Distinct Splice Variants of Dynamin-related Protein 1 Differentially Utilize Mitochondrial Fission Factor as an Effector of Cooperative GTPase Activity

Abstract

Multiple isoforms of the mitochondrial fission GTPase dynamin-related protein 1 (Drp1) arise from the alternative splicing of its single gene-encoded pre-mRNA transcript. Among these, the longer Drp1 isoforms, expressed selectively in neurons, bear unique polypeptide sequences within their GTPase and variable domains, known as the A-insert and the B-insert, respectively. Their functions remain unresolved. A comparison of the various biochemical and biophysical properties of the neuronally expressed isoforms with that of the ubiquitously expressed, and shortest, Drp1 isoform (Drp1-short) has revealed the effect of these inserts on Drp1 function. Utilizing various biochemical, biophysical, and cellular approaches, we find that the A- and B-inserts distinctly alter the oligomerization propensity of Drp1 in solution as well as the preferred curvature of helical Drp1 self-assembly on membranes. Consequently, these sequences also suppress Drp1 cooperative GTPase activity. Mitochondrial fission factor (Mff), a tail-anchored membrane protein of the mitochondrial outer membrane that recruits Drp1 to sites of ensuing fission, differentially stimulates the disparate Drp1 isoforms and alleviates the autoinhibitory effect imposed by these sequences on Drp1 function. Moreover, the differential stimulatory effects of Mff on Drp1 isoforms are dependent on the mitochondrial lipid, cardiolipin (CL). Although Mff stimulation of the intrinsically cooperative Drp1-short isoform is relatively modest, CL-independent, and even counter-productive at high CL concentrations, Mff stimulation of the much less cooperative longest Drp1 isoform (Drp1-long) is robust and occurs synergistically with increasing CL content. Thus, membrane-anchored Mff differentially regulates various Drp1 isoforms by functioning as an allosteric effector of cooperative GTPase activity.

Cooperative and independent roles of the Drp1 adaptors Mff, MiD49 and MiD51 in mitochondrial fission

“…Disparity in function between adaptors also lies in the opposing effects on Drp1 GTPase activity at the membrane -Drp1 GTPase activity is inhibited by MiD proteins whereas Mff stimulates it. This is consistent with other recent studies (Clinton et al, 2015;Macdonald et al, 2015). Taken together, these results present intriguing roles for MiD49, MiD51 and Mff -although they can both recruit Drp1 to mitochondria, they have functional divergence in regulating Drp1 GTPase activity.…”

“…5A,B). In contrast to MiD51, and as recently reported (Clinton et al, 2015;Macdonald et al, 2015), Mff stimulated Drp1 GTPase activity (Drp1+Mff: k cat =40.4 min −1 ). The opposing effects of MiD51 and Mff on Drp1 GTPase activity suggest a divergence in the roles of the adaptors in fission foci.…”

“…This phenotype is significantly milder than the embryonic lethality seen for Drp1-knockout mice (Ishihara et al, 2009;Wakabayashi et al, 2009), suggesting that mitochondrial fission can take place in other tissues lacking Mff (Chen et al, 2015). Mff might also play an important role in neurons given its ability to efficiently stimulate the GTPase activity of the brainspecific Drp1 isoform compared to the ubiquitously expressed isoform (Macdonald et al, 2015).…”

Dynamin-related Protein 1 Oligomerization in Solution Impairs Functional Interactions with Membrane-anchored Mitochondrial Fission Factor

“…Thus, interactions between the VD and the membrane have the potential to stabilize Drp1 dimers and promote cooperative interactions with Mff. In fact, the accompanying article (42) clearly shows that Mff stimulation of Drp1 activity is synergistic with CL stimulation. These results are congruent with previous reports of VD interactions with CL that promote Drp1 recruitment to lipid bilayers (21,(23)(24)(25).…”

“…Therefore, the presence of the 37-amino acid B-insert within the VD can further regulate Drp1 interactions with Mff as shown in the accompanying article (42). Given that as many as eight isoforms of Drp1 (34,41) and at least nine splice variants of Mff have been identified (30), the potential complexity of interactions that are regulated by sequence changes is vast.…”