An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.
An intralysosomal hsp70 is required for a selective pathway of lysosomal protein degradation.
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DOI：
10.1083/jcb.137.4.825
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发表时间：
1997-05-19
期刊：
影响因子：
--
通讯作者：
Dice JF
中科院分区：
文献类型：
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作者：
Agarraberes FA;Terlecky SR;Dice JF
以前的研究表明，73 kD（hsc 73）的热休克同源蛋白（hsc）在刺激溶酶体途径的蛋白水解，是选择性的特定的胞质蛋白。该途径通过在汇合培养的人成纤维细胞中的血清剥夺来激活。我们现在显示，使用间接免疫荧光和激光扫描共聚焦显微镜，热休克蛋白（HSP）的70 kD的家庭（HSP 70）与溶酶体（ly-hsc 73）。命名为13 D3的mAb特异性识别hsc 73，并且该抗体与针对lgp 120（一种溶酶体标记蛋白）的抗体共定位。大多数，但不是全部，溶酶体含有ly-hsc 73，这些细胞器的形态学外观显着变化，以响应血清撤回;点状溶酶体融合形成小管。基于对胰蛋白酶消化的敏感性和分离的溶酶体和分离的溶酶体膜的二维电泳后的免疫印迹分析，大多数ly-hsc 73位于溶酶体腔内。我们通过用[3 H]亮氨酸放射性标记细胞蛋白质，然后允许细胞内吞过量的mAb 13 D3，然后在存在和不存在血清的情况下测量蛋白质降解，来确定ly-hsc 73的功能重要性。内吞的mAb 13 D3完全抑制了血清剥夺引起的蛋白质降解增加，而在血清存在下维持的细胞中的蛋白质降解不受影响。内吞的mAb 13 D3不影响内吞的[3 H]RNase A的溶酶体内消化。这些结果表明ly-hsc 73是溶酶体内蛋白质消化前降解途径中的一个步骤所必需的，最有可能是用于底物蛋白的输入。
Previous studies have implicated the heat shock cognate (hsc) protein of 73 kD (hsc73) in stimulating a lysosomal pathway of proteolysis that is selective for particular cytosolic proteins. This pathway is activated by serum deprivation in confluent cultured human fibroblasts. We now show, using indirect immunofluorescence and laser scanning confocal microscopy, that a heat shock protein (hsp) of the 70-kD family (hsp70) is associated with lysosomes (ly-hsc73). An mAb designated 13D3 specifically recognizes hsc73, and this antibody colocalizes with an antibody to lgp120, a lysosomal marker protein. Most, but not all, lysosomes contain ly-hsc73, and the morphological appearance of these organelles dramatically changes in response to serum withdrawal; the punctate lysosomes fuse to form tubules. Based on susceptibility to digestion by trypsin and by immunoblot analysis after two-dimensional electrophoresis of isolated lysosomes and isolated lysosomal membranes, most ly-hsc73 is within the lysosomal lumen. We determined the functional importance of the ly-hsc73 by radiolabeling cellular proteins with [3H]leucine and then allowing cells to endocytose excess mAb 13D3 before measuring protein degradation in the presence and absence of serum. The increased protein degradation in response to serum deprivation was completely inhibited by endocytosed mAb 13D3, while protein degradation in cells maintained in the presence of serum was unaffected. The intralysosomal digestion of endocytosed [3H]RNase A was not affected by the endocytosed mAb 13D3. These results suggest that ly-hsc73 is required for a step in the degradative pathway before protein digestion within lysosomes, most likely for the import of substrate proteins.