Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.
Clathrin heavy chain functions in sorting and secretion of lysosomal enzymes in Dictyostelium discoideum.
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DOI：
10.1083/jcb.126.2.343
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发表时间：
1994-07
期刊：
影响因子：
--
通讯作者：
O'Halloran TJ
中科院分区：
文献类型：
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作者：
Ruscetti T;Cardelli JA;Niswonger ML;O'Halloran TJ
网格蛋白重链是网格蛋白包被囊泡的主要组成部分，在真核细胞中具有选择性膜运输功能。为了获得稳定的网格蛋白重链缺陷细胞系，我们破坏了盘状盘基钢鞘的网格蛋白重链基因（chcA）。网格蛋白缺失突变体的胞饮量测量显示，液相标记物的内化有四到五倍的缺陷。一旦内化，这些标记以野生型的速率循环到突变细胞的细胞表面。我们还探讨了网格蛋白重链在溶酶体酶运输中的作用。脉冲追踪分析显示，网格蛋白缺失的细胞将大多数α -甘露糖苷酶加工成成熟的形式，然而，大约20-25%的前体分子保持未分裂，被错误分类，并通过组成分泌途径迅速分泌。剩余的细胞内α -甘露糖苷酶成功靶向成熟溶酶体。标准分泌试验表明，与生长培养基中的对照细胞相比，无网格蛋白的细胞分泌α -甘露糖苷酶的速率明显降低。有趣的是，另一种溶酶体酶，酸性磷酸酶的分泌率在无网格蛋白和野生型细胞中相似。像野生型细胞一样，网格蛋白缺失突变体对饥饿条件的反应是增加溶酶体酶的分泌。我们对突变细胞的研究为网格蛋白重链在(a)质膜液体内化中的作用提供了活体证据；(b)水解酶前体从组成分泌途径到溶酶体途径的分选；(c)溶酶体向细胞外空间分泌成熟的水解酶。
The clathrin heavy chain is a major component of clathrin-coated vesicles that function in selective membrane traffic in eukaryotic cells. We disrupted the clathrin heavy chain gene (chcA) in Dictyostelium discoideum to generate a stable clathrin heavy chain- deficient cell line. Measurement of pinocytosis in the clathrin-minus mutant revealed a four-to five-fold deficiency in the internalization of fluid-phase markers. Once internalized, these markers recycled to the cell surface of mutant cells at wild-type rates. We also explored the involvement of clathrin heavy chain in the trafficking of lysosomal enzymes. Pulse chase analysis revealed that clathrin-minus cells processed most alpha-mannosidase to mature forms, however, approximately 20-25% of the precursor molecules remained uncleaved, were missorted, and were rapidly secreted by the constitutive secretory pathway. The remaining intracellular alpha-mannosidase was successfully targeted to mature lysosomes. Standard secretion assays showed that the rate of secretion of alpha-mannosidase was significantly less in clathrin-minus cells compared to control cells in growth medium. Interestingly, the secretion rates of another lysosomal enzyme, acid phosphatase, were similar in clathrin-minus and wild-type cells. Like wild-type cells, clathrin-minus mutants responded to starvation conditions with increased lysosomal enzyme secretion. Our study of the mutant cells provide in vivo evidence for roles for the clathrin heavy chain in (a) the internalization of fluid from the plasma membrane; (b) sorting of hydrolase precursors from the constitutive secretory pathway to the lysosomal pathway; and (c) secretion of mature hydrolases from lysosomes to the extracellular space.