif. Despite the insights obtained at the cellular level on ERdj5, the role of this protein in vivo is still unclear. Here, we present a simple method to purify and identify the disulfide-linked complexes of this protein efficiently from a mouse tissue. By combining acid quenching and thiol-alkylation, we identified a number of potential redox partners of ERdj5 from the mouse epididymis. Further, we show that ERdj5 indeed interacted with two of the identified proteins via formation of intermolecular disulfide bond. Thus, this approach enabled us to detect and identify redox partners of a PDI family member from an animal tissue.
Graduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, Japan; Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Aoba-ku, Sendai, Miyagi 980-8577, Japan. Electronic address: kadokura@tagen.tohoku.ac.jp.
Hiroshi
H
Kadokura
Michiko
M
Saito
Akio
A
Tsuru
Akira
A
Hosoda
Takao
T
Iwawaki
Kenji
K
Inaba
Kenji
K
Kohno
D000818:Animals; D004220:Disulfides / Q000378:metabolism; D004822:Epididymis; D005033:Ethylmaleimide / Q000378:metabolism; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism*; D008297:Male; D051379:Mice; D018345:Mice, Knockout; D018832:Molecular Chaperones / Q000378:metabolism*; D010084:Oxidation-Reduction; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*
D004220:Disulfides; C530517:ERdj5 protein, mouse; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones; D019704:Protein Disulfide-Isomerases; D005033:Ethylmaleimide
ER; ER-associated degradation; ERAD; ERdj5; ERp72; JPDI; N-ethylmaleimide; NEM; PAGE; PDI; SDS; Thioredoxin; endoplasmic reticulum; polyacrylamide gel electrophoresis; protein disulfide isomerase; sodium dodecyl sulfate