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GTPase domain mutations block Fzo1p function. (A) Schematic representation of Fzo1p illustrating the location and sequence of the GTPase domain motifs (G1–G4; not to scale) with mutations changing conserved residues indicated. The mutant amino acids are depicted above the original residues. (B) Mutations in the G1 and G2 motifs disrupt the function of Fzo1p. Glycerol growth, mitochondrial morphology (anti-porin, n 400), and mtDNA nucleoid distribution (DAPI staining, n 100) analyzed in fzo1 cells (JSY2354) containing wild-type FZO1 (JSY2392) or mutated fzo1 genes (JSY2355-2358) carried on low copy plasmids (pRS414).
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Membrane fusion is required to establish the morphology and cellular distribution of the mitochondrial compartment. In Drosophila, mutations in the fuzzy onions (fzo) GTPase block a developmentally regulated mitochondrial fusion event during spermatogenesis. Here we report that the yeast orthologue of fuzzy onions, Fzo1p, plays a direct and conserv...
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... Fzo1p GTPase domain contains four conserved mo- tifs designated G1-G4 (Fig. 7 A). In most GTPases, these domains are required for GTP binding and hydrolysis as well as conformational changes elicited by nucleotide binding (Bourne et al., 1991). Conserved residues in three of the four G motifs in Fzo1p (K200A and S201N in G1, T221A in G2, and K371A in G4) were altered by site- directed mutagenesis (Fig. 7 A). All of ...
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... designated G1-G4 (Fig. 7 A). In most GTPases, these domains are required for GTP binding and hydrolysis as well as conformational changes elicited by nucleotide binding (Bourne et al., 1991). Conserved residues in three of the four G motifs in Fzo1p (K200A and S201N in G1, T221A in G2, and K371A in G4) were altered by site- directed mutagenesis (Fig. 7 A). All of these amino acid substitutions are known to disrupt either nucleotide bind- ing or interactions with effector proteins in other GTPases ( Sigal et al., 1986;Adari et al., 1988;Cales et al., 1988;Feig and Cooper, 1988;Farnsworth and Feig, 1991;Vojtek et al., 1993;Murphy et al., 1997). When low copy plasmids containing the ...
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... at wild-type levels and were targeted to the mitochondrial compartment as assayed by differential centrifugation and Western blotting with anti-Fzo1p antiserum (data not shown). However, the fzo1(K200A), fzo1(S201N), and fzo1(T221A) mutant genes failed to rescue the glycerol growth defect or the mitochondrial morphology defects in the fzo1 strain (Fig. 7 B). Interestingly, a significant per- centage (13 and 12%, respectively) of cells containing the fzo1(K200A) and fzo1(T221A) mutant genes contained detectable mtDNA nucleoids (Fig. 7 B), although the total number of nucleoids was reduced relative to wild type (1-5 instead of 25-50) (data not shown). It is possible that these mutant Fzo1 ...
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... the fzo1(K200A), fzo1(S201N), and fzo1(T221A) mutant genes failed to rescue the glycerol growth defect or the mitochondrial morphology defects in the fzo1 strain (Fig. 7 B). Interestingly, a significant per- centage (13 and 12%, respectively) of cells containing the fzo1(K200A) and fzo1(T221A) mutant genes contained detectable mtDNA nucleoids (Fig. 7 B), although the total number of nucleoids was reduced relative to wild type (1-5 instead of 25-50) (data not shown). It is possible that these mutant Fzo1 proteins retain some residual functions re- quired for mtDNA maintenance. Alternatively, cells con- taining these mutant proteins may simply lose their mito- chondrial genomes more ...
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... It is possible that these mutant Fzo1 proteins retain some residual functions re- quired for mtDNA maintenance. Alternatively, cells con- taining these mutant proteins may simply lose their mito- chondrial genomes more slowly than fzo1-null cells. Mutation of a conserved residue in the G4 domain (K371A) did not disrupt the function of FZO1 (Fig. 7 B). This result is somewhat surprising, since lysine 371 is con- served throughout the GTPase superfamily and is known to be required for high-affinity GTP binding by Ras (Der et al., 1988;Bourne et al., 1991). In addition, the same mu- tation in Drosophila fzo had a modest but significant ef- fect on the function of the protein (Hales ...
Citations
... Finally, we observed no obvious changes in the behavior of key components of the mitochondrial division or fusion machinery that could explain the observed mitochondrial division defect. Fzo1, a protein involved in outer mitochondrial membrane fusion, displayed typical mitochondrial outer membrane localization in both Num1 and Scs2 mutants (Hermann et al., 1998; Fig. S3 B, 100% of cells, n = 100 cells per replicate over three imaging replicates). Num1 has previously been shown to be required to maintain a stable population of cortical Dnm1 foci that may influence mitochondrial division events . ...
The mitochondria–ER–cortex anchor (MECA) forms a tripartite membrane contact site between mitochondria, the endoplasmic reticulum (ER), and the plasma membrane (PM). The core component of MECA, Num1, interacts with the PM and mitochondria via two distinct lipid-binding domains; however, the molecular mechanism by which Num1 interacts with the ER is unclear. Here, we demonstrate that Num1 contains a FFAT motif in its C-terminus that interacts with the integral ER membrane protein Scs2. While dispensable for Num1’s functions in mitochondrial tethering and dynein anchoring, the FFAT motif is required for Num1’s role in promoting mitochondrial division. Unexpectedly, we also reveal a novel function of MECA in regulating the distribution of phosphatidylinositol-4-phosphate (PI(4)P). Breaking Num1 association with any of the three membranes it tethers results in an accumulation of PI(4)P on the PM, likely via disrupting Sac1-mediated PI(4)P turnover. This work establishes MECA as an important regulatory hub that spatially organizes mitochondria, ER, and PM to coordinate crucial cellular functions.
... Here, we have tagged the native gene at the C-terminus with a DD tag for the regulatable knock-down of the target protein in the parasite. The DD system uses a ddFKBP gene fusion and is regulated by using Shld1 drug [51], [55], [56]. Transient knock-down of PfDyn2 showed significant growth reduction and disrupted nuclear division as well as parasite development into schizonts stages. ...
Malaria parasite harbors a single mitochondrion and its proper segregation during the parasite multiplication is crucial for propagation of the parasite within the host. Mitochondrial fission machinery consists of a number of proteins that associate with mitochondrial membrane during segregation. Here, we have identified a dynamin-like protein in P. falciparum , Pf Dyn2, and deciphered its role in mitochondrial division, segregation and homeostasis. GFP targeting approach combined with high resolution microscopy studies showed that the Pf Dyn2 associates with mitochondrial membrane to form a clip/hairpin loop like structure around it at specific sites during mitochondrial division. The C-terminal degradation tag mediated inducible knock-down (iKD) of Pf Dyn2 resulted in significant inhibition of parasite growth. Pf Dyn2-iKD hindered mitochondrial development and functioning, decreased mtDNA replication, and induced mitochondrial oxidative-stress, ultimately causing parasite death. Further, treatment of parasites with dynamin specific inhibitors disrupted the recruitment of Pf Dyn2 on the mitochondria, blocked mitochondrial development, and induced oxidative stress. Regulated overexpression of a phosphorylation mutant of Pf Dyn2 (Ser-612-Ala) had no effect on the recruitment of Pf Dyn2 on the mitochondria; normal mitochondrial division and parasite growth showed that phosphorylation/dephosphorylation of this conserved serine residue (Ser612) may not be responsible for regulating recruitment of Pf Dyn2 to the mitochondrion. Overall, we show essential role of Pf Dyn2 in mitochondrial dynamics and fission as well as in maintaining its homeostasis during asexual cycle of the parasite.
... In fact, screens conducted in the 1990s using temperature-sensitive mutants, as well as studies focusing on mitochondrial genome maintenance, led to the identification of molecular players regulating mitochondrial morphology (52,112). Thus, molecules regulating mitochondrial fission and fusion were discovered and validated by application of live-cell fluorescence microscopy, along with functional implications such as growth rates and loss of mitochondrial DNA (mtDNA) (53,145). At this point, the physiological importance of mitochondrial dynamic players in mammalian systems was not yet understood. ...
Mitochondria are essential organelles performing important cellular functions ranging from bioenergetics and metabolism to apoptotic signaling and immune responses. They are highly dynamic at different structural and functional levels. Mitochondria have been shown to constantly undergo fusion and fission processes and dynamically interact with other organelles such as the endoplasmic reticulum, peroxisomes, and lipid droplets. The field of mitochondrial dynamics has evolved hand in hand with technological achievements including advanced fluorescence super-resolution nanoscopy. Dynamic remodeling of the cristae membrane within individual mitochondria, discovered very recently, opens up a further exciting layer of mitochondrial dynamics. In this review, we discuss mitochondrial dynamics at the following levels: ( a) within an individual mitochondrion, ( b) among mitochondria, and ( c) between mitochondria and other organelles. Although the three tiers of mitochondrial dynamics have in the past been classified in a hierarchical manner, they are functionally connected and must act in a coordinated manner to maintain cellular functions and thus prevent various human diseases.
Expected final online publication date for the Annual Review of Biophysics, Volume 53 is May 2024. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.
... Yeast Dnm1 and Fzo1 are crucial proteins in mitochondrial fission and fusion, respectively (Hermann et al., 1998;Sesaki and Jensen, 1999). As expected, the number of mitochondrial profiles in thin sections of H. polymorpha dnm1 cells is strongly reduced ( Fig. 2A), whereas an increase is observed in fzo1 cell sections. ...
... This phenotype is most likely due to reduced mitochondrial fusion. In S. cerevisiae a block in mitochondrial fusion, caused by the absence of Fzo1, results in fragmented mitochondrial structures that form a cluster in one area of the cell (Hermann et al., 1998). We observed a similar phenotype for H. polymorpha fzo1 ( Fig. 2A), which highly resembled the phenotype of H. polymorpha pex23 and pex29 cells. ...
Pex23 family proteins are membrane proteins of the endoplasmic reticulum that play a role in peroxisome and lipid body formation. The yeast Hansenula polymorpha contains four members: Pex23, Pex24, Pex29 and Pex32. We previously showed that the loss of Pex24 or Pex32 results in severe peroxisomal defects, caused by reduced peroxisome-endoplasmic reticulum membrane contact sites. We now analyzed whether the absence of Pex23 proteins affects other organelles. Vacuoles were normal in all deletion strains. The number of lipid droplets was reduced in pex23 and pex29, but not in pex24 and pex32, indicating that peroxisome and lipid droplet formation require different Pex23 proteins. In pex23 and pex29 cells, mitochondria were fragmented and clustered. This phenotype was not suppressed by an artificial mitochondria-endoplasmic reticulum tether, indicating that the abnormalities were not caused by reduced membrane contact sites. Deletion of DNM1 in pex23 cells partially suppressed the phenotype. Also, the level of the mitochondrial fusion protein Fzo1 was reduced in pex23 and pex29 cells. These observations indicate that certain Pex23 family proteins are required for normal mitochondrial fusion.
... However, understanding the precise role of mitochondrial remodeling in asymmetric inheritance is challenging, due to the intricate interplay between individual facets of mitochondria dynamics. Whereas mutants lacking core elements of either mitochondria fission, fusion, or trafficking machinery exhibit significant abnormalities in mitochondrial inheritance [43][44][45][46], perturbations in the proteins involved can have confounding effects such as altering mitochondria morphology and distribution [47,48], ER-contact sites [49][50][51], and a wide spectrum of cellular processes [52][53][54]. This complexity necessitates the combination of modeling and experiments to identify quantitative determinants of asymmetric mitochondria inheritance. ...
Mitochondria are essential and dynamic eukaryotic organelles that must be inherited during cell division. In yeast, mitochondria are inherited asymmetrically based on quality, which is thought to be vital for maintaining a rejuvenated cell population; however, the mechanisms underlying mitochondrial remodeling and segregation during this process are not understood. We used high spatiotemporal imaging to quantify the key aspects of mitochondrial dynamics, including motility, fission, and fusion characteristics, upon aggregation of misfolded proteins in the mitochondrial matrix. Using these measured parameters, we developed an agent-based stochastic model of dynamics of mitochondrial inheritance. Our model predicts that biased mitochondrial fission near the protein aggregates facilitates the clustering of protein aggregates in the mitochondrial matrix, and this process underlies asymmetric mitochondria inheritance. These predictions are supported by live-cell imaging experiments where mitochondrial fission was perturbed. Our findings therefore uncover an unexpected role of mitochondrial dynamics in asymmetric mitochondrial inheritance.
... The mitofusins Mfn1 and Mfn2 are found in mammals [3,4]. Fzo1 (Fuzzy Onion 1 ) is the only mitofusin homolog in Saccharomyces cerevisiae [5]. The structure of Mfn1 was partially solved, but without its transmembrane domain [6,7], and no solved structures are available for either Mfn2 or Fzo1. ...
Outer mitochondrial membrane (OMM) fusion is an important process for the cell and organism survival, as its dysfunction is linked to neurodegenerative diseases and cancer. The OMM fusion is mediated by members of the dynamin-related protein (DRP) family, named mitofusins. The exact mechanism by which the mitofusins contribute to these diseases, as well as the exact molecular fusion mechanism mediated by mitofusin, remains elusive.
We have performed extensive multiscale molecular dynamics simulations using both coarse-grained and all-atom approaches to predict the dimerization of two transmembrane domain (TM) helices of the yeast mitofusin Fzo1. We identify specific residues, such as Lys716, that can modulate dimer stability. Comparison with a previous computational model reveals remarkable differences in helix crossing angles and interfacial contacts. Overall, however, the TM1-TM2 interface appears to be stable in the Martini and CHARMM force fields. Replica-exchange simulations further tune a detailed atomistic model, as confirmed by a remarkable agreement with an independent prediction of the Fzo1-Ugo1 complex by AlphaFold2. Functional implications, including a possible role of Lys716 that could affect membrane interactions during fusion, are suggested and consistent with experiments monitoring mitochondrial respiration of selected Fzo1 mutants.
... Tubular mitochondrial networks are formed by a balance between opposing fission and fusion processes (Bleazard et al., 1999;Nunnari et al., 1997;Sesaki and Jensen, 1999), and we reasoned that the net-like morphology caused by the loss of Mdi1 could be due either to a deficiency in mitochondrial fission or excessive mitochondrial fusion. To address this, we utilized a temperature-sensitive allele of the gene encoding the mitochondrial OMM fusion DRP Fzo1 (fzo1-1; Hermann et al., 1998). Consistent with published observations, the growth of fzo1-1 cells at a non-permissive temperature (37°C) caused a deficiency in Fzo1-dependent mitochondrial fusion, and within 20 min, mitochondria appeared fragmented and/or aggregated (Fig. 1,C and D). ...
... Mitochondrial fusion is required for the maintenance of mtDNA in yeast and is therefore required for mitochondrial respiration (Hermann et al., 1998). Thus, fzo1-1 cells are inviable at elevated temperatures when grown on media containing a carbon source that requires respiration (ethanol/glycerol;Fig. 1 E;Hermann et al., 1998). ...
... Mitochondrial fusion is required for the maintenance of mtDNA in yeast and is therefore required for mitochondrial respiration (Hermann et al., 1998). Thus, fzo1-1 cells are inviable at elevated temperatures when grown on media containing a carbon source that requires respiration (ethanol/glycerol;Fig. 1 E;Hermann et al., 1998). However, simultaneous loss of both division and fusion machinery prevents mitochondrial fragmentation and allows for genome maintenance, albeit with an increased rate of mtDNA mutation and loss (Bleazard et al., 1999;Mozdy et al., 2000;Osman et al., 2015;Tieu and Nunnari, 2000). ...
Mitochondria are highly dynamic double membrane–bound organelles that maintain their shape in part through fission and fusion. Mitochondrial fission is performed by a dynamin-related protein, Dnm1 (Drp1 in humans), that constricts and divides the mitochondria in a GTP hydrolysis–dependent manner. However, it is unclear whether factors inside mitochondria help coordinate the process and if Dnm1/Drp1 activity is sufficient to complete the fission of both mitochondrial membranes. Here, we identify an intermembrane space protein required for mitochondrial fission in yeast, which we propose to name Mdi1 (also named Atg44). Loss of Mdi1 causes mitochondrial hyperfusion due to defects in fission, but not the lack of Dnm1 recruitment to mitochondria. Mdi1 is conserved in fungal species, and its homologs contain an amphipathic α-helix, mutations of which disrupt mitochondrial morphology. One model is that Mdi1 distorts mitochondrial membranes to enable Dnm1 to robustly complete fission. Our work reveals that Dnm1 cannot efficiently divide mitochondria without the coordinated function of Mdi1 inside mitochondria.
... A puzzling aspect of nebenkern formation is how the mitochondria fuse to form such an enormous organelle. Mitochondrial fusion is known to be mediated by the GTPases mitofusin-1 and mitofusin-2 (Mfn1, Mfn2) which are located in the outer mitochondrial membrane [23,47]. In D. melanogaster it has been shown that fuzzy onions (fzo) mutants show defects in nebenkern formation and lead to sterility [22]. ...
Spermatogenesis leads to the formation of functional sperm cells. Here we have applied high-pressure freezing in combination with transmission electron microscopy (TEM) to study the ultrastructure of sperm development in subadult males of the praying mantid Hierodula membranacea, a species in which spermatogenesis had not previously been studied. We show the ultrastructure of different stages of sperm development in this species. Thorough examination of TEM data and electron tomographic reconstructions revealed interesting structural features of the nebenkern, an organelle composed of fused mitochondria that has been studied in spermatids of other insect species. We have applied serial-section electron tomography of the nebenkern to demonstrate in three dimensions (3D) that this organelle in H. membranacea is composed of two interwoven mitochondrial derivatives, and that the mitochondrial derivatives are connected by a zipper-like structure at opposing positions. Our approach will enable further ultrastructural analyses of the nebenkern in other organisms.
... The increase in peroxisome-mitochondrion contacts due to Pex34 overproduction was accompanied by enhanced transport of acetyl-CoA between peroxisomes and mitochondria (Shai et al., 2018). In contrast, overproduction of ScFzo1, a mitochondrial outer membrane protein involved in mitochondrial fusion (Hermann et al., 1998), did not alter acetyl-CoA transport, implying that Fzo1 is a component of another MCS with yet unknown function (Shai et al., 2018). ...
Membrane contact sites are defined as regions of close proximity between two membranes; this association is mediated by protein-protein and/or protein-lipid interactions. Contact sites are often involved in lipid transport, but also can perform other functions. Peroxisomal membrane contact sites have obtained little attention compared to those of other cell organelles. However, recent studies resulted in a big leap in our knowledge of the occurrence, composition and function of peroxisomal contact sites. Studies in yeast strongly contributed to this progress. In this Review, we present an overview of our current knowledge on peroxisomal membrane contact sites in various yeast species, including Hansenula polymorpha, Saccharomyces cerevisiae, Pichia pastoris and Yarrowia lipolytica. Yeast peroxisomes form contacts with almost all other cellular organelles and with the plasma membrane. The absence of a component of a yeast peroxisomal contact site complex results in a range of peroxisomal phenotypes, including metabolic and biogenesis defects and alterations in organelle number, size or position.
... The 3xMyc-Fzo1 variants newly created in this study and used for analysis of yeast mating were generated by point mutagenesis in plasmid #350, 62 under the control of the GAL1 promoter and in the backbone pRS415 63 : C381S (#531), C805S (#532). ...
Defects in mitochondrial fusion are at the base of many diseases. Mitofusins power membrane-remodeling events via self-interaction and GTP hydrolysis. However, how exactly mitofusins mediate fusion of the outer membrane is still unclear. Structural studies enable tailored design of mitofusin variants, providing valuable tools to dissect this stepwise process. Here, we found that the two cysteines conserved between yeast and mammals are required for mitochondrial fusion, revealing two novel steps of the fusion cycle. C381 is dominantly required for the formation of the trans-tethering complex, before GTP hydrolysis. C805 allows stabilizing the Fzo1 protein and the trans-tethering complex, just prior to membrane fusion. Moreover, proteasomal inhibition rescued Fzo1 C805S levels and membrane fusion, suggesting a possible application for clinically approved drugs. Together, our study provides insights into how assembly or stability defects in mitofusins might cause mitofusin-associated diseases and uncovers potential therapeutic intervention by proteasomal inhibition.
