Rudick V L, Rudick M J, Munoz-Medellin D A, Brun-Zinkernagel A M, Chang I F
Department of Anatomy and Cell Biology, University of North Texas Health Science Center at Fort Worth 76107-2699.
Cell Mol Biol Res. 1993;39(8):773-88.
Transfected Madin Darby canine kidney (MDCK) cells (3A) expressing human growth hormone (hGH) contain twice as many Golgi stacks as untransfected cells. How MDCK cells, lacking a regulated pathway, deal with (over)expression of a protein hormone, or any exogenous protein, has not been examined in detail. Since hGH constituted 10% of total secreted proteins, it was not apparent why Golgi amplification was needed, unless some enters a nonsecretory compartment. Studies were undertaken to determine hGH fate. By using an inhibitor of protein synthesis, or by analyzing pulse labeled immunoprecipitated hGH, 20-30% of hGH was shown to remain intracellular even after 4 h. That portion might be localized in the endosome/lysosome compartment, because it is post-Golgi. Immunoelectron microscopy with antibodies against hGH, clathrin, and cathepsin D demonstrated clathrin and hGH colocalized, as did hGH and cathepsin D. The latter were found in large vesicles, but no hGH appeared in lysosomes, due to its degradation. Analysis of isolated lysosome/endosomes revealed vesicles containing both hGH and cathepsin D, but more containing only cathepsin D. Endocytosis studies suggested the 3A basolateral endosomal compartment may be more capacious than normal. Thus, 3A Golgi amplification resulted in an expanded endosome compartment to accommodate secretory protein (over)expression.
表达人生长激素（hGH）的转染马-达二氏犬肾（MDCK）细胞（3A）所含的高尔基体堆叠数量是未转染细胞的两倍。MDCK细胞缺乏调节途径，其如何处理蛋白质激素或任何外源蛋白质的（过度）表达尚未得到详细研究。由于hGH占总分泌蛋白的10%，除非有一些进入非分泌区室，否则不清楚为何需要高尔基体扩增。开展了研究以确定hGH的命运。通过使用蛋白质合成抑制剂，或通过分析脉冲标记的免疫沉淀hGH，结果显示即使在4小时后仍有20%-30%的hGH保留在细胞内。那部分可能定位于内体/溶酶体区室，因为它是高尔基体后产物。用抗hGH、网格蛋白和组织蛋白酶D的抗体进行免疫电子显微镜观察显示，网格蛋白和hGH共定位，hGH和组织蛋白酶D也共定位。后者存在于大囊泡中，但由于hGH的降解，溶酶体中未出现hGH。对分离的溶酶体/内体的分析显示，囊泡中同时含有hGH和组织蛋白酶D，但更多的囊泡只含有组织蛋白酶D。内吞作用研究表明，3A基底外侧内体区室可能比正常情况更宽大。因此，3A高尔基体扩增导致内体区室扩大，以容纳分泌蛋白的（过度）表达。
