The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis
The Lysosome Pathway Degrades CD81 on the Cell Surface by Poly-ubiquitination and Clathrin-Mediated Endocytosis
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DOI：
10.1248/bpb.b19-01097
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发表时间：
2020-03-01
影响因子：
2
通讯作者：
Fujimuro, Masahiro
中科院分区：
文献类型：
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作者：
Hosokawa, Kohei;Ishimaru, Hanako;Fujimuro, Masahiro
CD81是哺乳动物中高度保守的四跨膜蛋白，广泛表达于多种组织中。它属于四联蛋白家族，与各种细胞表面膜蛋白形成复合物。它还在细胞迁移和b细胞活化中发挥作用，这是由CD81与CD19、CD21和b细胞受体络合诱导的。因此，CD81被认为在调节细胞功能和命运中起着关键作用。然而，对CD81的降解机制知之甚少。在这里，我们发现质膜上的CD81通过内吞作用被溶酶体途径降解。用流式细胞术分析CD81在蛋白酶体抑制剂（乳酸丝素）和溶酶体抑制剂（氯喹和巴菲霉素A1）处理HEK293T细胞中的表达水平。溶酶体抑制剂可增加细胞表面CD81的表达，而乳酸菌素不能增加细胞表面CD81的表达。下拉实验表明，CD81在降解前与K63和k29连接的多泛素链结合，多泛素化位点为CD81 n端胞内结构域的Lys8。此外，突变型CD81中Lys8被丙氨酸（Ala）取代，与野生型相比，CD8I的半衰期延长。在正常细胞中，CD81主要定位在质膜上，但在氯喹处理的细胞中，CD81也与溶酶体LAMPI和早期内体EEA1共定位。此外，一种网格蛋白介导的内吞抑制剂氯丙嗪稳定了CD8I在细胞表面的表达。因此，我们证明CD8I通过网格蛋白介导的内吞作用内化，随后通过溶酶体途径降解，这需要K63和k29相关的CD81多泛素化。
CD81 is a highly conserved four-transmembrane protein in mammals and widely expressed on many tissues. It belongs to the tetraspanin family and forms complexes with various cell surface membrane proteins. It also functions in cell migration and B-cell activation, which is induced by CD81 complexing with CD19, CD21 and the B-cell receptor. Thus, CD81 is thought to play a key role in regulating cell function and fate. However, little is known about the degradation mechanism of CD81. Here we found that CD81 on the plasma membrane is degraded by the lysosome pathway via endocytosis. The expression levels of CD81 in HEK293T cells treated with a proteasome inhibitor (lactacystin) and lysosome inhibitors (chloroquine and bafilomycin A1) were analyzed by flow cytometry. The expression of CD81 on the cell surface was increased by the lysosome inhibitors, but not lactacystin. A pulldown assay revealed that CD81 was conjugated with a K63- and K29-linked poly-ubiquitin chain before its degradation, and the poly-ubiquitination site was Lys8 at the N-terminal intracellular domain of CD81. Furthermore, mutant CD81, in which Lys8 was substituted with alanine (Ala), extended the CD8I half-life compared with wildtype. CD81 was mainly localized on the plasma membrane in normal cells, but also co-localized with lysosomal LAMPI and early endosomal EEA1 in chloroquine-treated cells. Furthermore, a clathrin-mediated endocytosis inhibitor, chlorpromazine, stabilized CD8I expression on the cell surface. Hence, we demonstrated that CD8I is internalized by clathrin-mediated endocytosis and subsequently degraded via a lysosome pathway requiring the K63- and K29-linked poly-ubiquitination of CD81.