Domain Interactions within Fzo1 Oligomers Are Essential for Mitochondrial Fusion

Abstract

Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion. Mitofusins are conserved GTPases essential for the fusion of mitochondria. These mitochondrial outer membrane proteins contain a GTPase domain and two or three regions with hydrophobic heptad repeats, but little is known about how these domains interact to mediate mitochondrial fusion. To address this issue, we have analyzed the yeast mitofusin Fzo1p and find that mutation of any of the three heptad repeat regions (HRN, HR1, and HR2) leads to a null allele. Specific pairs of null alleles show robust complementation, indicating that functional domains need not exist on the same molecule. Biochemical analysis indicates that this complementation is due to Fzo1p oligomerization mediated by multiple domain interactions. Moreover, we find that two non-overlapping protein fragments, one consisting of HRN/GTPase and the other consisting of HR1/HR2, can form a complex that reconstitutes Fzo1p fusion activity. Each of the null alleles disrupts the interaction of these two fragments, suggesting that we have identified a key interaction involving the GTPase domain and heptad repeats essential for fusion. The balance between fusion and fission maintains mitochondria as a dynamic tubular reticulum. In the absence of fusion, ongoing fission results in complete fragmentation of the mitochondrial network. Fragmentation leads to a loss of mtDNA and respiratory incompetence in yeast (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar, 2Rapaport D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar) and accumulation of poorly functional mitochondria in mammals (3Chen H. Chomyn A. Chan D.C. J. Biol. Chem. 2005; 280: 26185-26192Abstract Full Text Full Text PDF PubMed Scopus (1024) Google Scholar). Mice deficient for mitochondrial fusion die during mid gestation (4Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1837) Google Scholar), and mutations in the mitochondrial fusion genes Mfn2 and OPA1 cause the neurodegenerative diseases Charcot-Marie-Tooth disease type 2A and dominant optic atrophy, respectively (5Alexander C. Votruba M. Pesch U.E. Thiselton D.L. Mayer S. Moore A. Rodriguez M. Kellner U. Leo-Kottler B. Auburger G. Bhattacharya S.S. Wissinger B. Nat. Genet. 2000; 26: 211-215Crossref PubMed Scopus (1076) Google Scholar, 6Delettre C. Lenaers G. Griffoin J.M. Gigarel N. Lorenzo C. Belenguer P. Pelloquin L. 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Importantly, the mitochondrial fusion machinery is dedicated solely to mitochondrial fusion and does not include SNARE 2The abbreviations used are: SNARE, soluble NSF attachment protein receptors; HR, heptad repeat; SD, synthetic media with 2% dextrose; YP, yeast extract peptone; mito-GFP, mitochondrially targeted green fluorescent protein; mito-DsRed, mitochondrially targeted red fluorescent protein from Discosoma; HA, hemagglutinin. proteins or NSF (N-ethylmaleimide-sensitive protein), which mediate most other intracellular membrane fusion events. Thus, mitochondrial fusion likely involves a unique mechanism. There are three essential components of the fusion pathway in yeast. The yeast mitofusin Fzo1p is a 98-kDa transmembrane GTPase uniformly distributed in the mitochondria outer membrane (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar, 2Rapaport D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Both the N- and C-terminal portions of Fzo1p are oriented toward the cytosol, in position to mediate important steps during fusion (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar, 2Rapaport D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). Mgm1p is a dynamin-related protein in the mitochondrial intermembrane space (10Sesaki H. Southard S.M. Yaffe M.P. Jensen R.E. Mol. Biol. Cell. 2003; 14: 2342-2356Crossref PubMed Scopus (206) Google Scholar, 11Wong E.D. Wagner J.A. Gorsich S.W. McCaffery J.M. Shaw J.M. Nunnari J. J. Cell Biol. 2000; 151: 341-352Crossref PubMed Scopus (269) Google Scholar, 12Wong E.D. Wagner J.A. Scott S.V. Okreglak V. Holewinske T.J. Cassidy-Stone A. Nunnari J. J. Cell Biol. 2003; 160: 303-311Crossref PubMed Scopus (196) Google Scholar). It remains unclear if Mgm1p possesses the membrane severing activity of other dynamin family members, and if so, how this activity relates to its requirement for fusion. The third component of the fusion pathway is Ugo1p, a 58-kDa protein that spans the outer membrane once and interacts with both Fzo1p and Mgm1p (10Sesaki H. Southard S.M. Yaffe M.P. Jensen R.E. Mol. Biol. Cell. 2003; 14: 2342-2356Crossref PubMed Scopus (206) Google Scholar, 12Wong E.D. Wagner J.A. Scott S.V. Okreglak V. Holewinske T.J. Cassidy-Stone A. Nunnari J. J. Cell Biol. 2003; 160: 303-311Crossref PubMed Scopus (196) Google Scholar, 13Sesaki H. Jensen R.E. J. Cell Biol. 2001; 152: 1123-1134Crossref PubMed Scopus (193) Google Scholar, 14Sesaki H. Jensen R.E. J. Biol. Chem. 2004; 279: 28298-28303Abstract Full Text Full Text PDF PubMed Scopus (147) Google Scholar). In this capacity, Ugo1p may anchor a fusion complex that coordinates the mitochondrial outer and inner membranes during fusion. Fzo1p is the best candidate to directly mediate fusion of the mitochondrial outer membrane. Unlike Mgm1p, it is positioned in the outer membrane and unlike Ugo1p its requisite role is conserved from yeast to mammals. In addition, Fzo1p has three hydrophobic heptad repeat (HR) regions that are predicted to form coiled-coils. Heptad repeat regions are directly involved in SNARE and Class I viral membrane fusion. In both cases, membrane fusion is driven when a trans complex that spans both membranes undergoes a structural transition to a highly stable fusogenic conformation. These fusogenic structures use coiled-coil-like helical bundles to position the two membranes in close apposition, leading to fusion (15Bonifacino J.S. Glick B.S. Cell. 2004; 116: 153-166Abstract Full Text Full Text PDF PubMed Scopus (1313) Google Scholar, 16Eckert D.M. Kim P.S. Annu. Rev. Biochem. 2001; 70: 777-810Crossref PubMed Scopus (1151) Google Scholar, 17Skehel J.J. Wiley D.C. Cell. 1998; 95: 871-874Abstract Full Text Full Text PDF PubMed Scopus (369) Google Scholar, 18Ungar D. Hughson F.M. Annu. Rev. Cell Dev. Biol. 2003; 19: 493-517Crossref PubMed Scopus (232) Google Scholar). Given these similarities, it is likely that the heptad repeat regions form interactions critical for mitochondrial fusion. Indeed, the mammalian mitofusins Mfn1 and Mfn2 form homotypic and heterotypic complexes, and several intermolecular interactions have been identified. Mfn1 HR2 is known to form a trans anti-parallel, dimeric coiled-coil that tethers mitochondria together (19Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (677) Google Scholar). There is also evidence that Mfn1 HR1 and HR2 interact with each other (20Rojo M. Legros F. Chateau D. Lombes A. J. Cell Sci. 2002; 115: 1663-1674Crossref PubMed Google Scholar). Moreover, Fzo/mitofusins have similarity to dynamins, a family of large membrane-associated GTPases characterized by oligomerization-stimulated GTP hydrolysis and multiple inter- and intramolecular interactions (21Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1129) Google Scholar). It has not been established if Fzo1p forms similar intermolecular interactions that contribute to mitochondrial fusion. In this study, we show that Fzo1p functions as an oligomer during mitochondrial fusion. We demonstrate that each of the Fzo1p heptad repeats is essential and that the GTPase domains and heptad repeats from different Fzo1p molecules within an oligomer function together to mediate fusion. Finally, we define a novel interaction between the HRN/GTPase and HR1/HR2 regions of Fzo1p that is essential for mitochondrial fusion. Media and Yeast Genetic Techniques—Standard genetic techniques and media were used. SD and YP media supplemented with either 2% dextrose (YPD) or 3% glycerol (YPG) were prepared as described previously (22Gunthrie C. Fink G. Guide to Yeast Genetics and Molecular Biology. 1991; (Academic Press, San Diego, CA)Google Scholar). DCY538 (MATa, ura3–52, his3Δ200, leu2Δ1, trp1Δ63, lys2Δ202, fzo1::HIS3, leu2:: GPD-mitoGFP-LEU2, pRS416-FZO1) was derived from JSY2287 (kindly provided by J. Shaw) (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar) by integration of BstEII-linearized EG686 (pRS405 plus mito-GFP) into the leu2Δ1 locus. DCY328 (MATα, ura3–52, his3Δ200, leu2Δ1, trp1Δ63, fzo1::HIS3, rho0) was derived from JSY2793 (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar) by selecting for the loss of pRS414-fzo1–1. Because the loss of mtDNA in fzo1Δ yeast is a non-revertible phenotype, a plasmid-shuffle strategy was used to generate yeast strains for structure-function and intragenic allelic complementation analyses. Briefly, DCY538, a rho+ yeast strain containing a chromosomal deletion of FZO1 covered by an FZO1-expressing plasmid (pRS416-FZO1), was transformed with a plasmid encoding an FZO1 under TRP1 selection (structure-function experiments) or two FZO1 plasmids under TRP1 and LYS2 selection (intragenic complementation experiments). The transformation plates were replica-plated, and the pRS416-FZO1 plasmid was removed by counterselection with 5-fluoroorotic acid. The resulting yeast colonies were replica-plated onto YPD and YPG plates. The glycerol growth phenotype was reproducibly evident after 3 days of growth. In the intragenic complementation experiments, for example, all colonies showed growth on YPG plates when a pair of null alleles were complemented. For quantitative serial dilution and morphological analysis, representative colonies were isolated and re-analyzed. The possibility of recombination events was excluded by the presence of many non-complementing pairs of alleles (for example, fzo1(V172P) and fzo1(L819P)), the robust complementation between non-overlapping fragments (Fzo1p-HRN/GTPase plus Fzo1p-HR1/HR2), and the reproducibility of the glycerol growth phenotype among all colonies at the replica plating In addition, in the complementation involving and analysis the of the and strains the FZO1 were a plasmid strategy and at in SD were in YPD and during growth. For glycerol growth in SD were at a of and in 3 of the was to plates and for days (YPD) or days (YPG) at was as The FZO1 was from the and and as a The FZO1 was as a and the FZO1 to a the The FZO1 was the and and as a These fragments were all into to a was into the to The was as a into and of to generate The in was with a to generate The was as a into resulting in plus was the was with the were in the with the or by containing the are yeast strains were in the SD by together and in YPD at for The were for with and with of were for the complete of the and were in (MATα, his3Δ200, trp1Δ63, and (MATa, his3Δ200, trp1Δ63, (MATα, his3Δ200, trp1Δ63, and (MATa, were used for the two FZO1 and Fzo1p were in a fzo1Δ strain The use of a strain in mtDNA by functional and null alleles of were in the SD into YPD and at of was by with a in of supplemented with were by at for and for at was and of the was with to of protein for at The were with of and in for at analysis was with or were in DCY538 by a plasmid YPD at were for with The were with and with were for in containing and and for with in with the were with a for was to were at were on a with a and by were at to with an at and were were with at of Fzo1p has a transmembrane domain that in the mitochondrial outer membrane and the protein into N- and C-terminal both of which toward the (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar, 2Rapaport D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar, S. D. E. Neupert W. Westermann B. J. Cell Biol. 2001; 152: PubMed Scopus Google Scholar). The a GTPase domain that is for fusion (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar). Fzo1p also three domains with hydrophobic heptad other the mammalian Mfn1 and Fzo1p the hydrophobic heptad repeat regions HR1 and which the transmembrane domain Fzo1p also an heptad repeat the that we Because the role of these heptad repeats in mitochondrial fusion has not previously been we a structure-function analysis of Fzo1p HR1, and analysis use of the that loss of Fzo1p function complete fragmentation of the mitochondrial due to ongoing mitochondrial fission (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar, H. Jensen R.E. J. Cell Biol. PubMed Scopus Google Scholar). In addition, mitochondrial fragmentation leads to a loss of mitochondrial fusion can on the dextrose but not the glycerol (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar). Thus, we the function of Fzo1p by mitochondrial and growth on glycerol plates. HR1 is as predicted by and between the GTPase domain and transmembrane domain A. M. J. Science. 1991; PubMed Scopus Google Scholar). We HR1 to the predicted coiled-coil and a in the protein and are with a helical can by in The and were of growth on glycerol plates or fragmentation of the mitochondrial and In addition, we for at two a and and hydrophobic at the a and of the heptad repeat form a stable hydrophobic between the of a by the is predicted to this and as the as a null growth on glycerol and mitochondrial and of HR1 an allelic and that HR1 is essential for Fzo1p In mammalian HR2 forms an that tethers mitochondria together at an in fusion (19Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (677) Google Scholar). in Mfn1 HR2 mitochondrial and mitochondrial fusion (19Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Science. 2004; 305: 858-862Crossref PubMed Scopus (677) Google Scholar). at and of Fzo1p HR2 were with on FZO1 function and at and in HR2 a complete loss of function and in growth on glycerol plates and mitochondrial fragmentation and Thus, Mfn1 Fzo1p HR2 is essential for mitochondrial fusion. is also for FZO1 showed that that little on FZO1 that portions of as null Griffin and D. In addition, loss of fzo1(V172P) as a null and these results that HR1 and is essential for Fzo1p activity. We several in structure-function with mutations in the GTPase domain of FZO1 and all complete fragmentation of the mitochondrial (1Hermann G.J. Thatcher J.W. Mills J.P. Hales K.G. Fuller M.T. Nunnari J. Shaw J.M. J. Cell Biol. 1998; 143: 359-373Crossref PubMed Scopus (429) Google Scholar). in several of the null and the mitochondrial fragments The of this is but may the of Finally, the in the transmembrane domain are not essential for fusion. Fzo1p has three and in the of the transmembrane domain to form a in the intermembrane The was highly functional and Thus, the in the intermembrane space is important for Fzo1p function S. D. E. Neupert W. Westermann B. J. Cell Biol. 2001; 152: PubMed Scopus Google Scholar), its is not between FZO1 mitofusins have been to form (4Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1837) Google Scholar, Y. N. S. K. J. Biochem. 2003; PubMed Scopus Google Scholar), and Fzo1p has been to in a complex in yeast D. Brunner M. Neupert W. Westermann B. J. Biol. Chem. 1998; 273: 20150-20155Abstract Full Text Full Text PDF PubMed Scopus (296) Google Scholar). In cases, proteins that can show intragenic complementation, a genetic that results from of a functional complex from Y. D. PubMed Scopus Google Scholar). To yeast Fzo1p intragenic complementation, we used a plasmid-shuffle strategy to fzo1Δ strains two null The function of the null alleles was by growth on glycerol plates and mitochondrial on results we used and as representative null alleles with mutations in HR1, and respectively These were with the three GTPase these we and intragenic complementation was reproducibly in and of either of two GTPase with a heptad repeat (HR) Fzo1p In the GTPase or showed complementation with either or resulting in tubular mitochondrial that with a to on glycerol plates and Thus, Fzo1p activity can when the function of the GTPase and heptad repeat regions on different Fzo1p These complementation also that the null mutations not have on Fzo1p of intragenic complementation in in a In many pairs of alleles to The GTPase not any of the heptad repeat complementation was when an HR1 was with an HR2 or an and In addition, when an was with an HR2 FZO1 activity was not and Finally, of any two GTPase not mitochondrial fusion. The of these is that Fzo1p functions as an oligomer in which GTPase and heptad repeat activity can by different it is also that the GTPase and heptad repeat regions (for example, at different steps in the fusion We to these by directly for Fzo1p Fzo1p as an evidence for Fzo1p oligomerization by a In these experiments, and were at in fzo1Δ was of the mammalian Mfn1 and Mfn2 have previously been to form homotypic and heterotypic (4Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1837) Google Scholar). in with intragenic complementation these interaction that Fzo1p functions as an oligomer during mitochondrial fusion. intragenic complementation is due to oligomerization between Fzo1p we pairs to Indeed, the Fzo1p GTPase and with each of the heptad repeat and and In with all the heptad repeat and was to and suggesting oligomerization is for intragenic all of null the to form homotypic interactions. in HR1, or in both HR1 and HR2 little on Fzo1p of an was with the GTPase most which the of interaction Given that mutations in domain Fzo1p we that oligomerization of Fzo1p likely involves interactions between multiple and that the null of the heptad repeat and GTPase are not a of Fzo1p between and several domains likely in to Fzo1p we to a for domain interactions. We fragments of Fzo1p interact with each We analyzed an Fzo1p containing and the GTPase domain and a C-terminal that HR1, the and HR2 with Fzo1p 3 and interaction was also between two fragments the non-overlapping and fragments with each other as as the interaction Thus, has at two of a homotypic interaction with and a heterotypic interaction with We the interaction between and by that can to mitochondria. and not a mitochondrial in all In was and in all with the mitochondrial of to mitochondria in mitochondrial and of These the interaction between and and that this interaction on mitochondria. identified an interaction between these two Fzo1p fragments, we this interaction is The null mutations identified previously were for on the of to mutations in either HR1 or HR2 were into its interaction with was when mutations in or the GTPase domain were into its interaction with was null alleles that the interaction of with is essential for fusion. between and interaction on we if of and mitochondrial fusion. growth on glycerol or mitochondrial and the of and was to fusion. These on glycerol plates and tubular mitochondria and Moreover, the Fzo1p fragments show mitochondrial fusion in the fusion J. A. A. J.W. P. Mol. Biol. Cell. PubMed Scopus Google Scholar). These that and form a complex to mitochondrial used in this FZO1 fzo1Δ fzo1Δ fzo1Δ in a In this study, we show that Fzo1p functions as an oligomer during mitochondrial fusion. In of this Fzo1p forms homotypic by In addition, pairs of null alleles are of intragenic complementation can between pairs of if each a different domain of an the different functional domains are from resulting in the of activity Y. D. PubMed Scopus Google Scholar). for intragenic complementation is that the GTPase and heptad repeat domains are not on the same at different steps in the fusion we this the that Fzo1p molecules the intragenic complementation into the for the activity of different domains on Fzo1p molecules within these an Fzo1p one the activity of all three heptad repeats (for example, a GTPase GTPase activity but not heptad repeat activity (for example, an HR1, or HR2 of Fzo1p molecules with heptad repeat activity and Fzo1p molecules with GTPase activity fusion, of two heptad repeat or two GTPase does not fusion. Because Fzo1p is on mitochondria to fusion S. McCaffery J.M. Nunnari J. Science. 2004; 305: PubMed Scopus Google Scholar), an that remains to is the intragenic complementation we in a complex on one or in trans a complex that spans two mitochondria to In this an in fusion but the does not for mitochondria isolated from yeast that are for S. McCaffery J.M. Nunnari J. Science. 2004; 305: PubMed Scopus Google Scholar). We have also identified of to as an interaction critical for mitochondrial fusion. of and mitochondrial fusion. these Fzo1p fragments interact with each Finally, mutations in the GTPase domain or heptad repeat regions this interaction and cause complete loss of mitochondrial fusion activity. to the complex interactions between Fzo1p Fzo1p is a large membrane-associated GTPase with to the dynamin family of are characterized by of GTP Thus, it to the possibility that intermolecular interactions within an Fzo1p oligomer its GTPase activity. Given that of a functional GTPase domain on one Fzo1p and functional heptad repeat regions on is to fusion, it is that the heptad repeats a role in GTPase activity. For example, on its the it has been HR2 function to the GTPase domain of dynamin (21Praefcke G.J. McMahon H.T. Nat. Rev. Mol. Cell. Biol. 2004; 5: 133-147Crossref PubMed Scopus (1129) Google Scholar). In of Fzo1p fragments can used to address this structural to the of the Fzo1p We Shaw for on allelic