The CHMP4b- and Src-docking sites in the Bro1 domain are autoinhibited in the native state of Alix.
The Bro1 domain of Alix [ALG-2 (apoptosis-linked gene 2)-interacting protein X], which plays important roles in endosomal sorting and multiple ESCRT (endosomal sorting complex required for transport)-linked processes, contains the docking sites for the ESCRT-III component CHMP4b (charged multivesicular body protein 4b) and the regulatory tyrosine kinase, Src. Although the structural bases for these docking sites have been defined by crystallography studies, it has not been determined whether these sites are available in the native state of Alix. In the present study, we demonstrate that these two docking sites are unavailable in recombinant Alix under native conditions and that their availabilities can be induced by detergents. In HEK (human embryonic kidney)-293 cell lysates, these two docking sites are not available in cytosolic Alix, but are available in membrane-bound Alix. These findings show that the native state of Alix does not have a functional Bro1 domain and predict that Alix's involvement in endosomal sorting and other ESCRT-linked processes requires an activation step that relieves the autoinhibition of the Bro1 domain.
Zhou
Xi
X
Department of Experimental Therapeutics, The University of Texas, M.D. Anderson Cancer Center, Houston, TX 77030, USA.
Pan
Shujuan
S
Sun
Le
L
Corvera
Joe
J
Lee
Yu-Chen
YC
Lin
Sue-Hwa
SH
Kuang
Jian
J
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001666:Binding Sites, Antibody; D002135:Calcium-Binding Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D002478:Cells, Cultured; D015536:Down-Regulation / Q000502:physiology; D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes / Q000276:immunology; D006706:Homeostasis / Q000502:physiology; D006801:Humans; D008954:Models, Biological; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry / Q000276:immunology; D011489:Protein Denaturation / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D017386:Sequence Homology, Amino Acid; D014981:Xenopus; D029867:Xenopus Proteins / Q000737:chemistry / Q000276:immunology; D019061:src-Family Kinases / Q000378:metabolism*
C478397:CHMP4B protein, human; D002135:Calcium-Binding Proteins; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D056827:Endosomal Sorting Complexes Required for Transport; D000939:Epitopes; C117750:PDCD6IP protein, Xenopus; C117020:PDCD6IP protein, human; D010750:Phosphoproteins; D029867:Xenopus Proteins; D019061:src-Family Kinases