The cytosolic entry of diphtheria toxin catalytic domain requires a host-cell cytosolic translocation factor complex

被引:150
作者
Ratts, R
Zeng, HY
Berg, EA
Blue, C
McComb, ME
Costello, CE
VanderSpek, JC
Murphy, JR
机构
[1] Boston Univ, Sch Med, Dept Biochem, Boston, MA 02118 USA
[2] Boston Univ, Sch Med, Dept Med, Sect Mol Med, Boston, MA 02118 USA
[3] Boston Univ, Sch Med, Cell & Mol Biol Program, Boston, MA 02118 USA
[4] Boston Univ, Sch Med, Mass Spectrometry Resource, Boston, MA 02118 USA
[5] Boston Univ, Sch Med, Dept Microbiol, Boston, MA 02118 USA
关键词
endosome; Hsp; 90; thioredoxin reductase; geldanamycin; radicicol;
D O I
10.1083/jcb.200210028
中图分类号
Q2 [细胞生物学];
学科分类号
071009 ; 090102 ;
摘要
In vitro delivery of the diphtheria toxin catalytic (C) domain from the lumen of purified early endosomes to the external milieu requires the addition of both ATP and a cytosolic translocation factor (CTF) complex. Using the translocation of C-domain ADP-ribosyltransferase activity across the endosomal membrane as an assay, the CTF complex activity was 650-800-fold purified from human T cell and yeast extracts, respectively. The chaperonin heat shock protein (Hsp) 90 and thioredoxin reductase were identified by mass spectrometry sequencing in CTF complexes purified from both human T cell and yeast. Further analysis of the role played by these two proteins with specific inhibitors, both in the in vitro translocation assay and in intact cell toxicity assays, has demonstrated their essential role in the productive delivery of the C-domain from the lumen of early endosomes to the external milieu. These results confirm and extend earlier observations of diphtheria toxin C-domain unfolding and refolding that must occur before and after vesicle membrane translocation. in addition, results presented here demonstrate that thioredoxin reductase activity plays an essential role in the cytosolic release of the C-domain. Because analogous CTF complexes have been partially purified from mammalian and yeast cell extracts, results presented here suggest a common and fundamental mechanism for C-domain translocation across early endosomal membranes.
引用
收藏
页码:1139 / 1150
页数:12
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