title: Hsp47 and cyclophilin B traverse the endoplasmic reticulum with procollagen into pre-Golgi intermediate vesicles. A role for Hsp47 and cyclophilin B in the export of procollagen from the endoplasmic reticulum.
abstract: Hsp47 and cyclophilin B (CyPB) are residents of the endoplasmic reticulum (ER). Both of these proteins are closely associated with polysome-associated alpha 1(I) procollagen chains. Hsp47 possesses chaperone properties early during the translation of procollagen while the cis/trans-isomerase properties of CyPB facilitate procollagen folding. In this report, we further investigate the interaction of these proteins with procollagen I during export from the ER. To inhibit vesicular budding and retain procollagen within the ER, cells were treated with the heterotrimeric G protein inhibitor mastoparan or calphostin C, a specific inhibitor of diacylglycerol/phorbol ester binding proteins. To arrest procollagen in pre-Golgi intermediate vesicles, cells were treated with guanosine 5'-3-O-(thio)triphosphate. Pulse-chase experiments of cells labeled with [35S]methionine followed by immunoprecipitation during the chase period with anti-procollagen, anti-Hsp47, and anti-CyPB antibodies were performed to reveal the relationship between Hsp47/CyPB/procollagen I. The distribution of procollagen, Hsp47, and CyPB to the ER and/or pre-Golgi vesicles was verified by immunofluorescence. Hsp47 and CyPB remained associated with procollagen retained within the ER. Hsp47 and CyPB were also associated with procollagen exported from the ER into pre-Golgi intermediate vesicles. Treatment of cells with cyclosporin A diminished the levels of CyPB bound to procollagen and diminished the rate of Hsp47 released from procollagen and the rate of procollagen secretion, suggesting that Hsp47 release from procollagen may be driven by helix formation. Also, these studies suggest that Hsp47 may resemble protein disulfide isomerase and possess both chaperone and anti-chaperone properties. During translation, high levels of Hsp47 are seen to limit protein aggregation and facilitate chain registration. Later, Hsp47 and/or CyPB and protein disulfide isomerase act as anti-chaperones and provide the basis for concentration of procollagen for ER export.
authors.affiliation: Department of Pathology, School of Dentistry, University of Maryland at Baltimore 21201, USA.
authors.forename: T
authors.initials: T
authors.lastname: Smith
authors.forename: L R
authors.initials: LR
authors.lastname: Ferreira
authors.forename: C
authors.initials: C
authors.lastname: Hebert
authors.forename: K
authors.initials: K
authors.lastname: Norris
authors.forename: J J
authors.initials: JJ
authors.lastname: Sauk
mesh_terms: D000591:Amino Acid Isomerases / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism*; D002451:Cell Compartmentation; D002460:Cell Line; D003432:Cross-Linking Reagents; D021983:Cyclophilins; D016572:Cyclosporine / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000494:pharmacology; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins / Q000378:metabolism*; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D008969:Molecular Sequence Data; D009281:Naphthalenes / Q000494:pharmacology; D010455:Peptides; D019696:Peptidylprolyl Isomerase; D011132:Polyribosomes / Q000378:metabolism; D011233:Precipitin Tests; D011347:Procollagen / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D014176:Protein Biosynthesis; D011994:Recombinant Proteins / Q000378:metabolism; D014862:Wasp Venoms / Q000494:pharmacology
chemical_list: D002352:Carrier Proteins; D003432:Cross-Linking Reagents; D050958:HSP47 Heat-Shock Proteins; D006360:Heat-Shock Proteins; D036341:Intercellular Signaling Peptides and Proteins; D009281:Naphthalenes; D010455:Peptides; D011347:Procollagen; D011994:Recombinant Proteins; C494661:Serpinh1 protein, mouse; D014862:Wasp Venoms; C081786:cyclophilin B; D016244:Guanosine 5'-O-(3-Thiotriphosphate); C037500:mastoparan; D016572:Cyclosporine; D000591:Amino Acid Isomerases; D021983:Cyclophilins; D019696:Peptidylprolyl Isomerase; C058819:calphostin C