NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

Abstract

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.

Is Drp1 sufficient to catalyze membrane fission?

“…Mahajan et al. report that the dynamin-related protein1 (Drp1) can constrict but not sever membrane nanotubes ( 1 ), which contradicts our results showing that Drp1 is sufficient for membrane fission ( 2 ). Drp1 functions to divide mitochondria and peroxisomes.…”

“…Mahajan et al. ( 1 ) attribute differences to the fact that we employed nanotubes supported on PEGylated glass coverslips ( 4 ), as opposed to their use of free-standing nanotubes. Time-lapse imaging of supported nanotubes exposed to Drp1 with guanosine triphosphate (GTP) showed fission ( Fig.…”

“…White asterisks mark sites of fission. ( C ) Representative fields of supported nanotubes incubated with the Drp1 cloned in pRSET-C ( 1 ) or in pET15B ( 2 ). The pRSET-C construct carries an N-terminal 6xHis, XpressTM epitope and an enterokinase cleavage site, while the pET15b construct carries an N-terminal 6xHis tag with a TEV protease cleavage site and a C-terminal StrepII tag (Addgene plasmid #174421).…”

Metal-binding propensity in the mitochondrial dynamin-related protein 1

“…The previously reported interaction between Drp1 and DGS-NTA(Ni 2+ ) was assayed using liposome sedimentation (Koirala et al 2013). To confirm this interaction using sensitive methods, we Drp1 functions have been assayed in vitro based on its ability to bind the mitochondrial lipid cardiolipin (CL) (Macdonald et al 2014;Bustillo-Zabalbeitia et al 2014;Stepanyants et al 2015;Francy et al 2017;Kamerkar et al 2018a;Mahajan et al 2021). Remarkably, we find that liposomes containing 5 mol% of DGS-NTA(Ni 2+ ) recruited as much Drp1 as did liposomes containing 30 mol% CL (Fig.…”

“…Drp1 functions have been assayed in vitro based on its ability to bind the mitochondrial lipid cardiolipin (CL) (Macdonald et al 2014; Bustillo-Zabalbeitia et al 2014; Stepanyants et al 2015; Francy et al 2017; Kamerkar et al 2018a; Mahajan et al 2021). Remarkably, we find that liposomes containing 5 mol% of DGS-NTA(Ni 2+ ) recruited as much Drp1 as did liposomes containing 30 mol% CL (Fig.…”