Smith T, Ferreira L R, Hebert C, Norris K, Sauk J J
Department of Pathology, School of Dentistry, University of Maryland at Baltimore 21201, USA.
J Biol Chem. 1995 Aug 4;270(31):18323-8. doi: 10.1074/jbc.270.31.18323.
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.
热休克蛋白47（Hsp47）和亲环蛋白B（CyPB）是内质网（ER）中的驻留蛋白。这两种蛋白都与多核糖体相关的α1（I）前胶原链密切相关。在原胶原翻译早期，Hsp47具有分子伴侣特性，而CyPB的顺/反异构酶特性则促进原胶原折叠。在本报告中，我们进一步研究了这些蛋白在从内质网输出过程中与I型原胶原的相互作用。为了抑制囊泡出芽并将原胶原保留在内质网中，用异三聚体G蛋白抑制剂马斯托帕兰或钙磷蛋白C（一种二酰基甘油/佛波酯结合蛋白的特异性抑制剂）处理细胞。为了将原胶原阻滞在前高尔基体中间囊泡中，用鸟苷5'-3-O-（硫代）三磷酸处理细胞。对用[35S]甲硫氨酸标记的细胞进行脉冲追踪实验，随后在追踪期用抗原胶原、抗Hsp47和抗CyPB抗体进行免疫沉淀，以揭示Hsp47/CyPB/原胶原I之间的关系。通过免疫荧光验证原胶原、Hsp47和CyPB在内质网和/或前高尔基体囊泡中的分布。Hsp47和CyPB仍然与保留在内质网中的原胶原相关。Hsp47和CyPB也与从内质网输出到前高尔基体中间囊泡中的原胶原相关。用环孢素A处理细胞可降低与原胶原结合的CyPB水平，并降低Hsp47从原胶原释放的速率以及原胶原分泌的速率，这表明Hsp47从原胶原的释放可能由螺旋形成驱动。此外，这些研究表明Hsp47可能类似于蛋白质二硫键异构酶，兼具分子伴侣和抗分子伴侣特性。在翻译过程中，高水平的Hsp47可限制蛋白质聚集并促进链的对齐。之后，Hsp47和/或CyPB以及蛋白质二硫键异构酶作为抗分子伴侣发挥作用，为内质网输出的原胶原浓缩提供基础。
