Human Fis1 Research Articles

Human Fis1 directly interacts with Drp1 in an evolutionarily conserved manner to promote mitochondrial fission

Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) are the only two proteins evolutionarily conserved for mitochondrial fission, and directly interact in Saccharomyces cerevisiae to facilitate membrane scission. However, it remains unclear if a direct interaction is conserved in higher eukaryotes as other Drp1 recruiters, not present in yeast, are known. Using NMR, differential scanning fluorimetry, and microscale thermophoresis, we determined that human Fis1 directly interacts with human Drp1 (KD= 12-68μM), and appears to prevent Drp1 assembly, but not GTP hydrolysis. Similar to yeast, the Fis1-Drp1 interaction appears governed by two structural features of Fis1: its N-terminal arm and a conserved surface. Alanine scanning mutagenesis of the arm identified both loss-of-function and gain-of-function alleles with mitochondrial morphologies ranging from highly elongated (N6A) to highly fragmented (E7A), demonstrating a profound ability of Fis1 to govern morphology in human cells. An integrated analysis identified a conserved Fis1 residue, Y76, that upon substitution to alanine, but not phenylalanine, also caused highly fragmented mitochondria. The similar phenotypic effects of the E7A and Y76A substitutions, along with NMR data, support that intramolecular interactions occur between the arm and a conserved surface on Fis1 to promote Drp1-mediated fission as in S.cerevisiae. These findings indicate that some aspects of Drp1-mediated fission in humans derive from direct Fis1-Drp1 interactions that are conserved across eukaryotes.

Structural studies of human FIS1 reveal a dynamic region important for DRP1 recruitment and mitochondrial fission

Mitochondrial fission protein 1 (Fis1) and dynamin-related protein 1 (Drp1) were initially described as being evolutionarily conserved for mitochondrial fission. However, the role of human Fis1 in this process remains unclear. In budding yeast, where Fis1 helps to recruit Drp1 from the cytoplasm to mitochondria for fission, the N-terminal “arm” of Fis1 is required for function. The yeast Fis1 arm interacts intramolecularly with a conserved concave surface that governs in vitro interactions with yeast Drp1.

Intramolecular regulation of Fis1 activity in fission and mitophagy

Since its initial identification in yeast, Fis1, a 17kDa mitochondria-outer-membrane protein has been implicated in mitochondrial dynamics, mitophagy, and apoptosis. Despite this importance, the mechanisms by which Fis1 activity is governed in these processes remain unclear. Structural considerations suggests that a highly flexible N-terminal “arm” may mediate interactions with cognate binding partners. We recently demonstrated that the human Fis1 arm, contrary to previous reports, is capable of assuming conformations akin to its distant relative, yeast Fis1p, suggesting a conserved mode of activity regulation across kingdoms. With this premise, we searched for amino acid sequences using structure and homology-based sequence alignments, which identified a uniquely conserved region that we hypothesize to be important for regulating Fis1 function. Using MD simulations, IF-microscopy, and NMR, we show that this region is important for Fis1 fission and mitophagic function.

Structural studies of human Fis1 reveals a dynamic region important for Drp1 recruitment and mitochondrial fission

Fission protein 1 (Fis1) and dynamin‐related protein 1 (Drp1) were initially described as being evolutionarily conserved for mitochondrial fission, yet the role of Fis1 in human fission is unclear and disputed by many. In budding yeast where Fis1 helps to recruit Drp1 from the cytoplasm to mitochondria for fission, an N‐terminal “arm” of Fis1 is required for function. The yeast Fis1 arm interacts intramolecularly with a conserved surface that governs in vitro interactions with yeast Drp1. In human Fis1, NMR and x‐ray structures show a different arm conformation and its importance for human Drp1 recruitment is unknown. Here, we use MD simulations and comparisons to experimental NMR chemical shifts to show the human Fis1 arm can adopt an intramolecular conformation akin to that observed with the yeast molecule. This finding is further supported through intrinsic tryptophan fluorescence and NMR experiments on human Fis1 with and without the arm. Using NMR, the human Fis1 arm is also observed to be sensitive to environmental changes. The importance of these findings are revealed in cellular studies where the removal of the hFis1 arm reduces Drp1 recruitment and mitochondrial fission suggesting an important role for Fis1 in human mitochondrial fission.Support or Funding InformationThis project was supported by the following National Institutes of Health grants: R01GM067180 (to RBH), and R01HL128240 (to MEW).

Human Fis1 regulates mitochondrial dynamics through inhibition of the fusion machinery

Mitochondrial dynamics is important for life. At center stage for mitochondrial dynamics, the balance between mitochondrial fission and fusion is a set of dynamin‐related GTPases that drive mitochondrial fission and fusion. Fission is executed by the GTPases Drp1 and Dyn2, whereas the GTPases Mfn1, Mfn2, and OPA1 promote fusion. Recruitment of Drp1 to mitochondria is a critical step in fission. In yeast, Fis1p recruits the Drp1 homolog Dnm1p to mitochondria through Mdv1p and Caf4p, but whether human Fis1 (hFis1) promotes fission through a similar mechanism as in yeast is not established. Here, we show that hFis1‐mediated mitochondrial fragmentation occurs in the absence of Drp1 and Dyn2, suggesting that they are dispensable for hFis1 function. hFis1 instead binds to Mfn1, Mfn2, and OPA1 and inhibits their GTPase activity, thus blocking the fusion machinery. Consistent with this, disruption of the fusion machinery in Drp1−/− cells phenocopies the fragmentation phenotype induced by hFis1 overexpression. In sum, our data suggest a novel role for hFis1 as an inhibitor of the fusion machinery, revealing an important functional evolutionary divergence between yeast and mammalian Fis1 proteins.

Removal of a consensus proline is not sufficient to allow tetratricopeptide repeat oligomerization.

Tetratricopeptide repeat (TPR) domains are ubiquitous protein interaction domains that adopt a modular antiparallel array of α-helices. The TPR fold typically adopts a monomeric state, and consensus TPRs sequences successfully fold into the expected monomeric topology. The versatility of the TPR fold also supports different quaternary structures, which may function as regulatory switches. One example is yeast mitochondrial fission 1 (Fis1) that appears to interconvert between monomer and dimer states in regulating division of peroxisomes and mitochondria. Whether human Fis1 can also interconvert like the yeast molecule is unknown. A TPR consensus proline residue present in human Fis1 is absent in the yeast molecule and, when added, prevents yeast Fis1 dimerization suggesting that the TPR consensus proline might have persisted to prevent TPR oligomerization. Here, we address this question with human Fis1 and the consensus TPR protein CTPR3. We demonstrate that human Fis1 does not form a noncovalent dimer via its TPR domain, despite conditions that favor dimerization of the yeast protein. We also show that the presence of the consensus proline is not sufficient to forbid TPR dimerization. Lastly, an analysis of all available TPR protein structures (22 nonredundant structures, totaling 64 TPRs-42 with the consensus proline and 22 without) revealed that the consensus proline is not necessary for turn formation, but does favor shorter turns. This work suggests the TPR consensus proline is not to prevent oligomerization, but to favor tight turns between repeats.

FIS1 and DNM1L Cooperate in Mitochondrial Fission: Convergence of Evolution and Intelligent Design

Mitochondrial fission helps to maintain proper mitochondrial homeostasis in a poorly understood manner despite its association with human disease. Several proteins have been identified in this process, but only two -- FIS1 and DNM1L -- are found in every species that contain mitochondria. We asked whether these proteins might cooperate together in this process. We found that FIS1 has the ability to directly recruit DNM1L, but is auto-inhibited by an N-terminal arm. A point mutant was rationally designed to relieve auto-inhibition and found to increase DNM1L binding and impair mitochondrial fission. This point mutant also increased the population of a latent dimeric state of FIS1 highlighting the complex nature of protein-protein interactions in fission. To address this, we devised an unbiased and general method to rapidly identify residues critical to protein interfaces and applied this technology to yeast Fis1 interactions. Of the >3000 Fis1 alleles screened, ∼9% selectively disrupted interactions with one of the three protein partners including DNM1L. To test the functional consequences, each allele was parsed into its corresponding point mutation and tested for mitochondrial fission. Of 211 yeast Fis1 mutants tested to date, 97 resulted in nonfunctional fission indicating that our method identifies residues essential for mitochondrial fission. Orthologous mutations were introduced into human Fis1 and also found to impair interactions with DNM1L and mitochondrial morphology. Analysis of these data supports a new model for the assembly of the mitochondrial fission machinery.

Fis1 and Drp1 cooperate in mitochondrial homeostasis (756.8)

Defects in mitochondrial fission and fusion cause numerous human disorders yet how these defects affect mitochondrial function and contribute to disease is unknown. Mitochondrial fission requires the dynamin‐related GTPase Drp1 and a lethal mutation (A395D) was reported in a neonate with microcephaly, abnormal brain development, optic atrophy, and lactic acidemia (Waterham et al., 2007, N. Engl. J. Med. 356, 1736‐1741). Another Drp1 mutation (C446F) was reported to cause severe cardiomyopathy in mice (Ashrafian et al., 2010, PLoS Genet 6, e1001000). We are determining how these mutants affect mechanoenzyme activity and function. For A395D, we found impaired localization, recruitment, assembly, and GTP hydrolysis. For C446F, we found impaired GTP hydrolysis and assembly. Thus, these mutants impair higher order assembly of Drp1 at mitochondria, leading to decreased fission, elongated mitochondria, and altered cellular distribution of mitochondria. Curiously, these mutants show enhanced in vitro interactions with the mitochondrial protein Fis1, a possible recruiter of Drp1. To determine the consequences of disrupting these interactions, we devised an unbiased and general method to rapidly identify residues critical to protein interfaces and applied this technology to yeast Fis1 interactions. Of the >3000 Fis1 alleles screened, ~9% selectively disrupted interactions with one of the three protein partners including yeast Drp1. To test the functional consequences, each allele was parsed into its corresponding point mutation and tested for mitochondrial fission. Of 211 yeast Fis1 mutants tested to date, 97 resulted in nonfunctional fission indicating that our method identifies residues essential for mitochondrial fission. Orthologous mutations were introduced into human Fis1 and also found to impair interactions with Drp1 and mitochondrial morphology consistent with a role for Fis1 in mitochondrial homeostasis.Grant Funding Source: RO1‐GM067180

Identification and Characterization of Unique Proline-rich Peptides Binding to the Mitochondrial Fission Protein hFis1

Mammalian mitochondrial fission requires at least two proteins, hFis1 and the dynamin-like GTPase DLP1/Drp1. The mitochondrial protein hFis1 is anchored at the outer membrane by a C-terminal transmembrane domain. The cytosolic domain of hFis1 contains six alpha helices [alpha1-alpha6] out of which [alpha2-alpha5] form tetratricopeptide repeat (TPR)-like motifs. DLP1 and possibly other proteins are thought to interact with the hFis1 TPR region during the fission process. It has also been suggested that the alpha1-helix regulates protein-protein interactions at the TPR. We performed random peptide phage display screening using the hFis1[alpha2-alpha6] as the target and identified ten different peptide sequences. Phage ELISA using mutant hFis1 indicates that the peptide binding requires the alpha2 and alpha3 helices and the intact TPR structure. Competition experiments and surface plasmon resonance analyses confirmed that a subset of free peptides enriched with proline residues directly bind to the target. Two of these peptides bind to the alpha1-containing intact cytosolic domain of hFis1 with decreased affinity. Peptide microinjection into cells abolished the mitochondrial swelling induced by overexpression of alpha1-deleted hFis1, and significantly decreased cytochrome c release from mitochondria upon apoptotic induction. Our data demonstrate that hFis1 can bind to multiple amino acid sequences selectively, and that the TPR constitutes the main binding region of hFis1, providing a first insight into the hFis1 TPR as a potential therapeutic target.

Endophilin B1/Bif-1 Stimulates BAX Activation Independently from Its Capacity to Produce Large Scale Membrane Morphological Rearrangements

Endophilin B1/BAX-interacting factor 1 (Bif-1) is a protein that cooperates with dynamin-like protein 1 (DLP1/Drp1) to maintain normal mitochondrial outer membrane (MOM) dynamics in healthy cells and also contributes to BAX-driven MOM permeabilization (MOMP), the irreversible commitment point to cell death for the majority of apoptotic stimuli. However, despite its importance, exactly how Bif-1 fulfils its proapoptotic role is unknown. Here, we demonstrate that the stimulatory effect of Bif-1 on BAX-driven MOMP and on BAX conformational activation observed in intact cells during apoptosis can be recapitulated in a simplified system consisting of purified proteins and MOM-like liposomes. In this reconstituted model system the N-BAR domain of Bif-1 reproduced the stimulatory effect of Bif-1 on functional BAX activation. This process was dependent on physical interaction between Bif-1 N-BAR and BAX as well as on the presence of the mitochondrion-specific lipid cardiolipin. Despite that Bif-1 N-BAR produced large scale morphological rearrangements in MOM-like liposomes, this phenomenon could be separated from functional BAX activation. Furthermore, DLP1 also caused global morphological changes in MOM-like liposomes, but DLP1 did not stimulate BAX-permeabilizing function in the absence or presence of Bif-1. Taken together, our findings not only provide direct evidence for a functional interplay between Bif-1, BAX, and cardiolipin during MOMP but also add significantly to the growing body of evidence indicating that components of the mitochondrial morphogenesis machinery possess proapoptotic functions that are independent from their recognized roles in normal mitochondrial dynamics.

Caenorhabditis elegans drp-1 and fis-2 Regulate Distinct Cell-Death Execution Pathways Downstream of ced-3 and Independent of ced-9

The dynamin family of GTPases regulate mitochondrial fission and fusion processes and have been implicated in controlling the release of caspase activators from mitochondria during apoptosis. Here we report that profusion genes fzo-1 and eat-3 or the profission gene drp-1 are not required for apoptosis activation in C. elegans. However, minor proapoptotic roles for drp-1 and fis-2, a homolog of human Fis1, are revealed in sensitized genetic backgrounds. drp-1 and fis-2 function independent of one another and the Bcl-2 homolog CED-9 and downstream of the CED-3 caspase to promote elimination of mitochondria in dying cells, an event that could facilitate cell-death execution. Interestingly, CED-3 can cleave DRP-1, which appears to be important for DRP-1's proapoptotic function, but not its mitochondria fission function. Our findings demonstrate that mitochondria dynamics do not regulate apoptosis activation in C. elegans and reveal distinct roles for drp-1 and fis-2 as mediators of cell-death execution downstream of caspase activation.

Novel Structure of the N Terminus in Yeast Fis1 Correlates with a Specialized Function in Mitochondrial Fission

Mitochondrial fission is facilitated by a multiprotein complex assembled at the division site. The required components of the fission machinery in Saccharomyces cerevisiae include Dnm1, Fis1, and Mdv1. In the present study, we determined the protein structure of yeast Fis1 using NMR spectroscopy. Although the six alpha-helices, as well as their folding, in the yeast Fis1 structure are similar to those of the tetratricopeptide repeat (TPR) domains of the human Fis1 structure, the two structures differ in their N termini. The N-terminal tail of human Fis1 is flexible and unstructured, whereas a major segment of the longer N terminus of yeast Fis1 is fixed to the concave face formed by the six alpha-helices in the TPR domains. To investigate the role of the fixed N terminus, exogenous Fis1 was expressed in yeast lacking the endogenous protein. Expression of yeast Fis1 protein rescued mitochondrial fission in delta fis1 yeast only when the N-terminal TPR binding segment was left intact. The presence of this segment is also correlated to the recruitment of Mdv1 to mitochondria. The conformation of the N-terminal segment embedded in the TPR pocket indicates an intra-molecular regulation of Fis1 bioactivity. Although the TPR-like helix bundle of Fis1 mediates the interaction with Dnm1 and Mdv1, the N terminus of Fis1 is a prerequisite to recruit Mdv1 to facilitate mitochondrial fission.

Establishment of Stable hFis1 Knockdown Cells with an siRNA Expression Vector

Yeast Fis1p participates in mitochondrial fission, together with Dnm1p and Mdv1p. Recently, human Fis1 (hFis1) was reported to be involved in mitochondrial fission, together with Drp1. We established stable transformants with an hFis1 siRNA expression vector. In the stable hFis1 knockdown cells, hFis1 expression was suppressed to approximately 10%, and mitochondrial fission, induced by cisplatin treatment, was delayed. In addition, mouse Fis1 (mFis1) expression promoted mitochondrial fission and cell death in the hFis1 knockdown cells, suggesting that mFis1 complements the function of hFis1. These hFis1 siRNA expression vectors may be useful for studying the molecular function of mammalian Fis1.

Levels of human Fis1 at the mitochondrial outer membrane regulate mitochondrial morphology.

Mitochondria undergo balanced fission and fusion events that enable their appropriate networking within the cell. In yeast, three factors have been identified that co-ordinate fission events at the mitochondrial outer membrane. Fis1p acts as the outer membrane receptor for recruitment of the dynamin member, Dnm1p and the WD40-repeat-containing protein Mdv1p. In mammals, the Dnm1p counterpart Drp1 has been characterized, but other components have not. Here, we report the characterization of human Fis1 (hFis1). hFis1 is inserted into the mitochondrial outer membrane via a C-terminal transmembrane domain that, along with a short basic segment, is essential for its targeting. Although expression of hFis1 does not complement the phenotype of yeast cells lacking Fis1p, overexpression of hFis1 in tissue culture cells nevertheless causes mitochondrial fragmentation and aggregation. This aggregation could be suppressed by expressing a dominant-negative Drp1 mutant (Drp1(K38A)). Knockdown of hFis1 in COS-7 cells using RNA interference results in mitochondrial morphology defects with notable extensions in the length of mitochondrial tubules. These results indicate that the levels of hFis1 at the mitochondrial surface influences mitochondrial fission events and hence overall mitochondrial morphology within the cell.

Cytosolic domain of the human mitochondrial fission protein fis1 adopts a TPR fold.

Fis1 is an integral membrane protein that acts in the fission of mitochondria by controlling the assembly, membrane distribution, and function of the mitochondrial fission machinery. Here we report the 2.0 A resolution crystal structure of the cytosolic domain of human Fis1. The structure reveals an antiparallel array of α-helices homologous to tetratricopeptide repeat (TPR) proteins. Structure-based sequence alignments of Fis1 uncovered two divergent TPR motifs; the first TPR motif differs from the TPR consensus sequence by a three-residue insertion in a loop that may be important for function. These TPR helices create an amphiphilic, concave surface that can accommodate a helix or, possibly, an extended segment. Indeed, this putative binding surface mediates homodimer formation of Fis1 in the crystal. The structure of Fis1 provides insight into the architecture of the proposed binding interactions that mediate mitochondrial fission.

The Solution Structure of Human Mitochondria Fission Protein Fis1 Reveals a Novel TPR-like Helix Bundle

Fis1 in yeast localizes to the outer mitochondrial membrane and facilitates mitochondrial fission by forming protein complexes with Dnm1 and Mdv1. Fis1 orthologs exist in higher eukaryotes, suggesting that they are functionally conserved. In the present study, we cloned the human Fis1 ortholog that was predicted in a database, and determined the protein structure using NMR spectroscopy. Following a flexible N-terminal tail, six α-helices connected with short loops construct a single core domain. The C-terminal tail containing a transmembrane segment appears to be disordered. In the core domain, each of two sequentially adjacent helices forms a hairpin-like conformation, resulting in a six helix assembly forming a slightly twisted slab similar to that of a tandem array of tetratrico-peptide repeat (TPR) motif folds. Within this TPR-like core domain, no significant sequence similarity to the typical TPR motif is found. The structural analogy to the TPR-containing proteins suggests that Fis1 binds to other proteins at its concave hydrophobic surface. A simple composition of Fis1 comprised of a binding domain and a transmembrane segment indicates that the protein may function as a molecular adaptor on the mitochondrial outer membrane. In HeLa cells, however, increased levels in mitochondria-associated Fis1 did not result in mitochondrial translocation of Drp1, a potential binding partner of Fis1 implicated in the regulation of mitochondrial fission, suggesting that the interaction between Drp1 and Fis1 is regulated.