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Molecular and Cellular Biology, February 2004, p. 1758-1768, Vol. 24, No. 4
0270-7306/04/$08.00+0     DOI: 10.1128/MCB.24.4.1758-1768.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

TRAM2 Protein Interacts with Endoplasmic Reticulum Ca²⁺ Pump Serca2b and Is Necessary for Collagen Type I Synthesis

Branko Stefanovic,^1* Lela Stefanovic,¹ Bernd Schnabl,² Ramon Bataller,³ and David A. Brenner^3,4

Department of Biomedical Science, College of Medicine, Florida State University, Tallahassee, Florida,¹ Department of Internal Medicine I, University of Regensburg, Regensburg, Germany,² Departments of Medicine, Biochemistry, and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina,³ Department of Medicine, Columbia University College of Physicians and Surgeons, New York, New York 10032-3784⁴

Received 30 April 2003/ Returned for modification 10 June 2003/ Accepted 3 November 2003

Cotranslational insertion of type I collagen chains into the lumen of the endoplasmic reticulum (ER) and their subsequent folding into a heterotrimeric helix is a complex process which requires coordinated action of the translation machinery, components of translocons, molecular chaperones, and modifying enzymes. Here we describe a role for the protein TRAM2 in collagen type I expression in hepatic stellate cells (HSCs) and fibroblasts. Activated HSCs are collagen-producing cells in the fibrotic liver. Quiescent HSCs produce trace amounts of type I collagen, while upon activation collagen synthesis increases 50- to 70-fold. Likewise, expression of TRAM2 dramatically increases in activated HSCs. TRAM2 shares 53% amino acid identity with the protein TRAM, which is a component of the translocon. However, TRAM2 has a C terminus with only a 15% identity. The C-terminal part of TRAM2 interacts with the Ca²⁺ pump of the ER, SERCA2b, as demonstrated in a Saccharomyces cerevisiae two-hybrid screen and by immunoprecipitations in human cells. TRAM2 also coprecipitates with anticollagen antibody, suggesting that these two proteins interact. Deletion of the C-terminal part of TRAM2 inhibits type I collagen synthesis during activation of HSCs. The pharmacological inhibitor of SERCA2b, thapsigargin, has a similar effect. Depletion of ER Ca²⁺ with thapsigargin results in inhibition of triple helical collagen folding and increased intracellular degradation. We propose that TRAM2, as a part of the translocon, is required for the biosynthesis of type I collagen by coupling the activity of SERCA2b with the activity of the translocon. This coupling may increase the local Ca²⁺ concentration at the site of collagen synthesis, and a high Ca²⁺ concentration may be necessary for the function of molecular chaperones involved in collagen folding.

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* Corresponding author. Mailing address: Department of Biomedical Science, Florida State University College of Medicine, Tallahassee, FL 32306. Phone: (850) 644-7600. Fax: (850) 644-8924. E-mail: branko.stefanovic{at}med.fsu.edu.

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Molecular and Cellular Biology, February 2004, p. 1758-1768, Vol. 24, No. 4
0022-538X/04/$08.00+0     DOI: 10.1128/MCB.24.4.1758-1768.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

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