Dynamin-Related Protein 1 (Drp1), a large GTPase of the dynamin superfamily, is 
required for mitochondrial fission in healthy and apoptotic cells. Drp1 
activation is a complex process that involves translocation from the cytosol to 
the mitochondrial outer membrane (MOM) and assembly into rings/spirals at the 
MOM, leading to membrane constriction/division. Similar to dynamins, Drp1 
contains GTPase (G), bundle signaling element (BSE) and stalk domains. However, 
instead of the lipid-interacting Pleckstrin Homology (PH) domain present in the 
dynamins, Drp1 contains the so-called B insert or variable domain that has been 
suggested to play an important role in Drp1 regulation. Different proteins have 
been implicated in Drp1 recruitment to the MOM, although how MOM-localized Drp1 
acquires its fully functional status remains poorly understood. We found that 
Drp1 can interact with pure lipid bilayers enriched in the 
mitochondrion-specific phospholipid cardiolipin (CL). Building on our previous 
study, we now explore the specificity and functional consequences of this 
interaction. We show that a four lysine module located within the B insert of 
Drp1 interacts preferentially with CL over other anionic lipids. This 
interaction dramatically enhances Drp1 oligomerization and assembly-stimulated 
GTP hydrolysis. Our results add significantly to a growing body of evidence 
indicating that CL is an important regulator of many essential mitochondrial 
functions.