A role for Fis1 in both mitochondrial and peroxisomal fission in mammalian cells

The mammalian dynamin-like protein DLP1/Drp1 has been shown to mediate both mitochondrial and peroxisomal fission. In this study, we have examined whether hFis1, a mammalian homologue of yeast Fis1, which has been shown to participate in mitochondrial fission by an interaction with DLP1/Drp1, is also involved in peroxisomal growth and division. We show that hFis1 localizes to peroxisomes in addition to mitochondria. Through differential tagging and deletion experiments, we demonstrate that the transmembrane domain and the short C-terminal tail of hFis1 is both necessary and sufficient for its targeting to peroxisomes and mitochondria, whereas the N-terminal region is required for organelle fission. hFis1 promotes peroxisome division upon ectopic expression, whereas silencing of Fis1 by small interfering RNA inhibited fission and caused tubulation of peroxisomes. These findings provide the first evidence for a role of Fis1 in peroxisomal fission and suggest that the fission machinery of mitochondria and peroxisomes shares common components.     

Targeting of hFis1 to peroxisomes is mediated by Pex19p

The processes of peroxisome formation and proliferation are still a matter of debate. We have previously shown that peroxisomes share some components of their division machinery with mitochondria. hFis1, a tail-anchored membrane protein, regulates the membrane fission of both organelles by DLP1/Drp1 recruitment, but nothing is known about the mechanisms of the dual targeting of hFis1. Here we demonstrate for the first time that peroxisomal targeting of hFis1 depends on Pex19p, a peroxisomal membrane protein import factor. hFis1/Pex19p binding was demonstrated by expression and co-immunoprecipitation studies. Using mutated versions of hFis1 an essential binding region for Pex19p was located within the last 26 C-terminal amino acids of hFis1, which are required for proper targeting to both mitochondria and peroxisomes. The basic amino acids in the very C terminus are not essential for Pex19p binding and peroxisomal targeting, but are instead required for mitochondrial targeting. Silencing of Pex19p by small interference RNA reduced the targeting of hFis1 to peroxisomes, but not to mitochondria. In contrast, overexpression of Pex19p alone was not sufficient to shift the targeting of hFis1 to peroxisomes. Our findings indicate that targeting of hFis1 to peroxisomes and mitochondria are independent events and support a direct, Pex19p-dependent targeting of peroxisomal tail-anchored proteins.     

Adaptor Proteins MiD49 and MiD51 Can Act Independently of Mff and Fis1 in Drp1 Recruitment and Are Specific for Mitochondrial Fission

Drp1 (dynamin-related protein 1) is recruited to both mitochondrial and peroxisomal membranes to execute fission. Fis1 and Mff are Drp1 receptor/effector proteins of mitochondria and peroxisomes. Recently, MiD49 and MiD51 were also shown to recruit Drp1 to the mitochondrial surface; however, different reports have ascribed opposing roles in fission and fusion. Here, we show that MiD49 or MiD51 overexpression blocked fission by acting in a dominant-negative manner by sequestering Drp1 specifically at mitochondria, causing unopposed fusion events at mitochondria along with elongation of peroxisomes. Mitochondrial elongation caused by MiD49/51 overexpression required the action of fusion mediators mitofusins 1 and 2. Furthermore, at low level overexpression when MiD49 and MiD51 form discrete foci at mitochondria, mitochondrial fission events still occurred. Unlike Fis1 and Mff, MiD49 and MiD51 were not targeted to the peroxisomal surface, suggesting that they specifically act to facilitate Drp1-directed fission at mitochondria. Moreover, when MiD49 or MiD51 was targeted to the surface of peroxisomes or lysosomes, Drp1 was specifically recruited to these organelles. Moreover, the Drp1 recruitment activity of MiD49/51 appeared stronger than that of Mff or Fis1. We conclude that MiD49 and MiD51 can act independently of Mff and Fis1 in Drp1 recruitment and suggest that they provide specificity to the division of mitochondria.     

Dynamin-like protein 1 at the Golgi complex: A novel component of the sorting/targeting machinery en route to the plasma membrane

The final step in the liberation of secretory vesicles from the trans-Golgi network (TGN) involves the mechanical action of the large GTPase dynamin as well as conserved dynamin-independent fission mechanisms, e.g. mediated by Brefeldin A-dependent ADP-ribosylated substrate (BARS). Another member of the dynamin family is the mammalian dynamin-like protein 1 (DLP1/Drp1) that is known to constrict and tubulate membranes, and to divide mitochondria and peroxisomes. Here, we examined a potential role for DLP1 at the Golgi complex. DLP1 localized to the Golgi complex in some but not all cell lines tested, thus explaining controversial reports on its cellular distribution. After silencing of DLP1, an accumulation of the apical reporter protein YFP-GL-GPI, but not the basolateral reporter VSVG-SP-GFP at the Golgi complex was observed. A reduction in the transport of YFP-GL-GPI to the plasma membrane was confirmed by surface immunoprecipitation and TGN-exit assays. In contrast, YFP-GL-GPI trafficking was not disturbed in cells silenced for BARS, which is involved in basolateral sorting and trafficking of VSVG-SP-GFP in COS-7 cells. Our data indicate a new role for DLP1 at the Golgi complex and thus a role for DLP1 as a novel component of the apical sorting machinery at the TGN is discussed.     

The mitochondrial protein hFis1 regulates mitochondrial fission in mammalian cells through an interaction with the dynamin-like protein DLP1

The yeast protein Fis1p has been shown to participate in mitochondrial fission mediated by the dynamin-related protein Dnm1p. In mammalian cells, the dynamin-like protein DLP1/Drp1 functions as a mitochondrial fission protein, but the mechanisms by which DLP1/Drp1 and the mitochondrial membrane interact during the fission process are undefined. In this study, we have tested the role of a mammalian homologue of Fis1p, hFis1, and provided new and mechanistic information about the control of mitochondrial fission in mammalian cells. Through differential tagging and deletion experiments, we demonstrate that the intact C-terminal structure of hFis1 is essential for mitochondrial localization, whereas the N-terminal region of hFis1 is necessary for mitochondrial fission. Remarkably, an increased level of cellular hFis1 strongly promotes mitochondrial fission, resulting in an accumulation of fragmented mitochondria. Conversely, cell microinjection of hFis1 antibodies or treatment with hFis1 antisense oligonucleotides induces an elongated and collapsed mitochondrial morphology. Further, fluorescence resonance energy transfer and coimmunoprecipitation studies demonstrate that hFis1 interacts with DLP1. These results suggest that hFis1 participates in mitochondrial fission through an interaction that recruits DLP1 from the cytosol. We propose that hFis1 is a limiting factor in mitochondrial fission and that the number of hFis1 molecules on the mitochondrial surface determines fission frequency.     

Elongation of peroxisomes as an indicator for efficient dynamin-like protein 1 knock down in mammalian cells.

RNA interference has become a valuable tool to identify and investigate proteins involved in the formation of peroxisomes. We demonstrate that the elongation of peroxisomes serves as an excellent indicator for efficient knock down of dynamin-like protein 1 (DLP1) in mammalian cells. We took advantage of the silencing-dependent morphological changes of peroxisomes to compare different transfection methods and show that a single transfection of DLP1 siRNA by electroporation is sufficient to effectively silence DLP1. We present a fast, easy, and convenient protocol for efficient gene silencing in a large number of cells, which can be used for quantitative and biochemical studies.     

6-Hydroxydopamine (6-OHDA) induces Drp1-dependent mitochondrial fragmentation in SH-SY5Y cells

Mitochondrial alterations have been associated with the cytotoxic effect of 6-hydroxydopamine (6-OHDA), a widely used neurotoxin to study Parkinson's disease. Herein we studied the potential effects of 6-OHDA on mitochondrial morphology in SH-SY5Y neuroblastoma cells. By immunofluorescence and time-lapse fluorescence microscopy we demonstrated that 6-OHDA induced profound mitochondrial fragmentation in SH-SY5Y cells, an event that was similar to mitochondrial fission induced by overexpression of Fis1p, a membrane adaptor for the dynamin-related protein 1 (DLP1/Drp1). 6-OHDA failed to induce any changes in peroxisome morphology. Biochemical experiments revealed that 6-OHDA-induced mitochondrial fragmentation is an early event preceding the collapse of the mitochondrial membrane potential and cytochrome c release in SH-SY5Y cells. Silencing of DLP1/Drp1, which is involved in mitochondrial and peroxisomal fission, prevented 6-OHDA-induced fragmentation of mitochondria. Furthermore, in cells silenced for Drp1, 6-OHDA-induced cell death was reduced, indicating that a block in mitochondrial fission protects SH-SY5Y cells against 6-OHDA toxicity. Experiments in mouse embryonic fibroblasts deficient in Bax or p53 revealed that both proteins are not essential for 6-OHDA-induced mitochondrial fragmentation. Our data demonstrate for the first time an involvement of mitochondrial fragmentation and Drp1 function in 6-OHDA-induced apoptosis.     

Peroxisome division is impaired in a CHO cell mutant with an inactivating point-mutation in dynamin-like protein 1 gene

We earlier isolated a Chinese hamster ovary cell line ZP121 showing morphologically abnormal, tubular peroxisomes, and apparent dysmorphogenesis of mitochondria. Here, we identified an inactivating point-mutation in dynamin-like protein 1 gene, DLP1, responsible for the phenotype of ZP121. One allele of DLP1 possessed a point missense mutation resulting in G363D in the middle region of 699-amino-acid long DLP1, termed DLP1G363D, while the other allele was normal. DLP1G363D was apparently expressed at a higher level than DLP1. Abnormal morphogenesis of peroxisomes as well as mitochondria was restored when wild-type DLP1 was transfected. The GTPase activity of DLP1G363D was barely detectable, indicating that the G363D mutation severely affected the GTPase activity. Moreover, a higher level of DLP1G363D expression in CHO-K1 cells reproduced the ZP121-type phenotype, hence indicating its dominant-negative activity to the wild-type DLP1, most likely by forming a heteromeric tetramer. The G363D mutation also gave rise to a temperature-sensitive phenotype showing normal morphogenesis of peroxisomes and mitochondria at 40 degrees C. Microtubule organization was most likely involved in the elongation of peroxisomes. Furthermore, ZP121 was lowered in the level of phospholipids, plasmalogens, and phosphatidylethanolamine and was less sensitive to oxidative stresses. Thus, ZP121 is the first dlp1 mutant in mammalian cells.     

Dynamin-like protein 1 is involved in peroxisomal fission

The mammalian dynamin-like protein 1 (DLP1), a member of the dynamin family of large GTPases, possesses mechanochemical properties known to constrict and tubulate membranes. In this study, we have combined two experimental approaches, induction of peroxisome proliferation by Pex11pbeta and expression of dominant-negative mutants, to test whether DLP1 plays a role in peroxisomal growth and division. We were able to localize DLP1 in spots on tubular peroxisomes in HepG2 cells. In addition, immunoblot analysis revealed the presence of DLP1 in highly purified peroxisomal fractions from rat liver and an increase of DLP1 after treatment of rats with the peroxisome proliferator bezafibrate. Expression of a dominant negative DLP1 mutant deficient in GTP hydrolysis (K38A) either alone or in combination with Pex11pbeta caused the appearance of tubular peroxisomes but had no influence on their intracellular distribution. In co-expressing cells, the formation of tubulo-reticular networks of peroxisomes was promoted, and peroxisomal division was completely inhibited. These findings were confirmed by silencing of DLP1 using siRNA. We propose a direct role for the dynamin-like protein DLP1 in peroxisomal fission and in the maintenance of peroxisomal morphology in mammalian cells.     

Mitochondria-specific function of the dynamin family protein DLP1 is mediated by its C-terminal domains

The dynamin superfamily of large GTPases has been implicated in a variety of distinct intracellular membrane remodeling events. One of these family members, DLP1/Drp1, is similar to conventional dynamins as it contains an N-terminal GTPase domain followed by a middle region (MID), an unconserved region (UC), and a coiled-coil (CC) domain. DLP1 has been shown to function in membrane-based processes distinct from conventional dynamin, most notably mitochondrial fission. In this study, we tested whether the functional specificities of DLP1 and dynamin stems from differences in the individual domains of these proteins by generating dynamin/DLP1 chimeras in which correlate domains had been interchanged. Here we report that three consecutive C-terminal domains of DLP1 (MID-UC-CC) contain information necessary for DLP1-specific function and removing any one of these domains results in a loss of DLP1 function. Importantly, the coiled-coil (CC) domain of DLP1 alone targets specifically and exclusively to mitochondria, implicating its involvement in localizing DLP1 to this organelle in vivo. The mitochondrial targeting information within the DLP1 CC domain is not sufficient to retarget dynamin to mitochondria but is still able to adequately function as an assembly domain in a dynamin background. These data suggest that whereas the GTPase domain of DLP1 provides an enzymatic function, other domains contain information for intermolecular assembly and mitochondrial targeting.     

Fis1, DLP1, and Pex11p coordinately regulate peroxisome morphogenesis

Dynamin-like protein 1 (DLP1) and Pex11pbeta function in morphogenesis of peroxisomes. In the present work, we investigated whether Fis1 is involved in fission of peroxisomes. Endogenous Fis1 was morphologically detected in peroxisomes as well as mitochondria in wild-type CHO-K1 and DLP1-defective ZP121 cells. Subcellular fractionation studies also revealed the presence of Fis1 in peroxisomes. Peroxisomal Fis1 showed the same topology, i.e., C-tail anchored membrane protein, as the mitochondrial one. Furthermore, ectopic expression of FIS1 induced peroxisome proliferation in CHO-K1 cells, while the interference of FIS1 RNA resulted in tubulation of peroxisomes, hence reducing the number of peroxisomes. Fis1 interacted with Pex11pbeta, by direct binding apparently involving the C-terminal region of Pex11pbeta in the interaction. Pex11pbeta also interacted with each other, whereas the binding of Pex11pbeta to DLP1 was not detectable. Moreover, ternary complexes comprising Fis1, Pex11pbeta, and DLP1 were detected by chemical cross-linking. We also showed that the highly conserved N-terminal domain of Pex11pbeta was required for the homo-oligomerization of Pex11pbeta and indispensable for the peroxisome-proliferating activity. Taken together, these findings indicate that Fis1 plays important roles in peroxisome division and maintenance of peroxisome morphology in mammalian cells, possibly in a concerted manner with Pex11pbeta and DLP1.     

Structural and functional analysis of MiD51, a dynamin receptor required for mitochondrial fission

Mitochondrial fission is important for organelle transport, inheritance, and turnover, and alterations in fission are seen in neurological disease. In mammals, mitochondrial fission is executed by dynamin-related protein 1 (Drp1), a cytosolic guanosine triphosphatase that polymerizes and constricts the organelle. Recruitment of Drp1 to mitochondria involves receptors including Mff, MiD49, and MiD51. MiD49/51 form foci at mitochondrial constriction sites and coassemble with Drp1 to drive fission. Here, we solved the crystal structure of the cytosolic domain of human MiD51, which adopts a nucleotidyltransferase fold. Although MiD51 lacks catalytic residues for transferase activity, it specifically binds guanosine diphosphate and adenosine diphosphate. MiD51 mutants unable to bind nucleotides were still able to recruit Drp1. Disruption of an additional region in MiD51 that is not part of the nucleotidyltransferase fold blocked Drp1 recruitment and assembly of MiD51 into foci. MiD51 foci are also dependent on the presence of Drp1, and after scission they are distributed to daughter organelles, supporting the involvement of MiD51 in the fission apparatus.     

The dynamin-like GTPase DLP1 is essential for peroxisome division and is recruited to peroxisomes in part by PEX11

Peroxisome division involves the conserved PEX11 peroxisomal membrane proteins and in yeast has been shown to require Vps1p, a dynamin-like protein. We show here that DLP1, the human homolog of the yeast DNM1 and VPS1 genes, plays an important role in peroxisome division in human cells. Disruption of DLP1 function by either RNA interference or overexpressing dominant negative DLP1 mutants causes a dramatic reduction in peroxisome abundance, although overexpression of functional DLP1 has no effect on peroxisome abundance. Overexpression of PEX11 induces peroxisome division in a multistep process involving elongation of preexisting peroxisomes followed by their division. We find that DLP1 is dispensable for the first phase of this process but essential for the second. Furthermore, we show that DLP1 associates with peroxisomes and that PEX11 overexpression recruits DLP1 to peroxisome membranes. However, we were unable to detect physical interaction between PEX11 and DLP1, and the stoichiometry of PEX11 and peroxisome-associated DLP1 was far less than 1:1. Based on these and other aspects, we propose that DLP1 performs an essential but transient role in peroxisome division and that PEX11 promotes peroxisome division by recruiting DLP1 to peroxisome membranes through an indirect mechanism.     

Charcot‐Marie‐Tooth disease‐associated mutants of GDAP1 dissociate its roles in peroxisomal and mitochondrial fission

Mitochondria and peroxisomes can be fragmented by the process of fission. The fission machineries of both organelles share a set of proteins. GDAP1 is a tail‐anchored protein of mitochondria and induces mitochondrial fragmentation. Mutations in GDAP1 lead to Charcot‐Marie‐Tooth disease (CMT), an inherited peripheral neuropathy, and affect mitochondrial dynamics. Here, we show that GDAP1 is also targeted to peroxisomes mediated by the import receptor Pex19. Knockdown of GDAP1 leads to peroxisomal elongation that can be rescued by re‐expressing GDAP1 and by missense mutated forms found in CMT patients. GDAP1‐induced peroxisomal fission is dependent on the integrity of its hydrophobic domain 1, and on Drp1 and Mff, as is mitochondrial fission. Thus, GDAP1 regulates mitochondrial and peroxisomal fission by a similar mechanism. However, our results reveal also a more critical role of the amino‐terminal GDAP1 domains, carrying most CMT‐causing mutations, in the regulation of mitochondrial compared to peroxisomal fission.     

Control of mitochondrial morphology through differential interactions of mitochondrial fusion and fission proteins

Mitochondria in mammals are organized into tubular networks that undergo frequent shape change. Mitochondrial fission and fusion are the main components mediating the mitochondrial shape change. Perturbation of the fission/fusion balance is associated with many disease conditions. However, underlying mechanisms of the fission/fusion balance are not well understood. Mitochondrial fission in mammals requires the dynamin-like protein DLP1/Drp1 that is recruited to the mitochondrial surface, possibly through the membrane-anchored protein Fis1 or Mff. Additional dynamin-related GTPases, mitofusin (Mfn) and OPA1, are associated with the outer and inner mitochondrial membranes, respectively, and mediate fusion of the respective membranes. In this study, we found that two heptad-repeat regions (HR1 and HR2) of Mfn2 interact with each other, and that Mfn2 also interacts with the fission protein DLP1. The association of the two heptad-repeats of Mfn2 is fusion inhibitory whereas a positive role of the Mfn2/DLP1 interaction in mitochondrial fusion is suggested. Our results imply that the differential binding of Mfn2-HR1 to HR2 and DLP1 regulates mitochondrial fusion and that DLP1 may act as a regulatory factor for efficient execution of both fusion and fission of mitochondria.     

Mitochondria and peroxisomes: Are the ‘Big Brother’ and the ‘Little Sister’ closer than assumed?

Mitochondria and peroxisomes are essential subcellular organelles in mammals. Despite obvious differences, both organelles display certain morphological and functional similarities. Recent studies have elucidated that these highly dynamic and plastic organelles share components of their division machinery. Mitochondria and peroxisomes are metabolically linked organelles, which are cooperating and cross-talking. This review addresses the dynamics and division of mitochondria and peroxisomes as well as their functional similarities to provide insight as to why these organelles share the fission machinery in evolutionary aspects.     

Yeast peroxisomes multiply by growth and division

Peroxisomes can arise de novo from the endoplasmic reticulum (ER) via a maturation process. Peroxisomes can also multiply by fission. We have investigated how these modes of multiplication contribute to peroxisome numbers in Saccharomyces cerevisiae and the role of the dynamin-related proteins (Drps) in these processes. We have developed pulse-chase and mating assays to follow the fate of existing peroxisomes, de novo-formed peroxisomes, and ER-derived preperoxisomal structures. We find that in wild-type (WT) cells, peroxisomes multiply by fission and do not form de novo. A marker for the maturation pathway, Pex3-GFP, is delivered from the ER to existing peroxisomes. Strikingly, cells lacking peroxisomes as a result of a segregation defect do form peroxisomes de novo. This process is slower than peroxisome multiplication in WT cells and is Drp independent. In contrast, peroxisome fission is Drp dependent. Our results show that peroxisomes multiply by growth and division under our assay conditions. We conclude that the ER to peroxisome pathway functions to supply existing peroxisomes with essential membrane constituents.     

The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.

In healthy cells, fusion and fission events participate in regulating mitochondrial morphology. Disintegration of the mitochondrial reticulum into multiple punctiform organelles during apoptosis led us to examine the role of Drp1, a dynamin-related protein that mediates outer mitochondrial membrane fission. Upon induction of apoptosis, Drp1 translocates from the cytosol to mitochondria, where it preferentially localizes to potential sites of organelle division. Inhibition of Drp1 by overexpression of a dominant-negative mutant counteracts the conversion to a punctiform mitochondrial phenotype, prevents the loss of the mitochondrial membrane potential and the release of cytochrome c, and reveals a reproducible swelling of the organelles. Remarkably, inhibition of Drp1 blocks cell death, implicating mitochondrial fission as an important step in apoptosis.     

An Arabidopsis dynamin-related protein, DRP3A, controls both peroxisomal and mitochondrial division

Peroxisomes undergo dramatic changes in size, shape, number, and position within the cell, but the division process of peroxisomes has not been characterized. We screened a number of Arabidopsis mutants with aberrant peroxisome morphology (apm mutants). In one of these mutants, apm1, the peroxisomes are long and reduced in number, apparently as a result of inhibition of division. We showed that APM1 encodes dynamin-related protein 3A (DRP3A), and that mutations in APM1/DRP3A also caused aberrant morphology of mitochondria. The transient expression analysis showed that DRP3A is associated with the cytosolic side of peroxisomes. These findings indicate that the same dynamin molecule is involved in peroxisomal and mitochondrial division in higher plants. We also report that the growth of Arabidopsis, which requires the cooperation of various organelles, including peroxisomes and mitochondria, is repressed in apm1, indicating that the changes of morphology of peroxisomes and mitochondria reduce the efficiency of metabolism in these organelles.     

The Mechanoenzymatic Core of Dynamin-related Protein 1 Comprises the Minimal Machinery Required for Membrane Constriction

Mitochondria are dynamic organelles that continually undergo cycles of fission and fusion. Dynamin-related protein 1 (Drp1), a large GTPase of the dynamin superfamily, is the main mediator of mitochondrial fission. Like prototypical dynamin, Drp1 is composed of a mechanochemical core consisting of the GTPase, middle, and GTPase effector domain regions. In place of the pleckstrin homology domain in dynamin, however, Drp1 contains an unstructured variable domain, whose function is not yet fully resolved. Here, using time-resolved EM and rigorous statistical analyses, we establish the ability of full-length Drp1 to constrict lipid bilayers through a GTP hydrolysis-dependent mechanism. We also show the variable domain limits premature Drp1 assembly in solution and promotes membrane curvature. Furthermore, the mechanochemical core of Drp1, absent of the variable domain, is sufficient to mediate GTP hydrolysis-dependent membrane constriction.