Ripley C R, Bienkowski R S
Department of Biology, Queens College of the City University of New York, Flushing 11367, USA.
Exp Cell Res. 1997 Oct 10;236(1):147-54. doi: 10.1006/excr.1997.3707.
A significant amount of newly synthesized collagen is degraded intracellularly rather than secreted, but there is controversy about whether this process occurs in the lysosomes. We addressed this problem using confocal microscopy and immunofluorescence imaging to study the distribution of procollagen I in the Golgi and the lysosome/endosome system of cultured human fibroblasts. Cells were incubated under basal conditions and then permeabilized and exposed to fluorescently tagged probes for procollagen, Golgi markers (Helix pomatia binding protein or beta-coatamer protein), and lysosome/endosome markers (cathepsin B or LAMP-2). Strong signals for procollagen codistributed with the Golgi and lysosome/endosome markers. Of note, many structures were positive for procollagen and lysosome/endosome markers but not for Golgi markers. When cells were incubated with the proline analog cis-hydroxyproline, which inhibits correct triple helix formation and increases intracellular degradation, the amount of procollagen codistributing with the lysosome/endosome markers increased greatly. Similar results were obtained in I-cells, which do not have functioning lysosomal hydrolases. These findings strongly indicate that the lysosome/endosome system participates in the intracellular degradation of newly synthesized procollagen and that trafficking of procollagen to the lysosome/endosome system does not depend on the cells having active lysosomal hydrolases. We present a model that integrates our findings with other work and resolves inconsistencies in the literature. This model postulates the existence of three separate degradation paths for newly synthesized procollagen. In addition to the endosome/lysosome system, degradation also takes place in the proximal region of the secretory pathway such as the endoplasmic reticulum, cis-Golgi network, or cis-Golgi and in a distal region of the secretory pathway such as the trans-Golgi or trans-Golgi network.
大量新合成的胶原蛋白在细胞内被降解而非分泌，但关于这一过程是否发生在溶酶体中存在争议。我们利用共聚焦显微镜和免疫荧光成像技术研究了原胶原蛋白I在培养的人成纤维细胞的高尔基体以及溶酶体/内体系统中的分布，从而解决了这个问题。细胞在基础条件下孵育，然后进行通透处理，并暴露于针对原胶原蛋白、高尔基体标记物（蜗牛凝集素结合蛋白或β-包被蛋白）以及溶酶体/内体标记物（组织蛋白酶B或溶酶体相关膜蛋白2）的荧光标记探针。原胶原蛋白的强信号与高尔基体和溶酶体/内体标记物共分布。值得注意的是，许多结构对原胶原蛋白和溶酶体/内体标记物呈阳性，但对高尔基体标记物呈阴性。当细胞与脯氨酸类似物顺式羟脯氨酸孵育时，顺式羟脯氨酸会抑制正确的三螺旋形成并增加细胞内降解，与溶酶体/内体标记物共分布的原胶原蛋白量大幅增加。在缺乏功能性溶酶体水解酶的I型细胞中也获得了类似结果。这些发现有力地表明，溶酶体/内体系统参与了新合成原胶原蛋白的细胞内降解，并且原胶原蛋白向溶酶体/内体系统的运输并不依赖于细胞具有活性溶酶体水解酶。我们提出了一个模型，将我们的发现与其他研究工作整合起来，并解决了文献中的不一致之处。该模型假定新合成的原胶原蛋白存在三条独立的降解途径。除了内体/溶酶体系统外，降解还发生在分泌途径的近端区域，如内质网、顺式高尔基体网络或顺式高尔基体，以及分泌途径的远端区域，如反式高尔基体或反式高尔基体网络。
