title: Retarded PDI diffusion and a reductive shift in poise of the calcium depleted endoplasmic reticulum.
abstract: BACKGROUND
  Endoplasmic reticulum (ER) lumenal protein thiol redox balance resists dramatic variation in unfolded protein load imposed by diverse physiological challenges including compromise in the key upstream oxidases. Lumenal calcium depletion, incurred during normal cell signaling, stands out as a notable exception to this resilience, promoting a rapid and reversible shift towards a more reducing poise. Calcium depletion induced ER redox alterations are relevant to physiological conditions associated with calcium signaling, such as the response of pancreatic cells to secretagogues and neuronal activity. The core components of the ER redox machinery are well characterized; however, the molecular basis for the calcium-depletion induced shift in redox balance is presently obscure.
  
  
  RESULTS
  In vitro, the core machinery for generating disulfides, consisting of ERO1 and the oxidizing protein disulfide isomerase, PDI1A, was indifferent to variation in calcium concentration within the physiological range. However, ER calcium depletion in vivo led to a selective 2.5-fold decline in PDI1A mobility, whereas the mobility of the reducing PDI family member, ERdj5 was unaffected. In vivo, fluorescence resonance energy transfer measurements revealed that declining PDI1A mobility correlated with formation of a complex with the abundant ER chaperone calreticulin, whose mobility was also inhibited by calcium depletion and the calcium depletion-mediated reductive shift was attenuated in cells lacking calreticulin. Measurements with purified proteins confirmed that the PDI1A-calreticulin complex dissociated as Ca(2+) concentrations approached those normally found in the ER lumen ([Ca(2+)]K(0.5max) = 190 μM).
  
  
  CONCLUSIONS
  Our findings suggest that selective sequestration of PDI1A in a calcium depletion-mediated complex with the abundant chaperone calreticulin attenuates the effective concentration of this major lumenal thiol oxidant, providing a plausible and simple mechanism for the observed shift in ER lumenal redox poise upon physiological calcium depletion.
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. ea347@cam.ac.uk.
authors.forename: Edward
authors.initials: E
authors.lastname: Avezov
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. tk431@medschl.cam.ac.uk.
authors.forename: Tasuku
authors.initials: T
authors.lastname: Konno
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. az310@cam.ac.uk.
authors.forename: Alisa
authors.initials: A
authors.lastname: Zyryanova
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK. wc269@cam.ac.uk.
authors.forename: Weiyue
authors.initials: W
authors.lastname: Chen
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK. rfl30@cam.ac.uk.
authors.forename: Romain
authors.initials: R
authors.lastname: Laine
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. ac880@cam.ac.uk.
authors.forename: Ana
authors.initials: A
authors.lastname: Crespillo-Casado
authors.affiliation: Center for Biomedical Research, Universidade do Algarve, Faro, Portugal. emelo@ualg.pt.
authors.forename: Eduardo Pinho
authors.initials: EP
authors.lastname: Melo
authors.affiliation: Faculty of Life Sciences, Kyoto Sangyo University, Kita-Ku, Kyoto-City, 603-8555, Japan. ryo3ussy3@cc.kyoto-su.ac.jp.
authors.forename: Ryo
authors.initials: R
authors.lastname: Ushioda
authors.affiliation: Faculty of Life Sciences, Kyoto Sangyo University, Kita-Ku, Kyoto-City, 603-8555, Japan. nagata@cc.kyoto-su.ac.jp.
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Nagata
authors.affiliation: Department of Chemical Engineering and Biotechnology, University of Cambridge, Cambridge, CB2 3RA, UK. cfk23@cam.ac.uk.
authors.forename: Clemens F
authors.initials: CF
authors.lastname: Kaminski
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. hph23@cam.ac.uk.
authors.forename: Heather P
authors.initials: HP
authors.lastname: Harding
authors.affiliation: University of Cambridge, Cambridge Institute for Medical Research, Biomedical Campus, Wellcome Trust/MRC Building, Hills Road, Cambridge, CB2 0XY, United Kingdom. dr360@medschl.cam.ac.uk.
authors.forename: David
authors.initials: D
authors.lastname: Ron
mesh_terms: D000818:Animals; D019556:COS Cells; D002118:Calcium / Q000172:deficiency* / Q000378:metabolism; D037282:Calreticulin / Q000378:metabolism; D002522:Chlorocebus aethiops; D004058:Diffusion; D004220:Disulfides / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D057809:HEK293 Cells; D050956:HSP40 Heat-Shock Proteins / Q000378:metabolism; D006801:Humans; D051379:Mice; D018832:Molecular Chaperones / Q000378:metabolism; D010084:Oxidation-Reduction; D011485:Protein Binding; D019704:Protein Disulfide-Isomerases / Q000378:metabolism*
chemical_list: D037282:Calreticulin; C471179:DNAJC10 protein, human; D004220:Disulfides; D050956:HSP40 Heat-Shock Proteins; D018832:Molecular Chaperones; D019704:Protein Disulfide-Isomerases; D002118:Calcium