Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (ER) membrane through a protein-conducting channel called translocon. Sec61a, a multispanning membrane translocon protein, has been implicated as being essential for translocation of polypeptide chains into the cisterns of the ER. Sec61a forms a protein complex with collagen and Hsp47, an ERresident heat shock protein that binds specifically to collagen. However, it is not known whether Sec61a is ubiquitously produced in collagen-producing F9 teratocarcinoma cells or under heat shock treatment. Furthermore, the production and utilization of Sec61a may depend on the stage of cell differentiation. Cultured F9 teratocarcinoma cells are capable of differentiation in response to low concentrations of retinoic acid. This differentiation results in loss of tumorigenicity. Mouse F9 cells were grown in culture medium at 37ºC and 43ºC (heat shock treatment) treated or not with retinoic acid, and labeled in certain instances with 35 S-methionine. Membrane-bound polysomes of procollagen IV were then isolated. Immunoprecipitation and Western blot analysis were performed using polyclonal antibodies against collagen IV, Hsp47 and Sec61a. Under retinoic acid-untreated conditions, F9 cells produced undetectable amounts of Sec61a. Sec61a, Hsp47 and type IV collagen levels were increased after retinoic acid treatment. Heat shock treatment did not alter Sec61a levels, suggesting that Sec61a production is probably not affected by heat shock. These data indicate that the enhanced production of Sec61a in retinoic acid-induced F9 teratocarcinoma cells parallels the increased synthesis of Hsp47 and collagen type IV.
Nascent procollagen peptides and other secretory proteins are transported across the endoplasmic reticulum (RE) membrane through a protein-conducting channel called the translocon. Sec61 alpha, a multispanning membrane translocon protein, has been implicated as essential for translocation of polypeptides chains into the cisterns of the ER. However, it is not known whether Sec61 alpha is ubiquitously expressed in collagen producing teratocarcinoma cells. Furthermore, the production, expression, and utilization of Sec61 alpha may depend on the cell differentiation stage. Stem cells from many cultured teratocarcinoma cell lines such as F9 and P19 cells are capable of differentiation in response to low retinoic acid concentrations. This differentiation of the tumorigenic stem cells results in tumorigenicity loss. For this study, mouse F9 and P19 teratocarcinoma cells were grown in culture medium treated with or without retinoic acid. Expression of Sec61 alpha was determined by reverse trancriptase polimerase chain reaction (RT-PCR). In untreated conditions, F9 cells expressed undetected Sec61 alpha amounts. It was also demonstrated that Sec61 alpha expression is stimulated in F9 cells after retinoic acid treatment for 72 hours. No changes were found in Sec61 alpha expression in P19 cells after retinoic acid treatment. These data indicate that the expression of Sec61 alpha is enhanced with retinoic acid induced differentiation of F9 teratocarcinoma cells.
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