title: Lipid-dependent bimodal MCL1 membrane activity.
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.
authors.affiliation: Unidad de Biofisica, Centro Mixto Consejo Superior de Investigaciones Científicas (CSIC)-Euskal Herriko Unibertsitatea/Universidad del Pais Vasco (EHU/UPV) , Barrio Sarriena s/n, Leioa 48940, Spain.
authors.forename: Olatz
authors.initials: O
authors.lastname: Landeta
authors.forename: Juan
authors.initials: J
authors.lastname: Garcia Valero
authors.forename: Hector
authors.initials: H
authors.lastname: Flores-Romero
authors.forename: Itsasne
authors.initials: I
authors.lastname: Bustillo-Zabalbeitia
authors.forename: Ane
authors.initials: A
authors.lastname: Landajuela
authors.forename: Miguel
authors.initials: M
authors.lastname: Garcia-Porras
authors.forename: Oihana
authors.initials: O
authors.lastname: Terrones
authors.forename: Gorka
authors.initials: G
authors.lastname: Basañez
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D051023:BH3 Interacting Domain Death Agonist Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D002308:Cardiolipins / Q000737:chemistry* / Q000378:metabolism; D002784:Cholesterol / Q000737:chemistry* / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D006801:Humans; D008081:Liposomes / Q000737:chemistry / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D053078:Membrane Potential, Mitochondrial / Q000235:genetics; D051379:Mice; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D064549:Myeloid Cell Leukemia Sequence 1 Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010716:Phosphatidylinositols / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D051028:bcl-2-Associated X Protein / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: C494826:BAX protein, human; C000595707:BCL2 protein, human; D051023:BH3 Interacting Domain Death Agonist Protein; C494814:BID protein, human; D002308:Cardiolipins; D008081:Liposomes; C579095:MCL1 protein, human; D008563:Membrane Lipids; D064549:Myeloid Cell Leukemia Sequence 1 Protein; D010716:Phosphatidylinositols; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D051028:bcl-2-Associated X Protein; D002784:Cholesterol