A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.
A lysosomal targeting signal in the cytoplasmic tail of the beta chain directs HLA-DM to MHC class II compartments.
复制标题
DOI：
10.1083/jcb.131.2.351
复制
发表时间：
1995-10
期刊：
影响因子：
--
通讯作者：
Bonifacino JS
中科院分区：
文献类型：
--
作者：
Marks MS;Roche PA;van Donselaar E;Woodruff L;Peters PJ;Bonifacino JS
在人B细胞中，主要组织相容性复合体（MHC-II）的II类分子积聚在内体/溶酶体区室（MIIC）中，在其中它们可能遇到并结合肽。MHC-II肽结合所需的另一种分子HLA-DM（DM）也定位于MIIC。MIIC与内体系统的关系以及DM定位于MIIC的机制都不清楚。为了解决这些问题，在表达或不表达MHC-II的细胞中分析DM定位。DM α β异二聚体在转染的MHC-II阴性HeLa和NRK细胞中，在MHC-II相关不变链的情况下，通过免疫荧光显微镜定位于前溶酶体/溶酶体区室。为了鉴定潜在的靶向决定簇，我们分析了嵌合蛋白T-T-Mb的定位，其中鼠DM β（Mb）的胞质尾被附加到细胞表面蛋白Tac的内腔和跨膜结构域。像完整的DM，T-T-Mb定位于HeLa和NRK细胞的溶酶体区室，如通过免疫荧光和免疫电子显微镜所判断的。T-T-Mb在该隔室中通过被溶酶体蛋白水解抑制剂阻断的过程快速降解。DM β胞质尾区还介导抗Tac抗体从细胞表面内化并递送至溶酶体。从DM β胞质尾的酪氨酸基序，YTPL的删除，导致T-T-Mb的细胞表面表达和降解和内化的损失;丙氨酸扫描诱变表明，Y和L残基是这些功能的关键。类似地，全长DM β内相同Y残基的突变导致DM α β异二聚体的细胞表面表达。最后，通过免疫电镜将T-T-MB b定位于人B淋巴母细胞样细胞系中的MIIC。我们的研究结果表明，基序，YTPL，在DM的β链的胞质尾是足够的靶向溶酶体或MIIC。
In human B cells, class II molecules of the major histocompatibility complex (MHC-II) accumulate in an endosomal/lysosomal compartment, the MIIC, in which they may encounter and bind peptides. An additional molecule required for MHC-II peptide binding, HLA-DM (DM), has also been localized to the MIIC. Neither the relationship of the MIIC to the endosomal system nor the mechanisms by which DM localizes to the MIIC are understood. To address these issues, DM localization was analyzed in cells that do or do not express MHC-II. DM alpha beta heterodimers were localized in transfected MHC-II-negative HeLa and NRK cells, in the absence of the MHC-II-associated invariant chain, to a prelysosomal/lysosomal compartment by immunofluorescence microscopy. To identify a potential targeting determinant, we analyzed the localization of a chimeric protein, T-T-Mb, in which the cytoplasmic tail of murine DM beta (Mb) was appended to the lumenal and transmembrane domains of a cell surface protein, Tac. Like intact DM, T- T-Mb was localized to a lysosomal compartment in HeLa and NRK cells, as judged by immunofluorescence and immunoelectron microscopy. T-T-Mb was rapidly degraded in this compartment by a process that was blocked by inhibitors of lysosomal proteolysis. The DM beta cytoplasmic tail also mediated internalization of anti-Tac antibody from the cell surface and delivery to lysosomes. Deletion from the DM beta cytoplasmic tail of the tyrosine-based motif, YTPL, resulted in cell surface expression of T-T-Mb and a loss of both degradation and internalization; alanine scanning mutagenesis showed that the Y and L residues were critical for these functions. Similarly, mutation of the same Y residue within full- length DM beta resulted in cell surface expression of DM alpha beta heterodimers. Lastly, T-T-Mb was localized by immunoelectron microscopy to the MIIC in a human B lymphoblastoid cell line. Our results suggest that a motif, YTPL, in the cytoplasmic tail of the beta chain of DM is sufficient for targeting either to lysosomes or to the MIIC.