The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B
The penta-EF-hand protein ALG-2 interacts directly with the ESCRT-I component TSG101, and Ca2+-dependently co-localizes to aberrant endosomes with dominant-negative AAA ATPase SKD1/Vps4B
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DOI：
10.1042/bj20050398
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发表时间：
2005-11-01
影响因子：
4.1
通讯作者：
Maki, M
中科院分区：
文献类型：
--
作者：
Katoh, K;Suzuki, H;Maki, M
ALG-2（aptosis-linked gene 2）是属于PEF（penta-EF-hand）蛋白家族的钙结合蛋白。阿利克斯（ALG-2-interacting protein X）/AIPI（ALG-2- interacting protein 1）是其结合配偶体之一，与TSG 101和CHMP 4（charged multisecular body protein 4）相互作用，TSG 101和CHMP 4分别是ESCRT-I（转运所需的内体分选复合物1）和ESCRT-III的组分。在本研究中，我们调查了ALG-2和ESCRT-I之间的关联。通过使用HEK-293 T（人胚肾293 T）细胞裂解物的GST（谷胱甘肽S-转移酶）下拉测定，内源性TSG 101和其他两种外源表达的ESCRT-I组分[hVps 28（人空泡蛋白分选28）和hVps 37 A]显示在Ca 2+存在下与GST-ALG-2缔合。然而，通过酵母双杂交测定，在三种ESCRT-1组分中仅观察到与TSG 101的正相互作用，表明ALG-2通过TSG 101间接地与hVps 28和hVps 37 A缔合。使用TSG 101的各种缺失突变体，通过GST-下拉测定发现中心PRR（富含脯氨酸的区域）足以与ALG-2相互作用。通过使用生物素标记的ALG-2作为探针的覆盖测定证明ALG-2与TSG 101 PRR的直接结合。在对过表达GFP（绿色荧光蛋白）融合的ATP酶缺陷型显性阴性形式的SKD 1/Vps 4 B（GFP-SKD 1（E235 Q））的HeLa细胞的免疫荧光显微镜分析中，ALG-2在核周区域表现出点状分布，并与GFP-SKD 1（E235 Q）共定位于异常的核内体。这种点状分布的ALG-2显着减少了HeLa细胞与膜渗透性钙螯合剂的治疗。此外，ALG-2的Ca 2+结合缺陷突变体：不与GFP-SKD 1（E235 Q）共定位。我们的研究结果表明，ALG-2可能作为一个Ca 2+依赖的辅助蛋白的内体分选机直接与TSG 101以及与阿利克斯相互作用。
ALG-2 (apoptosis-linked gene 2) is a Ca2+-binding protein that belongs to the PEF (penta-EF-hand) protein family. Alix (ALG-2-interacting protein X)/AIPI (ALG-2- interacting protein 1), one of its binding partners, interacts with TSG101 and CHMP4 (charged multivesicular body protein 4), which are components of ESCRT-I (endosomal sorting complex required for transport 1) and ESCRT-III respectively. In the present study, we investigated the association between ALG-2 and ESCRT-I. By a GST (glutathione S-transferase) pull-down assay using HEK-293T (human embryonic kidney 293T) cell lysates, endogenous TSG101 and two other exogenously expressed ESCRT-I components [hVps28 (human vacuolar protein sorting 28) and hVps37A] were shown to associate with GST-ALG-2 in the presence of Ca2+. By the yeast two-hybrid assay, however, a positive interaction was observed with only TSG101 among the three ESCRT-I components, suggesting that ALG-2 associates with hVps28 and hVps37A indirectly through TSG101 Using various deletion mutants of TSG101, the central PRR (proline-rich region) was found to be sufficient for interaction with ALG-2 by the GST-pull-down assay. Direct binding of ALG-2 to the TSG 101 PRR was demonstrated by an overlay assay using biotin-labelled ALG-2 as a probe. In immunofluorescence microscopic analysis of HeLa cells that overexpressed a GFP (green fluorescent protein)-fused ATPase-defective dominant-negative form of SKD 1/Vps4B (GFP-SKD1(E235Q)), ALG-2 exhibited a punctate distribution at the perinuclear area and co-localized with GFP-SKD1(E235Q) to aberrant endosomes. This punctate distribution of ALG-2 was markedly diminished by treatment of HeLa cells with a membrane-permeant Ca2+ chelator. Moreover, a Ca2+-binding-defective mutant of ALG-2 :did not co-localize with GFP-SKD1(E235Q). Our findings suggest that ALG-2 may function as a Ca2+-dependent accessory protein of the endosomal sorting machinery by interacting directly with TSG101 as well as with Alix.