Pulmonary fibroblasts: an in vitro model of emphysema. Regulation of elastin gene expression

Disruption and degradation of interstitial elastic fibers are significant characteristics of pulmonary emphysema. In order to examine the responses of elastogenic cells to the conditions mimicking degradation of interstitial pulmonary elastin, rat pulmonary fibroblast cultures were used as an in vitro model. Second passage fibroblasts were divided into two different environmental situations to represent cells adjacent to and remote from the site of elastase-digested matrix. One set of cell cultures was briefly digested with pancreatic elastase. The resultant digest was then added back incrementally to the medium of elastase-digested cell cultures and to the medium of a second set of undigested cultures. Both sets of cell cultures remained viable and metabolically active during these treatments (96 h of incubation) as judged by protein synthesis, cell number, and steady-state levels of beta-actin mRNA. However, the two sets of cultures exhibited opposite responses in elastin gene expression with addition of increasing amounts of the elastase digest. The elastase-digested cultures exhibited a 200% increase in extractable soluble elastin and a 186% increase in tropoelastin mRNA with the addition of increasing amounts of the elastase digest to the medium. Conversely, the amount of soluble elastin recovered from the undigested cultures decreased 75%, and the steady-state level of tropoelastin mRNA decreased 63%. Soluble elastin peptides generated from oxalic acid treatment of purified elastin were shown to decrease tropoelastin mRNA in undigested cell cultures in the same manner as the elastase digest. Based on these data, we propose that pulmonary fibroblast elastin gene expression can be controlled coordinately by the state of the extracellular matrix and solubilized peptides derived from that matrix. Such integrated regulation may serve to localize elastin repair mechanisms.     

Role of tropoelastin fragmentation in elastogenesis in rat smooth muscle cells

Neonatal rat aortic smooth muscle cell cultures produce two major soluble elastin molecules termed protropoelastin (77 kDa) and tropoelastin (71 kDa). Cell layer extracts are protroproelastin-enriched, while protropoelastin, tropoelastin, and significant amounts of discrete elastin fragments (Mr of 66,000, 61,000, 56,000, and 45,000) are present in preparations from the medium of these cultures. To determine the role of the various elastin molecules in the metabolism of elastin in neonatal rat aortic smooth muscle cell cultures, the amino termini of these proteins were sequenced. All soluble elastin components present in the medium were purified as a single peak by high performance liquid chromatography; further separation of the components was achieved by polyacrylamide gel electrophoresis and electroblotting. The bands were excised and sequenced. The amino-terminal sequences of protropoelastin, tropoelastin, and the 66-kDa, 61-kDa, and 56-kDa fragments were identical: Gly-Gly-Val-Pro-Gly-Ala-Val-Pro-Gly-Gly. This sequence is identical with published amino-terminal sequences of tropoelastins from several other species. As expected, when the cell cultures were pulsed with [3H]valine, all the soluble elastin molecules were radioactive, while only protropoelastin appeared radioactive after [35S] cysteine pulsing. Since cysteine is present only in the carboxyl-terminal end of the molecule, all the data indicate that the cleavage of the elastin fragments identified in the culture are occurring at the carboxyl end of protropoelastin. These results are consistent with the original hypothesis that a precursor-product relationship exists between the 77-kDa and 71-kDa soluble elastin molecules. Based on known tropoelastin sequences and the molecular weights of the discrete fragments, additional fragmentation of protropoelastin and/or tropoelastin most likely occurs at the lysine/alanine-enriched domains presumably involved in cross-link formation.     

Effect of monoclonal antibodies to defined regions of tropoelastin on elastogenesis in vitro.

Primary cultures of chick embryo aorta cells were grown for one week in the presence of mouse monoclonal antibodies directed against defined regions of chick tropoelastin. This treatment did not significantly alter cell proliferation, cell viability and incorporation of labeled amino acids into total protein or tropoelastin compared with control cultures in which antibodies were either omitted or substituted with an unrelated monoclonal antibody. Tropoelastin-reactive material in the cell layer was revealed by immunologic staining with rabbit antibodies against the chick protein both at the optical and ultrastructural level. Immunofluorescence of control cultures showed that tropoelastin was incorporated into thin and straight fibrils which were sometimes associated with spot-like elements. In the electron microscope tropoelastin-reactive sites were found mainly on the amorphous core of typical, small elastic fibers. The morphological picture of tropoelastin deposits in cultures exposed to anti-tropoelastin monoclonal antibodies depended on the molecular form (whole antibody or Fab fragments) and the binding specificity of the antibody used. Although alterations common to different antibodies were observed, the main structural features were peculiar for each antibody. Two antibodies which bound epitopes present in two regions of tropoelastin grossly altered the formation of amorphous elastin. Moreover, two antibodies directed against the region of tropoelastin containing the polypentapeptide-repeat (VPGVG)n stimulated the deposition of the protein into the amorphous core of normal-looking elastic fibers and disorganized the compact bundles of parallel microfibrils seen in controls. Finally, one antibody which recognized a unique epitope close to the carboxy-terminal end of tropoelastin and Fab fragments from all antibodies apparently inhibited the formation of the amorphous nuclei of elastic fibers, but not the association of tropoelastin with microfibrils. The data suggest that the association of tropoelastin molecules during fiber assembly is not random, but follows an ordered alignment process which the antibodies alter by imposing a different molecular packing.     

Alteration of the extracellular matrix of smooth muscle cells by ascorbate treatment

The protein composition in the extracellular matrix of cultured neonatal rat aortic smooth muscle cells has been monitored over time in culture. The influence of ascorbate on insoluble elastin and collagen has been described. In the absence of ascorbate, the cells accumulate an insoluble elastin component which can account for as much as 50% of the total protein in the extracellular matrix. In the presence of ascorbate, the amount of insoluble collagen increases, while the insoluble elastin content is significantly less. When ascorbate conditions are varied at different times during the culture, the extracellular matrices are altered with respect to collagen and elastin ratios. The decrease in elastin accumulation in the presence of ascorbate may be explained by an overhydroxylation of tropoelastin. Approximately 1/3 of the prolyl residues in the soluble elastin fractions isolated from cultures grown in the presence of ascorbate are hydroxylated. Since the insoluble elastin accumulated in these cultures contain the unique lysine-derived cross-links in amounts comparable to aortic tissue, this culture system proves ideal for studying the influence of extracellular matrix elastin on cell growth and metabolism.     

Deposition of tropoelastin into the extracellular matrix requires a competent elastic fiber scaffold but not live cells.

The initial steps of elastic fiber assembly were investigated using an in vitro assembly model in which purified recombinant tropoelastin (rbTE) was added to cultures of live or dead cells. The ability of tropoelastin to associate with preexisting elastic fibers or microfibrils in the extracellular matrix was then assessed by immunofluorescence microscopy using species-specific tropoelastin antibodies. Results show that rbTE can associate with elastic fiber components in the absence of live cells through a process that does not depend on crosslink formation. Time course studies show a transformation of the deposited protein from an initial globular appearance early in culture to a more fibrous structure as the matrix matures. Deposition required the C-terminal region of tropoelastin and correlated with the presence of preexisting elastic fibers or microfibrils. Association of exogenously added tropoelastin to the cellular extracellular matrix was inhibited by the addition of heparan sulfate but not chondroitin sulfate sugars. Together, these results suggest that the matrix elaborated by the cell is sufficient for the initial deposition of tropoelastin in the extracellular space and that elastin assembly may be influenced by the composition of sulfated proteoglycans in the matrix.     

Elastin production by cultured calf pulmonary artery endothelial cells

Calf pulmonary artery (CPA) endothelial cells synthesize and secrete soluble elastin when incubated in medium conditioned by arterial smooth muscle cells. Endothelial cell tropoelastin cross-reacts with antiserum to bovine ligamentum nuchae elastin and comigrates on SDS-PAGE with tropoelastins from fetal bovine ligamentum nuchae fibroblasts, aortic smooth muscle cells, and ear chondroblasts at an apparent molecular weight of 70,000. Endothelial cells synthesize only one-third as much elastin as these other cell types, however. Approximately 80% of the elastin synthesized by endothelial cells in confluent culture is released into the culture medium. The remaining 20% remains associated with the cell layer and is readily extractable with dilute acetic acid as un-cross-linked, 70,000-dalton tropoelastin. The addition of beta-aminopropionitrile to culture medium did not alter the ratio of tropoelastin in the medium and cell layer, suggesting that cross-linking of tropoelastin does not occur in culture. Immunofluorescent staining of confluent endothelial cell cultures with antielastin serum demonstrated elastin occurring as a web-like network of fine filaments extending throughout the extracellular space. The fibrous elastin was different in organization and distribution from fibers stained with antifibronectin serum, which were localized primarily beneath the cell layer and in regions of cell-cell contact. Extracellular matrix remaining after solubilization of cellular material with Triton X-100 stained positive for fibronectin, but not for elastin.     

The tissue distribution of murine Abcc6 (Mrp6) during embryogenesis indicates that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly

Summary Mutations in the gene coding for the ABC transporter, ABCC6, in humans cause Pseudoxanthoma elasticum, which is characterized by the deposition of aberrant elastic fibers. To investigate whether the presence of ABCC6 in tissues synthesizing elastin is required for elastin deposition and elastic fiber assembly, we have compared the steady-state levels and tissue distribution of Abcc6 and tropoelastin mRNAs during mouse embryogenesis. Whereas tropoelastin mRNA levels rose during embryogenesis and were the highest in neonatal mice, Abcc6 mRNA levels remained constantly low throughout embryogenesis. In some tissues, both Abcc6 and tropoelastin mRNA were detected. However, Abcc6 mRNA and protein were not detected in neonatal aorta and arteries, which produce large amounts of elastin indicating that the presence of Abcc6 in elastic tissues is not required for elastic fiber assembly.     

Identification of multiple tropoelastins secreted by bovine cells

High resolution gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, cell-free translation, and elastin-specific antibodies were used to identify three tropoelastin isoforms secreted by bovine tissue and cells. Tropoelastin isolated from nuchal ligament and from conditioned culture medium or cell-matrix extracts of ligament fibroblasts and auricular chondrocytes resolved as three distinct bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of approximately 67,500 (tropoelastin I), 65,000 (tropoelastin II), and 62,000 (tropoelastin III). Three tropoelastin polypeptides with molecular mass 2-3 kDa higher than their corresponding tissue forms were also evident in cell-free translation products of ligamentum nuchae RNA, suggesting that each tropoelastin species is encoded by a unique mRNA. The presence of cysteine in all three tropoelastin isoforms was demonstrated by the incorporation of [35S]cysteine into newly synthesized tropoelastin polypeptides and by immunoreactivity with an antibody raised against a synthetic peptide that defines the cysteine-containing carboxyl-terminal region of tropoelastin. Immunological co-localization of the carboxyl-terminal antibody with insoluble elastin in lung vasculature and parenchyma suggests that intact tropoelastin and not a processed form is incorporated into the elastin fiber.     

Tropoelastin production and tropoelastin messenger RNA activity. Relationship to copper and elastin cross-linking in chick aorta.

The elastin content of the chick thoracic aorta increases 2--3-fold during the first 3 weeks post-hatching. The deposition of elastin requires the covalent cross-linking of tropoelastin by means of lysine-derived cross-links. This process is sensitive to dietary copper intake, since copper serves as cofactor for lysyl oxidase, the enzyme that catalyses the oxidative deamination of the lysine residues involved in cross-link formation. Disruption of cross-linking alters tissue concentrations of both elastin and tropoelastin and results in a net decrease in aortic elastin content. Autoregulation of tropoelastin synthesis by changes in the pool sizes of elastin or tropoelastin has been suggested as a possible mechanism for the diminished aortic elastin content. Consequently, dietary copper deficiency was induced to study the effect of impaired elastin cross-link formation on tropoelastin synthesis. Elastin in aortae from copper-deficient chicks was only two-thirds to one-half the amount measured in copper-supplemented chicks, whereas copper-deficient concentrations of tropoelastin in aorta were at least 5-fold higher than normal. In spite of these changes, however, increased amounts of tropoelastin, copper deficiency and decreased amounts of elastin did not influence the amounts of functional elastin mRNA in aorta. Likewise, the production of tropoelastin in aorta explants was the same whether the explants were taken from copper-sufficient or -deficient birds. The lower accumulation of elastin in aorta from copper-deficient chicks appeared to be due to extracellular proteolysis, rather than to a decrease in the rate of synthesis. Electrophoresis of aorta extracts, followed by immunological detection of tropoelastin-derived products, indicated degradation products in aortae from copper-deficient birds. In extracts of aortae from copper-sufficient chicks, tropoelastin was not degraded and appeared to be incorporated into elastin without further proteolytic processing.     

Insulin-like growth factor I stimulates elastin synthesis by bovine pulmonary arterial smooth muscle cells

Insulin-like growth factor I stimulates mitogenesis in smooth muscle cells, and upregulates elastin synthesis in embryonic aortic tissue. Increased smooth muscle elastin synthesis may play an important role in vascular remodeling in chronic pulmonary hypertension. Therefore, we studied the effect of IGF-I on elastin and total protein synthesis by pulmonary arterial smooth muscle cells in vitro. Tropoelastin synthesis was measured by enzyme immunoassay, and total protein synthesis was measured by [3H]-leucine incorporation. In addition, the steady-state levels of tropoelastin mRNA were determined by slot blot hybridization. Incubation of confluent cultures with various concentrations of IGF-I resulted in a dose-dependent stimulation of elastin synthesis, with a 2.4-fold increase over control levels at 1000 ng/ml of IGF. The increase in elastin synthesis was reflected by a stimulation of the steady-state levels of tropoelastin mRNA. We conclude that IGF-I has potent elastogenic effects on vascular smooth muscle cells, and speculate that it may contribute to vascular wall remodeling in chronic hypertension.     

Accumulation and regulation of elastin in the rat uterus

The relative levels of elastin-specific mRNA were used as a measure of tropoelastin expression in uteri from pregnant Sprague-Dawley rats. The levels of elastin-specific mRNA were also correlated with values for net tropoelastin production and net deposition of mature, crosslinked elastin. The total content of uterine elastin increased throughout gestation, reaching maximal levels at Day 19 of gestation, which were three times those of nongravid tissue. Following involution, the elastin content decreased rapidly to near baseline values by 5 days postpartum. The content of soluble elastin, estimated using an enzyme-linked immunosorbent assay, paralleled in part the increase in elastin deposition and elastin mRNA levels. Uterine elastin metabolism appears to be unlike that in other elastic tissues, e.g., lung and large blood vessels. In most elastin containing tissues, the protein is synthesized during discrete developmental periods and is not readily degraded. However, uterine elastin is continuously expressed, and appears to be in a continual cycle of degradation and replacement.     

Characterization of a signal peptide sequence in the cell-free translation product of sheep elastin mRNA

In vitro explant cultures of near-term sheep nuchal ligament secrete tropoelastin of approximate M_r 70,000–72,000 while the elastin cell-free product of sheep nuchal ligament RNA is 2000 to 3000 M_r larger. Automated Edman degradation of immunoprecipitates of radiolabeled cell-free elastin precursor demonstrated the presence of a 26-residue signal sequence which was absent from sheep tropoelastin secreted from explant cultures. In addition, a 20-residue overlap was established between the cell-free product and the secreted protein. This overlap region, representing the N-terminal sequence of ovine tropoelastin, demonstrated complete homology with the N-terminal sequence of porcine tropoelastin and near complete homology with chick tropoelastin. These findings suggest that cotranslational removal of this hydrophobic peptide extension is likely a correlate of vectorial transport of elastin into the secretory apparatus.     

Protein interaction studies of MAGP-1 with tropoelastin and fibrillin-1

Elastic fibers consist primarily of an amorphous elastin core associated with microfibrils, 10-12 nm in diameter, containing fibrillins and microfibril-associated glycoproteins (MAGPs). To investigate the interaction of MAGP-1 with tropoelastin and fibrillin-1, we expressed human MAGP-1 as a T7-tag fusion protein in Escherichia coli. Refolding of the purified protein produced a soluble form of MAGP-1 that displayed saturable binding to tropoelastin. Fragments of tropoelastin corresponding to the N-terminal, C-terminal, and central regions of the molecule were used to characterize the MAGP-1 binding site. Cleavage of tropoelastin with kallikrein, which cleaves after Arg(515) in the central region of the molecule, disrupted the interaction, suggesting that the separated N- and C-terminal fragments were insufficient to determine MAGP-1 binding to intact tropoelastin. In addition, no evidence of an interaction was observed between MAGP-1 and a tropoelastin construct consisting of domains 17-27 that brackets the kallikrein cleavage site, suggesting a complex mechanism of interaction between the two molecules. Binding of MAGP-1 was also tested with overlapping recombinant fibrillin-1 fragments. MAGP-1 bound to a region at the N terminus of fibrillin-1 in a calcium-dependent manner. In summary, these results suggest a model for the interaction of elastin with the microfibrillar scaffold.     

Terminal differentiation of nuchal ligament fibroblasts: characterization of synthetic properties and responsiveness to external stimuli

The temporal expression of elastogenesis is unique among connective tissues in that elastin production occurs primarily during late fetal and early neonatal periods and is essentially fully repressed once fiber assembly is completed. To test whether elastin synthesis in adult nuchal ligament fibroblasts is permanently repressed or whether the cells retain the ability to reinitiate production upon proper stimulation, we examined in adult ligament cells various parameters known to be involved in the regulation of elastin production. Elastin synthetic capacity, as determined by the levels of steady-state tropoelastin mRNA, of adult tissue was significantly decreased relative to fetal tissue. Likewise, fibroblasts grown from explants of adult ligament had about a fourfold decrease in elastin production and elastin-specific mRNA levels. On the other hand, adult cells were similar to fetal ligament cells in that they were sensitive to glucocorticoid stimulation and demonstrated chemotactic responsiveness to elastin peptides. Since our previous studies have shown that the extracellular matrix (ECM) plays an important role in influencing elastin phenotypic expression, fetal and adult fibroblasts were grown on slices of nonviable adult ligament to test if repression of elastin production was directed by factors in ECM of adult tissues. No change in elastin synthesis was detected with either cell type grown on adult ligament, whereas both fetal and adult cells demonstrated increased elastin production in response to contact with fetal ligament. These results suggest that adult ligament ECM does not provide a metabolic signal to shut off the elastin gene and that adult cells remain responsive to external stimuli that may reinitiate high levels of elastin synthesis.     

Recycling of the 67-kDa Elastin Binding Protein in Arterial Myocytes Is Imperative for Secretion of Tropoelastin

We have shown previously that the 67-kDa elastin binding protein (EBP) colocalizes intracellularly and extracellularly with tropoelastin in fetal sheep aorta, suggesting that these two proteins associate along the secretory pathway. Moreover, we have established that association with EBP protects tropoelastin from serine proteinases and from intracellular coacervation, and is necessary for its proper extracellular assembly. Since the production of tropoelastin by aortic smooth muscle cells (Ao SMC) exceeds production of the EBP, we speculated that this binding protein might recycle back into the cell, associating again with newly synthesized tropoelastin. In this report we labeled cultured Ao SMC externally with the F(ab′)₂ fragments of immunoglobulin which recognizes sheep EBP and followed trafficking of EBP by immunofluorescence and electron microscopy. Our results indicate that the majority of the EBP residing on the cell surface can be internalized to endocytic compartments (but not to lysosomes) and recycled back to the plasma membrane within 45-60 min. We have also determined that reagents disturbing pH of distinct endocytic compartments (chloroquine and bafilomycin A₁, but not ammonium chloride) arrest recycling of the EBP and, at the same time, strongly inhibit deposition of insoluble elastin in cultures of sheep Ao SMC and in organ cultures of chicken aorta. In contrast, neither chloroquine nor bafilomycin A₁ inhibit total protein synthesis or synthesis of tropoelastin. Our results suggest that the EBP serves as a reusable shuttle protein for tropoelastin and that its recycling is essential for effective deposition of insoluble elastin.     

Extracellular matrix-specific induction of elastogenic differentiation and maintenance of phenotypic stability in bovine ligament fibroblasts

We studied the process of elastogenic differentiation in the bovine ligamentum nuchae to assess the mechanisms that regulate elastin gene expression during development. Undifferentiated ( nonelastin - producing) ligament cells from early gestation animals initiate elastin synthesis when grown on an extracellular matrix (ECM) substratum prepared from late gestation ligamentum nuchae. ECM from ligaments of fetal calves younger than the time when elastin production occurs spontaneously in situ (i.e., beginning the last developmental trimester at approximately 180 d of gestation) does not stimulate elastin production in undifferentiated cells. Matrix-induced differentiation requires direct cell matrix interaction, is dependent upon cell proliferation after cell-matrix contact, and can be blocked selectively by incorporation of bromodeoxyuridine into the DNA of undifferentiated cells before (but not after) contact with inducing matrix. Quantitative analysis of elastin synthesis in young cells after matrix-induced differentiation indicates that the entire cell population is competent to respond to the matrix inducer, and continued synthesis of elastin after young cells are removed from the ECM substratum indicates that the phenotypic transition to elastin synthesis is stable and heritable. Although ligament cells do not require continuous contact with ECM to express the elastin phenotype, elastin synthesis is increased substantially when elastin-producing cells are grown on ligament matrix, suggesting that elastogenic differentiation is stabilized by ECM. The matrix substratum was also found to alter the distribution of tropoelastin between the medium and matrix cell layer. When grown on tissue culture plastic, ligament cells secrete greater than 80% of newly synthesized tropoelastin into the culture medium. When cultured on ECM, however, 50-70% of the newly synthesized tropoelastin remains associated with the cell layer and is cross-linked to form insoluble elastin as shown by the incorporation of radiolabeled lysine into desmosine.     

Sustained changes in lung expansion alter tropoelastin mRNA levels and elastin content in fetal sheep lungs

Our objective was to determine the effects of sustained alterations in fetal lung expansion on pulmonary elastin synthesis. In fetal sheep, lung expansion was either decreased between 111 and 131 days' gestation (term approximately 147 days) by tracheal drainage or increased for 2, 4, 7, or 10 days by tracheal obstruction, ending at 128 days' gestation. Lung tropoelastin mRNA levels were assessed by Northern blot analysis, total elastin content was measured biochemically, and staining of lung sections was used to assess the localization and form of elastic fibers. Tracheal obstruction significantly elevated pulmonary tropoelastin mRNA levels 2.5-fold at 2 days, but values were not different from controls at 4, 7, and 10 days; elastin content tended to be increased at all time points. A sustained decrease in lung expansion by tracheal drainage reduced pulmonary tropoelastin mRNA levels 2.5-fold; elastin content was also decreased compared with controls, and tissue localization was altered. Our results indicate that the degree of lung expansion in the fetus influences elastin synthesis, content, and tissue deposition.     

Analysis of the 3' region of the sheep elastin gene

The nucleotide sequences of a 1279-bp sheep elastin cDNA clone, pcSEL1 [Yoon et al. (1984) Biochem. Biophys. Res. Commun. 118, 261-269], and a 1230-bp sheep elastin genomic subclone, pSS1 [Davidson et al. (1984) Biochem. J. 220, 643-652], corresponding to a portion of the cDNA clone, were determined. These analyses permitted determination of the 100 amino acids at the carboxy terminus of sheep tropoelastin. A portion of this sequence showed strong homology to known sequences of pig tropoelastin, but most of the sequence had not been previously determined through protein sequencing. Novel aspects of the tropoelastin molecule which have been revealed by the present analyses are (i) the presence of an unusual sequence, KPPKP, which may contribute to crosslink formation; and (ii) the finding of cysteine within a sequence, CLGKSCGRKRK, at the putative carboxy terminus of tropoelastin. Because of the presence of these sequences, it is speculated that the carboxy-terminal region may be of importance in crosslinking tropoelastin molecules to themselves or to other matrix macromolecules. The nucleotide analyses revealed that sheep elastin mRNA contains a 974-bp untranslated sequence at the 3' end, which appears to be strongly conserved among species.     

Elastin biosynthesis in chick-embryo arteries. Studies on the intracellular site of synthesis of tropoelastin.

Electrophoretic analyses of the products of cell-free translation of elastin mRNA isolated from 17-day chick-embryo thoracic arteries have demonstrated that the elastin mRNA codes for polypeptides that are slightly larger than the cellular tropoelastin polypeptides synthesized and secreted by matrix-free artery cells. Pulse-chase experiments with cells labelled with [3H]proline established that newly synthesized tropoelastin polypeptides were associated solely with membrane-bound particulate fractions. Cell-free translation of membrane-bound and free polyribosomes isolated from artery cells revealed that the tropoelastin mRNA was associated predominantly with the membrane-bound fraction. When rough-microsomal fractions, isolated from cells labelled with [3H]proline for 10 min, were treated with proteinases in the presence and in the absence of detergent, the nascent tropoelastin polypeptides were shown to be susceptible to proteolysis only when the integrity of the membranes was destroyed by detergent treatment. In similar experiments tropoelastin polypeptides synthesized by membrane-bound polyribosomes in the nuclease-treated reticulocyte lysate were also resistant to the proteolytic-enzyme treatment. The results suggest that tropoelastin polypeptides are synthesized on membrane-bound polyribosomes and discharged into the lumen of the endoplasmic reticulum with co-translational removal of a signal peptide.