Evidence of proteolipid domain formation in an inner mitochondrial membrane mimicking model

Abstract

BackgroundMitochondrial creatine kinase (mtCK) is highly abundant in mitochondria; its quantity is equimolecular to the Adenylic Nucleotide Translocator and represents 1% of the mitochondrial proteins. It is a multitask protein localized in the mitochondria intermembrane space where it binds to the specific cardiolipin (CL) phospholipid. If mtCK was initially thought to be exclusively implicated in energy transfer between mitochondria and cytosol through a mechanism referred to as the phosphocreatine shuttle, several recent studies suggested an additional role in maintaining mitochondria membrane structure. MethodsTo further characterized mtCK binding process we used multiphoton excitation fluorescence microscopy coupled with Giant Unilamellar Vesicles (GUV) and laurdan as fluorescence probe. ResultsWe gathered structural and dynamical information on the molecular events occurring during the binding of mtCK to the mitochondria inner membrane.We present the first visualization of mtCK-induced CL segregation on a bilayer model forming micrometer-size proteolipid domains at the surface of the GUV. Those microdomains, which only occurred when CL is included in the lipid mixture, were accompanied by the formation of protein multimolecular assembly, vesicle clamping, and changes in both vesicle curvature and membrane fluidity ConclusionThose results highlighted the importance of the highly abundant mtCK in the lateral organization of the mitochondrial inner membrane. General significanceMicrodomains were induced in mitochondria-mimicking membranes composed of natural phospholipids without cholesterol and/or sphingolipids differing from the proposed cytoplasmic membrane rafts. Those findings as well as membrane curvature modification were discussed in relation with protein-membrane interaction and protein cluster involvement in membrane morphology.