Subcellular localization, oligomerization, and ATP-binding of Caenorhabditis elegans CED-4.
Caspase family cell death proteases are activated during apoptosis through the oligomerization of caspase-binding "adapter" proteins. In the nematode Caenorhabditis elegans one adapter protein, CED-4, exists. Here we report an analysis of CED-4 protein expressed in insect Sf9 cells by infection with recombinant baculovirus. During expression, CED-4 assumed a perinuclear spherical or reticular localization where it was partly resistant to extraction with nonionic detergents. Both purified FLAG-CED-4 and GST-FLAG-CED-4 proteins were present in solution as large complexes. FLAG-CED-4 complexes were estimated by gel filtration to have a molecular weight of approximately 500 kDa to >1.2 MDa, while GST-FLAG-CED-4 complexes appeared somewhat smaller. Unlike its mammalian homologue Apaf-1, CED-4 exhibited a marked preference for ATP over dATP in filter binding studies and in competition experiments. ATP hydrolysis was required neither for complex stability nor for binding of CED-3. These features are likely to be relevant for CED-4's function as a caspase adapter.
Seiffert
Barbara M
BM
Institute for Medical Microbiology, Immunology, and Hygiene, Technische Universität München, Trogerstrasse 9, D-81675 Munich, Germany.
Vier
Juliane
J
Häcker
Georg
G
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000818:Animals; D001667:Binding, Competitive; D017173:Caenorhabditis elegans / Q000378:metabolism*; D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D020169:Caspases / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D015801:Helminth Proteins / Q000096:biosynthesis / Q000378:metabolism*; D007313:Insecta; D011485:Protein Binding; D011994:Recombinant Proteins / Q000378:metabolism
D029742:Caenorhabditis elegans Proteins; D002135:Calcium-Binding Proteins; C089224:Ced-4 protein, C elegans; D003432:Cross-Linking Reagents; D003902:Detergents; D015801:Helminth Proteins; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D020169:Caspases; C089223:ced-3 protein, C elegans