ABSTRACT
Increasing evidence indicates that the mitochondrial lipid membrane environment directly modulates the BCL2 family protein function, but the underlying mechanisms are still poorly understood. Here, we used minimalistic reconstituted systems to examine the influence of mitochondrial lipids on MCL1 activity and conformation. Site-directed mutagenesis and fluorescence spectroscopic analyses revealed that the BCL2 homology region of MCL1 (MCL1ΔNΔC) inhibits permeabilization of MOM-like membranes exclusively via canonical BH3-into-groove interactions with both cBID-like activators and BAX-like effectors. Contrary to currently popular models, MCL1ΔNΔC did not require becoming embedded into the membrane to inhibit membrane permeabilization, and interaction with cBID was more productive for MCL1ΔNΔC inhibitory activity than interaction with BAX. We also report that membranes rich in cardiolipin (CL), but not phosphatidylinositol (PI), trigger a profound conformational change in MCL1ΔNΔC leading to membrane integration and unleashment of an intrinsic lipidic pore-forming activity of the molecule. Cholesterol (CHOL) reduces both the conformational change and the lipidic pore-forming activity of MCL1ΔNΔC in CL-rich membranes, but it does not affect the interaction of MCL1ΔNΔC with proapoptotic partners in MOM-like liposomes. In addition, we identified MCL1α5 as the minimal domain of the protein responsible for its membrane-permeabilizing function both in model membranes and at the mitochondrial level. Our results provide novel mechanistic insight into MCL1 function in the context of a membrane milieu and add significantly to a growing body of evidence supporting an active role of mitochondrial membrane lipids in BCL2 protein function.
Subject(s)
Cardiolipins/chemistry , Cholesterol/chemistry , Membrane Lipids/chemistry , Myeloid Cell Leukemia Sequence 1 Protein/metabolism , Phosphatidylinositols/chemistry , Amino Acid Sequence , Animals , BH3 Interacting Domain Death Agonist Protein/chemistry , BH3 Interacting Domain Death Agonist Protein/genetics , BH3 Interacting Domain Death Agonist Protein/metabolism , Cardiolipins/metabolism , Cholesterol/metabolism , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , HeLa Cells , Humans , Liposomes/chemistry , Liposomes/metabolism , Membrane Lipids/metabolism , Membrane Potential, Mitochondrial/genetics , Mice , Mitochondria/chemistry , Mitochondria/genetics , Mitochondria/metabolism , Models, Molecular , Molecular Sequence Data , Mutagenesis, Site-Directed , Myeloid Cell Leukemia Sequence 1 Protein/chemistry , Myeloid Cell Leukemia Sequence 1 Protein/genetics , Phosphatidylinositols/metabolism , Protein Structure, Tertiary , Proto-Oncogene Proteins c-bcl-2/chemistry , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Alignment , bcl-2-Associated X Protein/chemistry , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is required for mitochondrial fission in healthy and apoptotic cells. Drp1 activation is a complex process that involves translocation from the cytosol to the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, instead of the lipid-interacting Pleckstrin Homology (PH) domain present in the dynamins, Drp1 contains the so-called B insert or variable domain that has been suggested to play an important role in Drp1 regulation. Different proteins have been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 acquires its fully functional status remains poorly understood. We found that Drp1 can interact with pure lipid bilayers enriched in the mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous study, we now explore the specificity and functional consequences of this interaction. We show that a four lysine module located within the B insert of Drp1 interacts preferentially with CL over other anionic lipids. This interaction dramatically enhances Drp1 oligomerization and assembly-stimulated GTP hydrolysis. Our results add significantly to a growing body of evidence indicating that CL is an important regulator of many essential mitochondrial functions.
