Identification of tropoelastin as a ligand for the 65-kD FK506-binding protein, FKBP65, in the secretory pathway

Abstract

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.

Figure 3

Sedimentation analysis of DSP cross-linked protein complexes. Cells were labeled with [³⁵S]cysteine/ methionine for 18 h, cross-linked with DSP, lysed, and the lysate fractionated over a 5–25% sucrose density gradient. Fractions were immunoprecipitated for tropoelastin and analyzed by SDS-PAGE under reducing conditions and fluorography (2 wk exposure). Two pools of tropoelastin are evident; a population not cross-linked by DSP (fractions 6–10) and a second population cross-linked to p74 and p78 (fractions 14–24). Note that two additional bands are seen to codistribute with the tropoelastin-p74–p78 in the center of the gradient (arrowheads).

Figure 1

Cross-linking of tropoelastin into higher molecular weight complexes by DSS treatment. (A) Intact FBCs labeled for 4 h with [³H]leucine in the presence of BFA and ALLN were cross-linked with either DSP or DSS and then lysed and immunoprecipitated for tropoelastin. Analysis by SDS-PAGE under reducing conditions showed higher molecular weight complexes in the DSS-treated cells (arrows) and a reduced intensity of tropoelastin monomer at 70 kD (TE). (B) FBCs treated for 4 h with BFA and ALLN were cross-linked, lysed, and immunoprecipitated for tropoelastin. After SDS-PAGE, the proteins were transferred to nitrocellulose and blotted for tropoelastin. Immunoreactive bands in the DSS lane (arrows) indicate nonreducible cross-linked complexes of tropoelastin with other proteins. (PC) pre-clear.

Figure 4

Effect of secretion-disrupting agents on the association of p74 and p78 with tropoelastin. (A) FBCs were metabolically labeled with [³⁵S]cysteine/methionine for 16 h with either no treatment (control) or treatment with BFA + ALLN, monensin, or bafilomycin A1 for the final 4 h of the labeling period. After cross-linking with either DSP or DSS, the cells were lysed and immunoprecipitated for tropoelastin. Samples were analyzed by SDS-PAGE under reducing conditions. A concurrent increase in p74 and p78 was observed along with tropoelastin in cells treated with either BFA + ALLN or monensin. No increase in p74 and p78 was observed in bafilomycin A1-treated cells as compared to control cells despite an increase in tropoelastin. (B) Densitometric scanning of the bands in A confirm the lack of increased p74 and p78 coimmunoprecipitation in bafilomycin A1-treated cells. Note that the amount of p74 and p78 decreases as the secretion block moves progressively farther along the secretory pathway.

Figure 6

Bovine FKBP65 is cross-linked to tropoelastin in the secretory pathway of FBCs. FBCs were treated with BFA + ALLN for 4 h before cross-linking with either DSP or DSS. Cell lysates were immunoprecipitated for either tropoelastin or FKBP65, separated by SDS-PAGE under reducing conditions, transferred, and blotted for either the same protein or the opposite protein. Lane 2 shows that FKBP65 can be detected in the cross-linked cell lysate after immunoprecipitation with tropoelastin antibody and lane 5 shows that tropoelastin can be detected in the FKBP65 immunoprecipitation after cross-linking. Note that when the nonreducible cross-linker is used (lane 3), FKBP65 is detected in a higher molecular weight complex that must also contain tropoelastin (arrowhead). This experiment was designed to maximize the ability to detect the cross-linked proteins and should not be interpreted in a quantitative manner because the amount of cell lysate used was increased threefold for conditions where the blotting antibody was different than that used for immunoprecipitation. The ECL exposure times were also adjusted as follows: lanes 1–3, ∼30 s; lane 4, ∼30 s after waiting 2 h for the signal to decay; lane 5, ∼2 min.

Figure 2

³⁵S-labeling allows the identification of two proteins that are cross-linked to tropoelastin. FBCs were labeled with [³⁵S]cysteine/methionine for 16 h and (A) either left untreated (CONTROL) or (B) treated with BFA and ALLN (BFA + ALLN) for the final 4 h of the labeling period. After metabolic labeling, intracellular proteins were cross-linked with either DSP or DSS, lysed, and immunoprecipitated for tropoelastin. One of the BFA + ALLN samples was treated with an equivalent concentration of DMSO alone (vehicle) rather than cross-linker. Analysis by SDS-PAGE under reducing conditions showed the presence of two additional bands in the DSP-treated cells (p74 and p78). These bands were greatly enhanced after BFA-treatment. In the DSS cross-linked lysates, high molecular weight bands in the same position as were seen in Fig. 1 were observed (arrows), and as expected, the immunoprecipitated bands of tropoelastin (TE), p74 and p78, were all reduced in density. Note that p78 coimmunoprecipitates with tropoelastin in the absence of cross-linker.

Figure 5

Microsequencing identifies p78 as BiP and p74 as bovine FKBP65. (A) FBCs were treated with BFA + ALLN for 4 h, cross-linked with DSP, lysed and immunoprecipitated using an antibody to tropoelastin. Samples were separated by SDS-PAGE, transferred to PVDF membrane, and stained with Coomassie blue. Areas indicated on the right were cut from the blot and treated with CNBr before sequencing. Unique sequences from p78 obtain following OPA treatment identified p78 as BiP. Separate sequencing of the two p74 bands following CNBr digestion showed that both bands were the same protein. (B) Alignment of the amino acid sequence from a portion of each of the four PPIase domains in mouse FKBP65 as reported by Coss and colleagues (1995) with the sequence determined for p74 after CNBr treatment. Note the high degree of homology between the PPIase domains (bold) that allows a definitive sequence for p74 to be obtained from the peptide mixture.