Discovery of the membrane receptor for mitochondrial fission GTPase Drp1

Abstract

Mitochondria frequently change their morphology by fusion and fission, and these dynamic morphologic changes are essential for maintaining both mitochondrial and cellular functions. The cytoplasmic dynamin-related guanosine triphosphatase (GTPase) Drp1 (Dnm1 in yeast) is recruited to mitochondrial fission sites and severs mitochondria. Although the mitochondrial outer membrane (MOM) protein Fis1 functions as a membrane receptor for Dnm1 in yeast, it is not yet known whether the human homolog of yeast Fis1 (hFis1) is a membrane receptor for Drp1 in mammals. We recently identified the C-tail anchored MOM protein Mff as the bona fide receptor essential for recruiting Drp1 to mitochondrial fission sites. Here, we focus on this key molecule for mitochondrial fission after a brief description of the proteins involved in mitochondrial fission and fusion reactions. Finally, we discuss the expected role of hFis1 for regulating the mitochondrial dynamics in mammals.

Introduction

Mitochondria are essential organelles for the life and death of eukaryotic cells and participate in aerobic energy production, intermediary metabolism, Ca²⁺ signaling and apoptosis. They move along cytoskeletal tracks to sites of high-energy demand, or change their overall morphology by fusion and fission in response to the environment and cellular differentiation. Because mitochondria proliferate by the growth and division of pre-existing mitochondria, their fission is also important for maintaining mitochondrial number and function. High-molecular weight GTPases are key components for the regulation of mitochondrial morphologic dynamics. In vertebrates, mitofusin proteins (Mfn1 and Mfn2) of the mitochondrial outer membrane (MOM) and mitochondrial inner membrane protein OPA1 are involved in mitochondrial fusion probably in a coupled fashion, whereas dynamin-related protein Drp1 regulates mitochondrial fission.¹ Mitochondrial morphology regulation is important for maintaining cellular function, and its dysfunction causes aging, neuronal synaptic loss and cell death in several human neurologic diseases.² Genetic and biochemical studies in the budding yeast Saccharomyces cerevisiae, Drosophila and mammalian cells have contributed to the identification and characterization of the major components of the mitochondrial fission and fusion machineries.³

Drp1/Dnm1 is a conserved dynamin GTPase superfamily protein that mediates membrane remodeling in a variety of cellular membranes (Fig. 1A). It forms assemblies (foci) that primarily associate with the mitochondrial surface, occasionally at points of tubule constriction and fission.^4–6 Mice lacking Drp1 have developmental abnormalities that result in embryonic lethality.^7,8 Because mitochondria play essential roles in a variety of cellular processes as described above, most of the mutations that disrupt these functions are fatal to the organism rather than manifesting as disorders. To date, only a heterozygous, dominant-negative middle domain mutation (A395D) in human Drp1 has been reported in a lethal disorder with microcephaly, abnormal brain development, optic atrophy and hypoplasia.⁹ Similarly, Mfn1 or Mfn2 knockout results in embryonic lethality in mice and mutations in MFN2 and OPA1 genes cause Charcot Mary Tooth neuropathy 2A and optic atrophy type I, respectively.²

Figure 1.

Drp1 assembly on the mitochondrial outer membrane via the membrane receptor Mff in mammals. (A) Schematic representation of Drp1 and Mff. (B) FLAG-Mff and the Drp1 binding-incompetent mutant FLAG-MffΔN60 (deletion of N-terminal 60 amino acid residues) were expressed in Mff-RNAi HeLa cells and analyzed by immunofluorescence microscopy with anti-Drp1 (green) and anti-FLAG antibodies (red). Drp1 dispersed in the cytosol in Mff RNAi cells was recruited to mitochondria upon expression of FLAG-Mff, but not FLAG-MffΔN60. (C) Mitochondrial fission in mammals requires the membrane receptor Mff. Cytosolic Drp1 is recruited to the MOM by Mff. Mff seems to function simultaneously as an adaptor and receptor for the mitochondrial recruitment of Drp1, mimicking the function of the yeast Mdv1-Fis1 complex. After targeting, Drp1 self-assembles into large ring-like structures that hydrolyze GTP and sever the mitochondrial membrane. The function of hFis1, Endophilin B1 and GDAP1 in mammals remains to be elucidated. GDAP1 (Ganglioside induced differentiation associated protein 1) is a C-tail anchored mitochondrial fission factor of MOM. (D) Time course of GTPase activity of wild-type Drp1 proteins in the presence of the indicated amounts of Mff proteins.

Based mainly on studies in yeast, mitochondrial fission is thought to begin with Drp1/Dnm1 recruitment from the cytoplasm to the MOM, where it self-assembles into large ring-like structures in a GTP-dependent manner, and then severs the mitochondrial membrane GTP-hydrolysis dependently. In this context, dissipation of the mitochondrial membrane potential or apoptotic stimulus induces the mitochondrial recruitment of Drp1 to stimulate mitochondrial fission concomitant with inhibition of the mitochondrial fusion reaction, thus the mitochondrial morphology easily shifts toward fission.^10,11 Binding of Drp1 to the MOM-anchored receptor and subsequent formation of the functional fission complex are thought to be essential for the initial step of mitochondrial fission. Although several candidate proteins have been identified in mammals, their mechanistic roles in mitochondrial fission remain elusive.

Fis1-Mdv1 Complex Recruits Dnm1 to the MOM and Drives Mitochondrial Fission in Yeast

Based on structural and biochemical analyses of purified Dnm1 bound to phospholipid vesicles, it has been demonstrated that, upon GTP-binding the mitochondria-recruited Dnm1/Drp1 forms higher-order structures that wrap around the lipid membrane tubules, although the precise mechanism remains controversial.^5,6 These spiral higher-order structures are thought to constrict and eventually sever the mitochondrial membrane by a GTP hydrolysis-dependent mechanism.¹² Recent studies using cryo-electron microscopy, however, have revealed that, upon GTP hydrolysis Dnm1 constricts liposomes and subsequently dissociates from the lipid bilayer by larger conformational changes.¹³ In vivo, additional Dnm1-associated factors should regulate and facilitate membrane constriction and scission events.

Genetic and biochemical studies in yeast revealed that Dnm1-mediated mitochondrial fission requires a MOM protein Fis1 and a soluble adaptor protein Mdv1 or its paralogue Caf4. During mitochondrial fission, Fis1 transiently interacts via cytosolic adaptor proteins Mdv1/Caf4 with Dnm1 by its tetratricopeptide repeat (TPR) motif, indicating that Fis1 functions as the mitochondrial Dnm1 receptor.¹⁴ Mdv1 comprises an N-terminal extension containing a helix-loop-helix motif that interacts with Fis1, a central coiled-coil region that mediates Mdv1 homo-oligomerization and a C-terminal WD40 domain to form a multibladed β-propeller that interacts with Dnm1.^3,15 To date, Fis1 and Mdv1/Caf4 are the only components known to form the fission complex with Dnm1 in yeast. Unlike Fis1, the homologs of WD motif proteins Mdv1 and Caf4 have not been found in mammalian cells.

Mff is an Essential Factor for Recruiting Drp1 to the Prospective Fission Sites in Mammals

Mitochondrial fission factor (Mff) was previously identified by a search of the Drosophila RNA interference (RNAi) library for mitochondrial morphology alterations.¹⁶ It is a C-tail anchored MOM protein exposing the bulk N-terminal segment with a central coiled-coil motif into the cytosol (Fig. 1A). Mammalian mitochondria contain an Mff ortholog. Silencing this factor by RNAi induces mitochondrial network extension and, at the same time, the endogenous Drp1 observed as dotted structures on mitochondria disappears and is released into the cytoplasm (Fig. 1B). Conversely, Mff overexpression induces mitochondrial fission concomitant with increased Drp1 recruitment to the mitochondria, suggesting that Mff activity is rate-limiting in mitochondrial fission.¹⁷ It is noteworthy that no such changes are detected in the cells transfected with hFis1 expression plasmid, although hFis1 overexpression induces perinuclear accumulation of fragmented mitochondria.¹⁷

Both in vitro and in vivo experiments demonstrated that Mff transiently interacts with Drp1 through its N-terminal cytoplasmic region.¹⁷ Mff has two short repeats in the N-terminal half, which are conserved in metazoans and deletion of these repeats severely compromises Drp1 recruitment, suggesting that they function as the binding motifs of Drp1 during mitochondrial recruitment: i.e., they function like the WD40 motifs of Mdv1. Although Mff binds Drp1 directly, the interaction should be transient because the interaction is detectable only in the presence of cross-linking agents and thus, Drp1 might dynamically cycle on and off on the membrane, possibly regulated by GTP or other modifications (Fig. 1C). Interestingly, in this context, a mutation in the WD40 domain of Mdv1, Mdv1(N544R), that strengthens the Dnm1-Mdv1 interaction supports assembly of the division complex but cannot support the subsequent membrane scission step in yeast.¹⁸ Thus, simple mitochondrial recruitment of Drp1 seems to be insufficient for membrane scission, and Mff may facilitate membrane scission by promoting the self-assembly of Drp1, probably in conjunction with as-yet unknown factors. In this regard, we found that Mff stimulates the GTPase activity of Drp1, suggesting that Mff stimulates Drp1 self-assembly (Fig. 1D). In marked contrast, however, it was previously reported that the GTPase activity of Drp1 is not affected by hFis1.¹⁹ We also found that the GTPase middle domain mutant of Drp1 (A395D), identified from a human patient and with defects in higher order assembly and mitochondrial fission, failed to interact with Mff and to be recruited to mitochondria.¹⁷ These results indicate that not only the initial recruitment of Drp1 but its subsequent oligomeric assembly proceeds in a GTP-dependent manner. Although the significance of the highly conserved coiled-coil domain in Mff (Fig. 1A) is not yet clarified, deletion of this domain results in the decreased targeting of Mff to the MOM, suggesting that it is required for correct and efficient mitochondrial targeting of Mff, which may facilitate efficient Drp1 recruitment and subsequent formation of the division complexes on the mitochondrial surface.

Mff localizes on the MOM as punctate structures that colocalize mostly with the Drp1 foci.¹⁷ In marked contrast, hFis1 localizes uniformly on the MOM. Interestingly, the Mff punctate structures are still present on the extended tubular networks in Drp1 RNAi cells or Drp1-knockout MEFs. Together, these results strongly indicate that Mff present as preassembled structures on the MOM not only functions as the bona fide Drp1 receptor, but stimulates self-assembly of the recruited Drp1 at the prospective mitochondrial fission sites to drive the fission reaction (Fig. 1D). Of note, Ryan and collaborators have recently demonstrated that the N-terminal anchored MOM proteins, MiD49 and MiD51, forming foci or ring-like structures on the mitochondria are involved in mitochondrial recruitment of Drp1.²⁰ Functional division between MiD proteins and Mff remains to be analyzed.

Physiological Role of hFis1 in Mammalian Cells

What is the functional relation of Mff and hFis1? One possibility is that Mff functions as a Drp1 receptor and hFis1 functions downstream of Mff where hFis1 modulates the assembly of fission foci containing Drp1, Mfn2, endophilin B1 or other factors, and the subsequent severing process^21–23 (Fig. 1D).

Mff and hFis1 are both C-tail anchored in the MOM, although their other structural properties are distinct and they exist in separate 200-kDa complexes, suggesting that they have distinct functions in mammalian cells.¹⁶ As described already, hFis1 is the ortholog of the yeast mitochondrial fission factor Fis1 and is therefore thought to be involved in the mitochondrial recruitment of Drp1 as is the case in yeast,^12,14,19 although several functional inconsistencies have been noted (i.e., lack of hFis1-RNAi effect on localization of the Drp1 foci, uniform distribution of hFis1 on the MOM distinct from dot-like localization of Drp1 and failure of functional complementation of fis1Δ yeast cells). Overexpression of hFis1 results in mitochondrial fragmentation and perinuclear clustering,^17,19,24,25 seemingly consistent with the assumption that hFis1 is involved in mammalian mitochondrial fission. However, caution should be taken in interpreting the effects of overexpressed membrane proteins that were frequently used in the previous studies, because these manipulations sometimes induce non-physiologic stress in the cells, which seemed to lead to mitochondrial morphology changes. Conversely, in previous quantitative studies hFis1 RNAi has been reported to induce mitochondrial network extension.^19,24,26 In our experiments, however, mitochondrial recruitment of Drp1 to the foci and mitochondrial division were not significantly affected by hFis1 RNAi and the effects were further corroborated by conditional hFis1-knockout cells.¹⁷ We concluded that hFis1 is dispensable for mitochondrial fission in mammals, although the possibility remains that only a few Drp1 foci are actively engaged in fission at any one time. Or, the increased mitochondrial interconnectivity by hFis1 RNAi may be cell type specific.

Overexpression of hFis1 is reported to induce mitochondrial fragmentation concomitant with Bax/Bak-dependent release of cytochrome c into the cytosol, where it is recruited to the Apaf1 complex to activate effector caspases.²⁵ In contrast, however, Bax and Bak double knockout (DKO) cells revealed that hFis1 can promote mitochondrial fission but not cell death.²⁷ Interestingly, expression of sarco/endoplasmic reticulum-associated calcium transporting ATPase (SERCA), but not mitochondria-targeted Bax, specifically restored the apoptosis susceptibility in these cells, indicating that death by hFis1 relies on the ER gateway of apoptosis requiring adequate ER Ca²⁺ concentration. These results together might suggest that hFis1 has dual functions that independently regulate mitochondrial fission and the ER Ca²⁺-dependent apoptosis. In the latter case, hFis1 does not directly activate Bax and Bak, but induces Ca²⁺-dependent mitochondrial dysfunction.²⁷ In this relation, Iwasawa et al. recently demonstrated that hFis1 transmits an apoptosis signal from the mitochondria to the endoplasmic reticulum (ER) by interacting with Bap31 on the ER and facilitating its cleavage by procaspase 8 into the pro-apoptotic form (p20Bap31).²⁸ p20Bap31 causes rapid transmission of ER calcium signals to the mitochondria at close ER-mitochondria junctions (Fig. 2). This calcium influx into mitochondria is thought to stimulate Drp1-dependent mitochondrial fission and cytochrome c release. Thus, the hFis1-Bap31 complex bridging the mitochondria and ER functions as a platform to activate the initiator procaspase-8 in apoptosis signaling (Fig. 2). Although the exact mechanism of apoptosis regulation by hFis1 is unclear, hFis1 may function as the ER gateway for ER-mediated apoptosis rather than in mitochondrial division in mammals.^27,28 Importance of interorganellar organization between the ER and mitochondria in regulation of a number of physiological processes such as lipid transfer and Ca²⁺ signaling has been noted, i.e., a mitochondrial fusion protein Mfn2 localized both on mitochondria and on the mitochondria-associated ER membrane (MAMs) is involved in this organization.²⁹ If hFis1-Bap21 were involved in the regulation of mitochondria-ER tethering, the expression levels of hFis1 should affect mitochondrial morphology via lipid transfer or Ca²⁺ signaling.

Figure 2.

Hypothetical models of hFis1 function in mammalian cells. The hFis1/Bap31 platform transmits the mitochondrial stress signal to the ER via the activation of procaspase-8. The cytosolic region of the ER integral membrane protein Bap31 is cleaved by activated caspase-8 to generate proapoptotic p20Bap31, which causes rapid transmission of ER calcium signals to the mitochondria via the IP3 receptor. At close ER-mitochondria contact sites, mitochondria takes up calcium into the matrix via the mitochondrial calcium channels MICU1 or LETM1. The massive influx of calcium leads to mitochondrial fission, cristae remodeling and cytochrome c release. Moderate calcium influx levels may result in mitochondrial dysfunction and lead to mitophagy to eliminate the dysfunctional mitochondria. Another possibility is that hFis1 functions to generate mitochondria-derived vesicles by a Drp1-independent mechanism. A putative physiologic function of the new vesicular mitochondria in the peroxisomes transport pathway is presently unknown, but it may influence organelle morphology and functions. SERCA, sarco/endoplasmic reticulum Ca²⁺-ATPase. MICU1, mitochondrial calcium uptake 1. LETM1, leucine zipper/EF hand-containing transmembrane 1.

hFis1 exhibits dual localization in mitochondria and peroxisomes and is also thought to be involved in Drp1-dependent peroxisomal fission.^30,31 Our experiments revealed, however, that peroxisomes show no significant morphology alterations either in hFis1 RNAi cells or in hFis1 conditional knockout human colon tumor cells, indicating that hFis1 has no or minor role in peroxisomal fission.¹⁷ We do not have appropriate explanation for this discrepancy, although the possibility that hFis1 RNAi influenced a wide range of diverse metabolic activities and indirectly affected the perosisomal morphology cannot be ruled out.

Mitochondria and peroxisomes apparently share some biological functions, such as the oxidation of fatty acids. A novel vesicular transport pathway from the mitochondria to the peroxisomes was recently described.³² This pathway depends on the unique mitochondria-derived vesicles (MDVs), which bud from mitochondria in a Drp1-independent manner and are transported to peroxisomes (Fig. 2). Although the physiological function of the MDV transport pathway is presently unclear, it may contribute to the transport of metabolites, lipids or proteins to a peroxisome subpopulation. In this relation, mitochondrial-synthesized cardiolipin is present at significant levels within peroxisomes.³³ hFis1 might thus be involved in the generation of MDVs from mitochondria in a Drp1-independent manner and indirectly influence mitochondria morphology. Or, it might influence peroxisomal morphology through the ER-dependent de novo synthetic pathway of peroxisomal membrane proteins.³⁴

Mitochondrial abundance is regulated by mitochondrial fission and degradation. Mitochondrial degradation is mediated by autophagic processes (mitophagy; Fig. 2) thought to be closely linked to mitochondrial dynamics and division.³⁵ hFis1 has been reported to induce mitochondrial fragmentation and enhance mitophagy.^26,36 Furthermore, reduced hFis1 expression in β-cells by RNAi decreases mitophagy and results in the accumulation of oxidized mitochondrial proteins, reduced respiration and impaired insulin secretion.³⁵ Studies with overexpression of hFis1 and the mutants revealed that their ability to induce mitochondrial fragmentation is separable from their injurious effects on mitochondrial function, indicating that stimulation of autophagy by hFis1 correlates with mitochondrial dysfunction rather than with fission of the organelle.³⁶ Because no functional assay is available for the role of hFis1 in vitro or in vivo, it is not known whether its participation in mitophagy is direct or indirect. Further studies are needed to clarify the functional relevance of hFis1 in mitophagy.

Abbreviations

CCCP

carbonyl cyanide m-chlorophenyl hydrazone

Drp1

dynamin related protein 1

ER

endoplasmic reticulum

Fis1

fission 1

GDAP1

ganglioside-induced differentiation-associated protein 1

GED

GTPase effector domain

Mdv1

mitochondrial division protein 1

Mff

mitochondrial fission factor

Mfn1

mitofusin 1

Mfn2

mitofusin 2

MOM

mitochondrial outer membrane

RNAi

RNA interference

TPR

tetratricopeptide repeat

CMT2A

charcot-marie-tooth disease type 2A

Bap31

B cell receptor-associated protein

IP3R

inositol-1,4,5-triphosphate receptor

SERCA

sarco/endoplasmic reticulum calcium transporting ATPase

MICU1

mitochondrial calcium uptake 1

LETM1

leucine zipper-EF-hand containing transmembrane protein 1

MiD49 and MiD51

mitochondrial dynamics proteins of 49 and 51 kDa

Extra View to: Otera H, Mihara K. Molecular mechanisms and physiologic functions of mitochondrial dynamics. J Biochem. 2011;149:241–251. doi: 10.1093/jb/mvr002.