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1.
Nat Commun ; 15(1): 52, 2024 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-38168038

RESUMO

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.


Assuntos
Dinaminas , GTP Fosfo-Hidrolases , Dinaminas/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Mitocôndrias/metabolismo , Hidrólise , Fusão de Membrana , Dinâmica Mitocondrial , Proteínas Mitocondriais/metabolismo
3.
Proc Natl Acad Sci U S A ; 118(29)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-34261790

RESUMO

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.


Assuntos
Cardiolipinas/metabolismo , Dinaminas/química , Dinaminas/metabolismo , Dinâmica Mitocondrial/fisiologia , Motivos de Aminoácidos , Sítios de Ligação , Dinaminas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Espectroscopia de Ressonância Magnética , Mitocôndrias/metabolismo , Membranas Mitocondriais/metabolismo , Membranas Mitocondriais/patologia , Mitofagia , Mutação , Ligação Proteica , Conformação Proteica
4.
Methods Mol Biol ; 2159: 31-40, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32529361

RESUMO

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.


Assuntos
Desmoplaquinas/genética , Desmoplaquinas/isolamento & purificação , Escherichia coli/genética , Expressão Gênica , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Animais , Escherichia coli/metabolismo , Plasmídeos/genética , Solubilidade , Transformação Bacteriana
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