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. 2016 Jan 1;291(1):493-507.
doi: 10.1074/jbc.M115.680181. Epub 2015 Nov 17.

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

Patrick J Macdonald  1 , Christopher A Francy  2 , Natalia Stepanyants  1 , Lance Lehman  1 , Anthony Baglio  1 , Jason A Mears  2 , Xin Qi  3 , Rajesh Ramachandran  4
Affiliations

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

Patrick J Macdonald et al. J Biol Chem. .

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.

Keywords: GTPase; alternative splicing; cardiolipin; dynamin-related protein 1; fluorescence resonance energy transfer (FRET); isoforms; mitochondria; mitochondrial dynamics; mitochondrial fission factor.

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Figures

FIGURE 1.
FIGURE 1.
Drp1 isoforms exhibit differential GTPase activities. A, graphic illustration of the Drp1 primary structure showing the locations of the A- and B-insert segments present in select Drp1 isoforms. The respective locations of the A- and B-inserts within the Drp1 structure (Protein Data Bank ID: 4BEJ) and their sequences are shown. The six extra amino acid residues (aa 594–599) found in the VD of the rat Drp1-long clone are also indicated. GED, GTPase effector domain. B–E, time course of GTP hydrolysis for Drp1-short (B), Drp1-long (C), Drp1-A-only (D), and Drp1-B-only (E) (0.5 μm protein) in the absence and presence of 25 mol % CL-containing liposomes (150 μm total lipid) was measured as a function of Pi released over time using a malachite green-based colorimetric assay. The initial rates of GTP hydrolysis were fit to a linear equation. The calculated turnover number (kcat) for each condition is indicated above. F, a bar plot summarizing the progressive reduction of cooperative basal and liposome-stimulated GTPase activities upon inclusion of either A-inserts or B-inserts or both in Drp1. Error bars indicate means ± S.D.
FIGURE 2.
FIGURE 2.
Drp1 isoforms exhibit differential oligomerization propensities in solution and distinct helical geometry on CL-containing membranes. A, left panel, SEC-MALS analyses of Drp1-short, Drp1-long, Drp1-A-only, and Drp1-B-only fractionated on a Superose 6 10/300 GL column at a loading concentration of 10 μm. Normalized differential refractive indices (left axis; line traces) and molar mass profiles underneath the peak regions (right axis; dots) are plotted against elution volume (in ml). The slopes of the molar mass profiles were fitted to an exponential trace (dotted line) to model oligomerization propensities. Right panel, magnified negative-stain EM images of the corresponding Drp1 helical polymers (2 μm protein) formed in the presence of 1 mm GMP-PCP in solution. Scale bar, 100 nm. B, left panel, representative FRET emission spectra of Drp1 Trp (0.5 μm protein) in the absence (Donor only) and presence of 10 mol % dansyl-PE (Donor + Acceptor) in 25 mol % CL-containing liposomes (150 μm total lipid). The acceptor-only trace for an equivalent concentration of dansyl-PE-containing liposomes in the absence of protein is also shown. FRET is characterized by the decrease in donor emission intensity in the presence of acceptor and an increase in the sensitized emission of the acceptor upon donor excitation. Right panel, Trp-dansyl FRET efficiency (E) for the various Drp1 isoforms (1 μm protein) in the presence of 25 mol % CL-containing liposomes that contain 10 mol % dansyl-PE (100 μm total lipid). E is expressed as a percentage. Error bars indicate means ± S.D. C, representative negative-stain EM images of Drp1-short, Drp1-long, and Drp1-A-only helical polymers (2 μm protein) tubulating 25 mol % CL-containing liposomes (50 μm total lipid). Scale bar, 200 nm. Histograms of tube diameter size distributions are shown below for Drp1-short and Drp1-A-only. The small sample size precluded statistical analyses for Drp1-long. No polymers were visualized for Drp1-B-only.
FIGURE 3.
FIGURE 3.
Drp1 A- and B-inserts differentially affect membrane curvature. A–D, representative negative-stain EM images (right panels) and corresponding histograms of tube diameter size distribution (left panels) for Drp1-short (A), Drp1-long (B), Drp1-A-only (C), and Drp1-B-only (D) helical polymers (2 μm protein) tubulating and decorating unextruded pure DOPS liposomes (100 μm total lipid). Scale bar, 50 nm.
FIGURE 4.
FIGURE 4.
Drp1 A- and B-inserts do not affect GTP binding kinetics or affinity. A, representative stopped-flow FRET kinetic time course of the association of mant-GTP (7 μm) with Drp1-short (0.4 μm protein). The data were best fit to a single exponential rate equation (red fitted trace). F0 and F are the initial and at time t intensities of mant, respectively. B, representative stopped-flow FRET kinetic time course of the dissociation of preincubated mant-GTP (4 μm) from Drp1-short (0.4 μm protein) assembled on 25 mol % CL-containing liposomes (40 μm total lipid) upon rapid mixing with a vast molar excess of unlabeled GTP (400 μm final). The data were best fit to a single exponential rate equation (red fitted trace) and plotted as in panel A. C, concentration dependence of the apparent rate constants (kapp) of mant-GTP association for Drp1-long and -short in the absence and presence of 25 mol % CL-containing liposomes. Data from at least two independent experiments for each condition are averaged and plotted with the errors indicated. Association (kon) and dissociation (koff) rate constants were determined from the linear concentration dependence of kapp as described under “Experimental Procedures.” Dissociation rate constants were also determined directly from competition experiments with unlabeled GTP. The kinetic data are summarized in Table 1. Error bars indicate means ± S.D.
FIGURE 5.
FIGURE 5.
Drp1-short exhibits greater cooperative GTPase activities relative to Drp1-long. A–D, Michaelis-Menten kinetics of Drp1-long and -short (0.5 μm protein) in the absence and presence of 25 mol % CL-containing liposomes (150 μm total lipid). Data from four individual experiments are plotted as reaction rate versus GTP concentration in each panel. GTP concentration at half-maximal rate (Km or Michaelis-Menten constant) for each reaction is indicated above.
FIGURE 6.
FIGURE 6.
Membrane-reconstituted Mff differentially regulates the cooperative GTPase activities of distinct Drp1 isoforms. A, far-UV CD spectra for full-length Mff (15 μm) in the absence and presence of 25 mol % CL-containing liposomes (710 μm total lipid). mdeg, millidegrees. B, Trp-dansyl FRET efficiency (E) for Mff (1 μm protein) upon incubation with either 25 mol % CL-containing or 25 mol % DOPS-containing liposomes (100 μm total lipid). C, time course of GTP hydrolysis for Drp1-short (0.5 μm) upon self-assembly on 25 mol % CL-containing liposomes (150 μm total lipid) in the absence and presence of externally added full-length Mff (1 μm) that was preincubated with the liposomes prior to Drp1 addition. kcat values are indicated above. D, time course of GTP hydrolysis for Drp1-long (0.5 μm) upon self-assembly on 25 mol % CL-containing liposomes (150 μm total lipid) in the absence and presence of either externally added, liposome-preincubated full-length Mff or MffΔTM (1 μm). E, the dependence of -fold stimulation of Drp1-long by liposome-preincubated Mff on intrinsic Drp1-long lipid-stimulated GTPase activity in the absence of Mff. Data points represent multiple batches of either protein. F, same as in panel C but for Drp1-A-only and Drp1-B-only. Error bars indicate means ± S.D.
FIGURE 7.
FIGURE 7.
Mff and CL function differentially but cooperatively in regulating distinct Drp1 isoforms. A, sucrose density gradient flotation assay confirming the reconstitution of full-length Mff in proteoliposomes. The top panel shows the percentage of the fluorescent lipid tracer, RhPE, present in the different sucrose layers whose peak overlapped with that of Mff protein density as determined by SDS-PAGE analysis of the various fractions as shown in the bottom panel. MffΔTM treated similarly to full-length (FL) Mff served as a non-incorporating control and showed no overlap of liposome and protein density. MW marker, molecular weight marker. * marks a soluble contaminant that co-purifies with Mff but does not float with proteoliposomes validating our protocols for the specific reconstitution of TMD-anchored, full-length Mff. B, same as in Fig. 6D, but with full-length Mff reconstituted in proteoliposomes (1.5 μm total exposed Mff, 150 μm total lipid). The indicated concentration of Mff in proteoliposomes is the “effective” concentration assuming that only half of the reconstituted Mff, 3 μm final, is in the proper, soluble N-terminal domain-out orientation to be available for Drp1 binding on the exterior leaflet of the liposome. The other half of Mff resides with the soluble N-terminal domain facing the liposome lumen. Data with similarly treated liposomes that do not contain Mff (empty proteoliposomes) (150 μm total lipid) are shown as control. C, left panel, rate of GTP hydrolysis for Drp1-long (0.5 μm) incubated with liposomes (150 μm total lipid) containing varying mole percentages of CL in the absence and presence of externally added, preincubated, full-length Mff (1 μm). Right panel, same as left panel but plotted on a numerical scale. D, rate of Drp1-short (0.5 μm) GTP hydrolysis on CL-free liposomes (150 μm total lipid) preincubated with externally added full-length Mff (1.25 μm) and on proteoliposomes (150 μm total lipid) reconstituted with full-length Mff (1.25 μm) in the absence of CL. Error bars indicate means ± S.D.
FIGURE 8.
FIGURE 8.
Differential dependence of distinct Drp1 isoforms on Mff for function in vivo. A and C, Western blotting to detect expression levels of various Myc-tagged Drp1 isoforms in either Drp1 KO MEFS (A) or Mff KO MEFs (C). Actin served as loading control. B and D, quantification of mitochondrial fission in Drp1 KO (B) or Mff KO MEFs (D) expressing distinct Myc-tagged isoforms of Drp1. Vector Ctrl., vector control. Data are expressed as the percentage of total Myc-expressing cells that display fragmented mitochondria. **, p < 0.01. Error bars indicate means ± S.D.
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