Sec 61 alpha synthesis is enhanced during translocation of nascent chains of collagen type IV in F 9 teratocarcinoma cells after retinoic acid treatment

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Introduction
Secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel (1,2).This protein-conducting channel consists of ER-associated proteins forming a translocon, which includes the translocon-associated protein, the translocating chain-associating membrane protein (TRAM) and the Sec61p complex (1,2).TRAM and Sec61p are tightly associated with membrane-bound ribosomes, suggesting that polypeptide nascent chains pass directly from the ribosome into a protein-conducting channel (3,4).
The Sec61p complex is composed of three subunits: a, ß, g (5).Sec61a, a multispanning membrane protein and the largest component of the Sec61p complex, is essential for translocation of polypeptide chains through this channel into the cisterns of the ER (5).The Sec61p complex is homologous to SecYp present in bacteria (6).
The translocation process requiring the Sec61p complex in mammalian cells is associated with a specialized co-translational pathway in which the nascent polypeptide is continuously bound to polysome-bound ribosomes as it translocates through the ER membrane (7).Photocrosslinking experiments and studies involving chemical crosslinking reagents such as dithio-bis-succinimidyl propionate (DSP) or disuccinimidylsuberate have shown that Sec61a is closely associated with nascent polypeptides undergoing translocation across mammalian rough ER including collagen and Hsp47 (3,8).
Hsp47 is a 47-kDa collagen-binding glycoprotein localized in the ER of collagenproducing cells (9).Hsp47 binds primarily to collagens, procollagen in particular, but may also associate with fetuin (10).Hsp47 is constitutively expressed, as well as affected by environmental stresses such as heat shock (11,12).Following heat shock, Hsp47 protein levels rapidly increase through an acti-vation of heat shock transcription factors (13).Immunoprecipitation studies have revealed that Hsp47 is closely associated with the translation-translocation machinery during the production of procollagen I, suggesting that Hsp47 might function as a molecular chaperone for nascent chains of collagen (14,15).The levels of Hsp47 have been shown to vary with collagen concentration following retinoic acid-induced differentiation of mouse teratocarcinoma cells (16), in pathological conditions such as hepatic, renal and pulmonary fibrosis (17)(18)(19)(20), and in cultured fibroblasts grown from patients with hereditary gingival fibromatosis (21).
In the present study, we tested the proposition that the translocation of secretory proteins through the ER membrane is the result of the enhanced production and utilization of Sec61a and may depend on the stage of cell differentiation.However, it is not known whether Sec61a is produced ubiquitously in all collagen-producing cells or under heat shock treatment.Stem cells from many cultured teratocarcinoma cell lines such as mouse F9 and P19 cells are capable of differentiation in response to low concentrations of retinoic acid, a derivative of vitamin A (22).This differentiation of tumorigenic stem cells results in the loss of tumorigenicity (23).Therefore, teratocarcinoma cell lines provide a model to examine the association between the process of cell differentiation and the production of Sec61a.
We determined the synthesis of Sec61a and Hsp47 during translocation of collagen nascent chains in F9 cells following retinoic acid and heat shock treatment.

Material and Methods
Cell culture and metabolic labeling F9 teratocarcinoma cells, obtained from the American Type Tissue Culture Collection (Rockville, MD, USA), were grown to confluence in 75-cm 2 plastic flasks using Dulbeccos modified Eagles medium (DMEM), 10% fetal bovine serum, 1.16 g/l glutamine, and 100 mg/ml streptomycin at 37ºC in 5% CO 2 .For the cell growth experiments, F9 stem cells (1 x 10 5 cells) were plated onto gelatinized tissue culture plates containing 10 ml of medium.Mouse F9 cells were induced to differentiate into parietal endoderm cells after treatment with all-trans retinoic acid (10 µM) for 4 days.When it was necessary to label proteins, the medium was removed and replaced with fresh methionine-free DMEM containing 35 S-methionine (100 µCi/ml; New England Nuclear, Boston, MA, USA) for 20 min under normal conditions (37ºC) or for 90 min at 43ºC for heat shock. 35S-methionine was used to label nascent chains of collagen and Hsp47.

Immunoprecipitation and analysis by gel electrophoresis
The 35 S-methionine-labeled F9 cells were collected and incubated in lysis buffer (50 mM Tris-HCl, pH 8.0, 40 mM NaCl, 5 mM EDTA) at 4ºC for 30 min.Rabbit antibodies against mouse type IV collagen, Hsp47, and Sec61a (diluted 1:2000) were added to the samples.The samples (100 µl) were incubated for 2 h at 4ºC, then precipitated with 30 µl of protein A-Sepharose.The precipitated immune complexes were washed three times with 1 ml of lysis buffer, and once with 1 ml of 10 mM Tris-HCl, pH 6.8.The samples were suspended in Laemmlis sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) buffer (1.25 M Tris-HCl, pH 6.8, 10% SDS in distilled water, 30% bromophenol blue in glycerol and 0.5% mercaptoethanol), and boiled for 5 min.Twentyfive microliters of the supernatant was loaded onto 4-20% polyacrylamide gels by the method of Laemmli (24).The gels were fixed, dried and autoradiographed using the method of Bonner and Laskey (25).In some instances, treatment with a cleavable bifunctional membrane permeable cross-linking reagent, DSP, was performed prior to immunoprecipitation (16).After labeling, cells were harvested and rinsed with phosphatebuffered solution (PBS).Cells were then treated with 0.1% collagenase to remove the extracellular collagen.After rinsing in PBS, cells were combined with 2 ml DSP (0.1% in DMSO) and placed on ice for 30 min.After 30 min the cells were rinsed with 2 mM glycine in PBS and lysed in lysis buffer with 50 µg/ml gelatin on ice for 15 min.The supernatants were then divided into equal aliquots and immunoprecipitation was performed as described above.

Isolation of membrane-bound polysomes (microsomes) and collagenase digestion
Microsomes from F9 cells were isolated by the method of Vedeler et al. (26).After microsomes were collected, immunoprecipitation was performed as previously described.In some cases, collagenase digestion was performed before adding Laemmlis buffer.Microsome extracts were incubated with 2.7 U of bacterial collagenase in 50 µl collagenase buffer (10 mM Tris, pH 7.5, 40 mM NaCl, 5 mM CaCl 2 , 30 mM KCl, 10 mM PMSF, 50 µM N-ethylmaleimide, 2.5 mM benzamidine) and incubated for 1 h at 37ºC.Laemmlis buffer was then added to the samples, the samples were boiled for 5 min and the immunoprecipitated microsomes bound to protein A-Sepharose were released and collected.SDS-PAGE and autoradiography were performed as previously described.

Western blotting by enhanced chemiluminescence
F9 microsomes were isolated and analyzed by SDS-PAGE as described above.The microsomes were then electrotransferred to a nitrocellulose filter (27).The filter was blocked for 1 h with 10% dried milk in PBS and rinsed three times with PBS.Anti-bodies against TRAM or Sec61a (Biopolymer Laboratories, Baltimore, MD, USA) diluted 1:2,000 in 5% dried milk/1% BSA in PBS were added, and the filter was incubated overnight at room temperature.The filter was washed four times with PBS in 0.2% Tween 20 and incubated for 2 h at room temperature with a secondary goat anti-mouse IgG antibody (Amersham Inc., Arlington Heights, IL, USA) diluted 1:2000 in 1% BSA in PBS, in order to reduce nonspecific binding according to Towbin et al. (27).The filter was then washed in PBS and incubated with enhanced chemiluminescence reagents for 1 min according to the manufacturer protocol (Amersham).An X-ray film was exposed to the filter for less than a minute.

Results
It is not known whether Sec61a is ubiquitously expressed and produced in collagenproducing cells.Furthermore, it is also not clear whether this binding occurs through the enhanced production and utilization of Sec61a and whether it might be dependent on the stage of cell differentiation.In order to answer these questions, we treated F9 cells with all-trans retinoic acid for 4 days, followed by labeling with 35 S-methionine.Membrane-bound polysomes (microsomes) were extracted from the cells according to the method of Vedeler et al. (26).The effect of all-trans retinoic acid on the synthesis of Hsp47 (47 kDa), collagen IV (170-to 185-kDa bands which correspond to a2 and a1 chains, respectively) and Sec61a is shown in Figure 1.Anti-Sec61a antibodies immunoprecipitated a protein of approximately 37-38 kDa in samples treated with retinoic acid (Figure 1, lane 4), while no bands were detected in control samples (Figure 1, lane 3).Treatment of F9 cells with retinoic acid resulted in increased collagen and Hsp47 synthesis (Figure 1, lane 6).

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To confirm the presence of both TRAM and Sec61a in F9 cell membrane-bound polysomes after treatment with retinoic acid, enhanced chemiluminescence Western blotting using specific antibodies against TRAM (28) and Sec61a was employed (Figure 3).Sec61a was detected in retinoic acid-treated (Figure 3, lane 4) but not in untreated cells (Figure 3, lane 3).Interestingly, the other component of the protein-conducting channel, TRAM, was seen in both control and retinoic acid-treated samples (Figure 3, lanes 1 and 2).TRAM was previously shown to mediate protein translocation in mammalian cells (28).
It was also important to determine whether Sec61a production is influenced by heat shock.Hsp47 is constitutively expressed, and also has its production increased by stress including heat shock (29).After heat shock, Hsp47 protein levels rapidly increase through an activation of heat shock transcriptional factors (13).Western blots using anti-Sec61a antibodies demonstrated Sec61a in samples from both control and heat shocktreated cells following retinoic acid treatment (Figure 4, lanes 2 and 4).Sec61a was not detected in untreated control cells (incubated at 37ºC; Figure 4, lane 1) or in retinoic acid nontreated heat-shocked cells (incubated at 43ºC; Figure 4, lane 3).

Discussion
Retinoic acid is an established regulator of epithelial cell differentiation in vivo and in vitro (30).One of the major physiological functions of retinoic acid is to prevent squamous differentiation (23,31).Squamous metaplasia, which develops during vitamin A deficiency, can be reversed by supplementation with retinoic acid (23).Furthermore, retinoic acid suppresses squamous cell differentiation that occurs spontaneously or induced by calcium ions in cultured normal keratinocytes or tracheal cells (23).
F9 cells comprise a cultured teratocarci-noma cell line which can be induced to differentiate into parietal endodermal cells (primitive endoderm-like cells) in the presence of retinoic acid and dibutyryl cAMP (32).The phenotype of the new cell type is characterized by synthesis of plasminogen activator, laminin and type IV collagen and by low levels of alkaline phosphatase and lactase dehydrogenase.The cell type generated by this induction is not dependent upon - the continued presence of either compound, but the cAMP agents are active only on F9 cells that have been treated with retinoic acid (33,34).This differentiation of the tumorigenic F9 stem cells results in the loss of their tumorigenicity (32).This differentiation response of F9 cells is irreversible, fairly synchronous, and rapid within 24 to 48 h (34).
It is unclear at the present time whether an intracellular binding protein mediates all of the actions of retinoic acid in cells.There has also been no convincing evidence in any system that retinoic acid regulates transcription of specific genes including Sec61a.Although the mechanism whereby retinoic acid regulates gene expression and differentiation is not completely understood, it is likely that high-affinity retinoic acid receptors and/ or binding proteins may be involved.
The delineation of translocation mechanisms in mammalian cells is still incomplete.However, several investigators have provided evidence that the ER membrane glycoproteins TRAM and the Sec61p complex are components of the mammalian ER translocon that participates in protein translocation (2).These translocon proteins may associate with ER proteins during the translocation of secretory proteins.Candidates for this asso-Figure 3 -Distribution of translocating chain-associating membrane protein (TRAM) and Sec61a using the Western blotting technique with enhanced chemiluminescence following retinoic acid (RA) treatment.F9 cells were incubated at 37ºC with (lanes 2 and 4) or without (lanes 1 and 3) retinoic acid for 4 days.Antibodies against TRAM (lanes 1 and 2) and Sec61a (lanes 3 and 4) were applied and the nitrocellulose paper was then exposed to radiographic film.
Figure 4. Distribution of Sec61a using the Western blotting technique with enhanced chemiluminescence following retinoic acid (RA) and heat shock (HS) treatment.F9 cells were either incubated at 37ºC (control cells, lanes 1 and 2) or at 43ºC (heatshocked cells, lanes 3 and 4) for 90 min.In some cases, F9 cells were treated with all-trans retinoic acid for 4 days (lanes 2 and 4).Antibodies against Sec61a were added and the nitrocellulose paper was then exposed to radiographic film.-----ciation include components of the ribosomebinding site or the major component of the mammalian homolog of the Sec61p complex, Sec61a (2).The precise localization of Sec61a in the ER membrane has not been determined, although it is likely that portions of Sec61a project into the ER lumen.Sec61a was localized adjacent to nascent polypeptides that are being translocated through the ER membrane (8).Since Sec61a proteins were not associated with long translating polypeptide chains, it has been suggested that Sec61a proteins reside near nascent chains at the beginning of their membrane passage (2).Furthermore, Sec61a was essential for the translocation of certain nascent polypeptide chains through the ER membrane (2).It is not clear whether Sec61a binding to translocating proteins occurs through the enhanced production and utilization of Sec61a, or whether it is dependent on the stage of cell differentiation.It is also not known to date whether Sec61a is constitutively expressed and produced in collagen-producing cells.Accordingly, in the present study we investigated the production of Sec61a and other translocon components following retinoic acid treatment.The production of Sec61a markedly increased after retinoic acid treatment.Sec61a production was not detected in untreated F9 cells.However, another translocon component, TRAM, was present in both retinoic acid-treated and control samples.
The relevance of the undetectable levels of Sec61a in undifferentiated F9 cells is not known.Sec61a was essential for the translocation of secretory proteins (2).The mechanism whereby these tumor cells secrete proteins without detectable amounts of Sec61a is still not well understood.Further experiments are needed to clarify the role of Sec61a in collagen-producing cells.Among mammalian species, the cDNA sequence for the Sec61a gene is known for rats, dogs, and humans, but the cDNA sequence for the mouse Sec61a gene has not been reported.
A number of explanations currently exist for the source of the forces that drive the newly synthesized chains into the lumen of the ER.Topogenic sequences located in translocon components may influence translocation into the ER (35,36).However, another possibility is that simple elongation of the nascent chain drives extrusion of the polypeptide into the ER and the protein translocation channel is a passive pore (37).Consequently, this channel is without directional preference for polypeptide movement.Thus, the bias in random movement of a polypeptide resulting in vectorial translocation is driven by nascent chain elongation and is sustained by inclusion of a stop codon or by post-translational events, such as glycosylation, folding and/or binding to chaperones (38).While it is not known whether distinctive chaperones or translocons are utilized for specialized proteins such as procollagen, there is strong evidence to suggest that some classes of proteins use distinctive machinery for their organization.For example, although evidence strongly indicated the participation of TRAM in the translocation of ß-lactamase and prepro-a-factor, elimination of this translocon component only reduced the translocation efficiency for preprolactin (3).
Using a cross-linking reagent on membrane-bound polysome extracts from retinoic acid-induced F9 teratocarcinoma cells, Sec61a is shown in this study to co-precipitate with collagen IV, Hsp47, and Grp94.These results indicate that a major component of the translocon complex, Sec61a, may associate early with nascent collagen IV polypeptides and ER-resident molecular chaperones during translation/translocation.These data suggest that Sec61a may form complexes with Hsp47 and other ER proteins during the constitutive synthesis of collagen IV by F9 cells after retinoic acid treatment.
Hsp47, a collagen-bound heat shock protein, has been found associated with evolv-ing chains of procollagen associated with polysomes by binding to the Gly-X-Y regions of nascent collagen chains (39).Sauk et al. (15) have demonstrated that Hsp47 associates with Grp78, Grp94 and nascent procollagen I to form large complexes.Gorlich et al. (40) have shown the association of TRAM and Sec61a with complexes of proteins after photocrosslinking.High et al. (3) demonstrated the formation of protein complexes with Sec61a after chemical crosslinking with DSP.These data support the hypothesis of a successive action of molecular chaperones.
The production of Sec61a was shown to be markedly increased following retinoic acid treatment.Another translocon component, TRAM, was demonstrated to be present in both retinoic acid-treated and control samples.Therefore, the absence or limited production of Sec61a in F9 cells prior to retinoic acid treatment is not restricted to other members of the translocation machinery.The relevance of the diminished or absent production of Sec61a in undifferentiated F9 cells is not known.Also, the mechanism whereby these tumor cells secrete proteins without detectable amounts of Sec61a is still not well understood.