Developmental regulation of FKBP65. An ER-localized extracellular matrix binding-protein

Abstract

FKBP65 (65-kDa FK506-binding protein) is a member of the highly conserved family of intracellular receptors called immunophilins. All have the property of peptidyl-prolyl cis-trans isomerization, and most have been implicated in folding and trafficking events. In an earlier study, we identified that FKBP65 associates with the extracellular matrix protein tropoelastin during its transport through the cell. In the present study, we have carried out a detailed investigation of the subcellular localization of FKBP65 and its relationship to tropoelastin. Using subcellular fractionation, Triton X-114 phase separation, protease protection assays, and immunofluorescence microscopy (IF), we have identified that FKBP65 is contained within the lumen of the endoplasmic reticulum (ER). Subsequent IF studies colocalized FKBP65 with tropoelastin and showed that the two proteins dissociate before reaching the Golgi apparatus. Immunohistochemical localization of FKBP65 in developing lung showed strong staining of vascular and airway smooth muscle cells. Similar areas stained positive for the presence of elastic fibers in the extracellular matrix. The expression of FKBP65 was investigated during development as tropoelastin is not expressed in adult tissues. Tissue-specific expression of FKBP65 was observed in 12-d old mouse tissues; however, the pattern of expression of FKBP65 was not restricted to those tissues expressing tropoelastin. This suggests that additional ligands for FKBP65 likely exist within the ER. Remarkably, in the adult tissues examined, FKBP65 expression was absent or barely detectable. Taken together, these results support an ER-localized FKBP65-tropoelastin interaction that occurs specifically during growth and development of tissues.

Figure 1

Cytosol/membrane fractionation. FKBP65 is contained entirely within the membrane fraction. NIH3T3 cells were disrupted by nitrogen cavitation, unbroken cells were pelleted, and the resulting supernatant separated into cytosol and membrane fractions by a 200,000g spin. The membrane fraction was solubilized in Triton X-100, then proteins in both fractions were acetone precipitated. Pellets were dissolved in sample buffer, resolved by SDS-PAGE, and Western blotted. CNX, calnexin; Rib-1, ribophorin-1.

Figure 2

Triton X-114 phase separation. FKBP65 does not have a hydrophobic domain indicative of a transmembrane protein. NIH3T3 cells and FBCs were solubilized in Triton X-114, warmed to 30°C, and separated into detergent and aqueous phases by centrifugation. The two phases were collected and either immunoprecipitated (FBCs) or acetone precipitated (NIH3T3 cells). Samples were resolved by SDS-PAGE and Western blotted. A sample from the original solubilization (detergent + aqueous) was also analyzed. Tropo, tropoelastin; CNX, calnexin; Rib-1, ribophorin-1.

Figure 3

Protease protection assay. FKBP65 is entirely intravesicular. The 200,000g microsomal pellet from NIH3T3 cells was treated with buffer alone (control), 100 μg/ml proteinase K alone, or 100 μg/ml proteinase K + 1% Triton X-100. After 30 min at room temperature, excess PMSF was added to each sample to inhibit the enzyme, and the membranes in all samples were solubilized with Triton X-100. Proteins were acetone precipitated and Western blotted. CNX-CT = calnexin C-terminal antibody.

Figure 4

Indirect immunofluorescence labeling of FKBP65 is identical to that of Grp94. FBCs were fixed with 2% paraformaldehyde, permeabilized with 0.1% saponin in PBS containing 1% BSA, and immunolabeled for either FKBP65 or Grp94. Both antibodies show the same ER pattern of staining, and in many cells a negatively stained area representing the perinuclear Golgi cap can be seen.

Figure 5

FKBP65 is a resident protein of the ER. Subconfluent cultures of FBCs were treated for 3 h with either (A) 10 μg/ml brefeldin A + 10 μg/ml ALLN to “hold” the tropoelastin in the fused ER/Golgi compartment (ALLN is needed to prevent tropoelastin degradation), or (B, C) 10 μM monensin to “hold” the tropoelastin in the cis-Golgi cisternae. Cells were then fixed, permeabilized, and double labeled with a monoclonal antibody to tropoelastin (red) and a polyclonal antibody to either (A, B) FKBP65 (green) or (C) Grp94 (green). After treatment with BFA+ALLN, tropoelastin and FKBP65 were colocalized in the fused ER/Golgi apparatus. When tropoelastin was accumulated in the Golgi, however, the two proteins showed distinct patterns of immunolabeling with tropoelastin in the peri-nuclear Golgi region and FKBP65 remaining in the ER. The same distribution of labeling was seen when Grp94 was colocalized with tropoelastin after monensin treatment. Note that the production of tropoelastin by FBCs in culture is density-dependent, thus not all the cells are synthesizing tropoelastin at subconfluency.

Figure 6

FKBP65 and tropoelastin colocalize in developing lung. Immunostaining for FKBP65 and tropoelastin on serial sections of paraffin-embedded fetal bovine lung. (A) Tropoelastin staining, seen surrounding an airway and small blood vessel, represents elastic fibers in the extracellular matrix. (B, C) The SMCs that synthesize the tropoelastin to form these fibers stain strongly for FKBP65. Note that the blood vessel stains with equal intensity with both FKBP65 antibodies, but stronger staining of the airway SMCs is seen with the antipeptide FKBP65 antibody than with the commercial antibody. A, airway; BV, blood vessel.

Figure 7

FKBP65 is developmentally regulated. (A) Northern analysis of tissues from 12-d postnatal mice shows no expression of FKBP65 in liver, whereas elevated-to-moderate levels of expression are seen in aorta, brain, kidney, and lung. Expression of Grp94 is seen in all five tissues. (B) Northern analysis of the same tissues from adult mice shows developmental down-regulation of tropoelastin. FKBP65 expression was also found to be absent, or barely detectable, in all tissues examined. In contrast, Grp94 is constituitively expressed. Adult blots were exposed relative to a control lane of 12-d postnatal mouse lung RNA (control lane not shown). AO, aorta; BR, brain; KD, kidney; LV, liver; LU, lung.

Figure 8

FKBP65 is not expressed in liver. Northern analysis of kidney, liver, and lung from 14.5-d mouse embryos shows strong expression FKBP65 in kidney and lung but no expression in liver. KD, kidney; LV, liver; LU, lung.

Figure 9

Amino acid sequence of bovine tropoelastin (Indik et al., 1990). Proline residues make up ∼12% of the tropoelastin molecule. Prolines appear in repeating sequences (see line 11, dotted underline) and preceding cross-linking domains (double underlines). The sequence in this figure is arranged with the cross-linking domains and preceding proline aligned to the left.