Identification of tropoelastin as a ligand for the 65-kD FK506-binding protein, FKBP65, in the secretory pathway.
The folding and trafficking of tropoelastin is thought to be mediated by intracellular chaperones, although the identity and role of any tropoelastin chaperone remain to be determined. To identify proteins that are associated with tropoelastin intracellularly, bifunctional chemical cross-linkers were used to covalently stabilize interactions between tropoelastin and associated proteins in the secretory pathway in intact fetal bovine auricular chondrocytes. Immunoprecipitation of tropoelastin from cell lysates after cross-linking and analysis by SDS-PAGE showed the presence of two proteins of approximately 74 kD (p74) and 78 kD (p78) that coimmunoprecipitated with tropoelastin. Microsequencing of peptide fragments from a cyanogen bromide digest of p78 identified this protein as BiP and sequence analysis identified p74 as the peptidyl-prolyl cis-trans isomerase, FKPB65. The appearance of BiP and FKBP65 in the immunoprecipitations could be enhanced by the addition of brefeldin A (BFA) and N-acetyl-leu-leu-norleucinal (ALLN) to the culture medium for the final 4 h of labeling. Tropoelastin accumulates in the fused ER/Golgi compartment in the presence of BFA if its degradation is inhibited by ALLN (Davis, E.C., and R.P. Mecham. 1996. J. Biol. Chem. 271:3787-3794). The use of BFA and other secretion-disrupting agents suggests that the association of tropoelastin with FKBP65 occurs in the ER. Results from this study provide the first identification of a ligand for an FKBP in the secretory pathway and suggest that the prolyl cis-trans isomerase activity of FKBP65 may be important for the proper folding of the proline-rich tropoelastin molecule before secretion.
Davis
E C
EC
Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri 63110, USA. davis16@utsw.swmed.edu
Broekelmann
T J
TJ
Ozawa
Y
Y
Mecham
R P
RP
D000591:Amino Acid Isomerases / Q000378:metabolism*; D000818:Animals; D020126:Brefeldin A; D002352:Carrier Proteins / Q000378:metabolism*; D002417:Cattle; D002451:Cell Compartmentation; D002499:Centrifugation, Density Gradient; D003517:Cyclopentanes / Q000494:pharmacology; D003545:Cysteine / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006360:Heat-Shock Proteins / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D008715:Methionine / Q000378:metabolism; D008970:Molecular Weight; D017510:Protein Folding; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D016559:Tacrolimus / Q000378:metabolism*; D022021:Tacrolimus Binding Proteins; D014333:Tropoelastin / Q000378:metabolism*
D002352:Carrier Proteins; D003517:Cyclopentanes; D004268:DNA-Binding Proteins; D006360:Heat-Shock Proteins; D007976:Leupeptins; D008024:Ligands; D011500:Protein Synthesis Inhibitors; D014333:Tropoelastin; C063261:acetylleucyl-leucyl-norleucinal; D020126:Brefeldin A; D008715:Methionine; D000591:Amino Acid Isomerases; D022021:Tacrolimus Binding Proteins; D003545:Cysteine; D016559:Tacrolimus