RESUMEN
Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission.
Asunto(s)
Cardiolipinas/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Dinámicas Mitocondriales/fisiología , Secuencias de Aminoácidos , Sitios de Unión , Dinaminas/genética , Humanos , Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/genética , Proteínas Intrínsecamente Desordenadas/metabolismo , Espectroscopía de Resonancia Magnética , Mitocondrias/metabolismo , Membranas Mitocondriales/metabolismo , Membranas Mitocondriales/patología , Mitofagia , Mutación , Unión Proteica , Conformación ProteicaRESUMEN
The mechanochemical GTPase dynamin-related protein 1 (Drp1) catalyzes mitochondrial and peroxisomal fission, but the regulatory mechanisms remain ambiguous. Here we find that a conserved, intrinsically disordered, six-residue Short Linear Motif at the extreme Drp1 C-terminus, named CT-SLiM, constitutes a critical allosteric site that controls Drp1 structure and function in vitro and in vivo. Extension of the CT-SLiM by non-native residues, or its interaction with the protein partner GIPC-1, constrains Drp1 subunit conformational dynamics, alters self-assembly properties, and limits cooperative GTP hydrolysis, surprisingly leading to the fission of model membranes in vitro. In vivo, the involvement of the native CT-SLiM is critical for productive mitochondrial and peroxisomal fission, as both deletion and non-native extension of the CT-SLiM severely impair their progression. Thus, contrary to prevailing models, Drp1-catalyzed membrane fission relies on allosteric communication mediated by the CT-SLiM, deceleration of GTPase activity, and coupled changes in subunit architecture and assembly-disassembly dynamics.
Asunto(s)
Dinaminas , GTP Fosfohidrolasas , Dinaminas/metabolismo , GTP Fosfohidrolasas/metabolismo , Mitocondrias/metabolismo , Hidrólisis , Fusión de Membrana , Dinámicas Mitocondriales , Proteínas Mitocondriales/metabolismoRESUMEN
Mammalian DSPs have been historically isolated either from native tissue sources or from transfected insect cell cultures via time-consuming and cumbersome protocols often yielding protein of variable quality and quantity. A facile and highly reproducible alternative methodology involving the heterologous expression and purification of soluble mammalian DSPs from E. coli, which yields highly active and functional protein of a uniform quality and quantity, free of spurious posttranslational modifications inherent to mammalian and insect cell expression systems, is described in this chapter.