Chaperone function of calnexin for the folding intermediate of gp80, the major secretory protein in MDCK cells. Regulation by redox state and ATP.
The endoplasmic reticulum (ER) not only links the translational machinery to the endomembrane system in eukaryotic cells but also provides a protective environment for the folding of exoplasmic proteins translocated across the ER membrane. Here we describe that the lumenal surface of the ER membranes transiently tethers the folding intermediate of secretory proteins via a 90-kDa ER membrane protein, calnexin. We demonstrate that p70, the precursor to gp80, the major secretory protein in Madin-Darby canine kidney (MDCK) cells, was bound transiently to calnexin in the immediate post-synthetic period (0-10 min) and showed a t1/2 for dissociation from calnexin of 2.5 min. The bound p70 was found to be incompletely folded as assessed by susceptibility to proteinase K digestion. Perturbation of the redox state by 5 mM dithiothreitol or 1 mM diamide markedly inhibited the dissociation of p70 from calnexin (t1/2 > 30 min). Cellular depletion of ATP led to premature dissociation of p70 from calnexin and the formation of p70 aggregates that did not bind calnexin. These findings demonstrate that nascent unfolded p70 is tethered to calnexin during normal protein maturation, including the formation and editing of disulfide bonds and that ATP is required for the productive interaction of gp80 and calnexin.
Wada
I
I
Laboratory of Cell and Molecular Biology, Charles A. Dana Research Institute, Beth Israel Hospital, Boston, Massachusetts.
Ou
W J
WJ
Liu
M C
MC
Scheele
G
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D037281:Calnexin; D002478:Cells, Cultured; D018833:Chaperonins; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism; D008562:Membrane Glycoproteins / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D010084:Oxidation-Reduction; D017510:Protein Folding; D011498:Protein Precursors / Q000378:metabolism; D011506:Proteins / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011498:Protein Precursors; D011506:Proteins; D037281:Calnexin; D000255:Adenosine Triphosphate; D018833:Chaperonins