scholarly journals NMR identification of a conserved Drp1 cardiolipin-binding motif essential for stress-induced mitochondrial fission

2021 ◽  
Vol 118 (29) ◽  
pp. e2023079118
Author(s):  
Mukesh Mahajan ◽  
Nikhil Bharambe ◽  
Yutong Shang ◽  
Bin Lu ◽  
Abhishek Mandal ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Fission ◽  
Stress Conditions ◽  
Variable Domain ◽  
Mitochondrial Outer Membrane ◽  
Membrane Insertion ◽  
Binding Motif ◽  
Related Protein ◽  
Conformational Rearrangements ◽  
Nmr Identification

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane–localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD–CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce “donut” mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1–CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1–CL interactions in stress-induced mitochondrial fission.

scholarly journals Mutations in Fis1 disrupt orderly disposal of defective mitochondria

2014 ◽  
Vol 25 (1) ◽  
pp. 145-159 ◽  
Author(s):  
Qinfang Shen ◽  
Koji Yamano ◽  
Brian P. Head ◽  
Sumihiro Kawajiri ◽  
Jesmine T. M. Cheung ◽  
...  
Keyword(s):  
Endoplasmic Reticulum ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Couple Stress ◽  
Mitochondrial Fission ◽  
Mitochondrial Outer Membrane ◽  
Related Protein ◽  
Degradation Processes ◽  
Mitochondrial Toxins

Mitochondrial fission is mediated by the dynamin-related protein Drp1 in metazoans. Drp1 is recruited from the cytosol to mitochondria by the mitochondrial outer membrane protein Mff. A second mitochondrial outer membrane protein, named Fis1, was previously proposed as recruitment factor, but Fis1−/− cells have mild or no mitochondrial fission defects. Here we show that Fis1 is nevertheless part of the mitochondrial fission complex in metazoan cells. During the fission cycle, Drp1 first binds to Mff on the surface of mitochondria, followed by entry into a complex that includes Fis1 and endoplasmic reticulum (ER) proteins at the ER–mitochondrial interface. Mutations in Fis1 do not normally affect fission, but they can disrupt downstream degradation events when specific mitochondrial toxins are used to induce fission. The disruptions caused by mutations in Fis1 lead to an accumulation of large LC3 aggregates. We conclude that Fis1 can act in sequence with Mff at the ER–mitochondrial interface to couple stress-induced mitochondrial fission with downstream degradation processes.

scholarly journals Fis1, Mff, MiD49, and MiD51 mediate Drp1 recruitment in mitochondrial fission

2013 ◽  
Vol 24 (5) ◽  
pp. 659-667 ◽  
Author(s):  
Oliver C. Losón ◽  
Zhiyin Song ◽  
Hsiuchen Chen ◽  
David C. Chan
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Outer Membrane Proteins ◽  
Mitochondrial Outer Membrane ◽  
Related Protein ◽  
Immunofluorescence Analysis ◽  
Multiple Receptors ◽  
Mitochondrial Elongation

Several mitochondrial outer membrane proteins—mitochondrial fission protein 1 (Fis1), mitochondrial fission factor (Mff), mitochondrial dynamics proteins of 49 and 51 kDa (MiD49 and MiD51, respectively)—have been proposed to promote mitochondrial fission by recruiting the GTPase dynamin-related protein 1 (Drp1), but fundamental issues remain concerning their function. A recent study supported such a role for Mff but not for Fis1. In addition, it is unclear whether MiD49 and MiD51 activate or inhibit fission, because their overexpression causes extensive mitochondrial elongation. It is also unknown whether these proteins can act in the absence of one another to mediate fission. Using Fis1-null, Mff-null, and Fis1/Mff-null cells, we show that both Fis1 and Mff have roles in mitochondrial fission. Moreover, immunofluorescence analysis of Drp1 suggests that Fis1 and Mff are important for the number and size of Drp1 puncta on mitochondria. Finally, we find that either MiD49 or MiD51 can mediate Drp1 recruitment and mitochondrial fission in the absence of Fis1 and Mff. These results demonstrate that multiple receptors can recruit Drp1 to mediate mitochondrial fission.

scholarly journals The mitochondrial fission receptor Mff selectively recruits oligomerized Drp1

2015 ◽  
Vol 26 (24) ◽  
pp. 4466-4477 ◽  
Author(s):  
Raymond Liu ◽  
David C. Chan
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Fission ◽  
Higher Order ◽  
Mitochondrial Outer Membrane ◽  
Stable Complex ◽  
Related Protein ◽  
Biochemical Assays ◽  
Order Complexes

Dynamin-related protein 1 (Drp1) is the GTP-hydrolyzing mechanoenzyme that catalyzes mitochondrial fission in the cell. Residing in the cytosol as dimers and tetramers, Drp1 is recruited by receptors on the mitochondrial outer membrane, where it further assembles into a helical ring that drives division via GTP-dependent constriction. The Drp1 receptor Mff is a major regulator of mitochondrial fission, and its overexpression results in increased fission. In contrast, the alternative Drp1 receptors MiD51 and MiD49 appear to recruit inactive forms of Drp1, because their overexpression inhibits fission. Using genetic and biochemical assays, we studied the interaction of Drp1 with Mff. We show that the insert B region of Drp1 inhibits Mff–Drp1 interactions, such that recombinant Drp1 mutants lacking insert B form a stable complex with Mff. Mff cannot bind to assembly-deficient mutants of Drp1, suggesting that Mff selectively interacts with higher-order complexes of Drp1. In contrast, the alternative Drp1 receptors MiD51 and MiD49 can recruit Drp1 dimers. Therefore Drp1 recruitment by Mff versus MiD51 and MiD49 may result in different outcomes because they recruit different subpopulations of Drp1 from the cytosol.

scholarly journals Structural Insights into Bak Activation and Oligomerisation

2014 ◽  
Vol 70 (a1) ◽  
pp. C1166-C1166
Author(s):  
Jason Brouwer ◽  
Adeline Robin ◽  
Geoff Thompson ◽  
Ahmad Wardak ◽  
Ruth Kluck ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Cell Death ◽  
Outer Membrane ◽  
Crystal Structures ◽  
Conformational Changes ◽  
Mitochondrial Outer Membrane ◽  
Related Protein ◽  
Mitochondrial Outer Membrane Permeabilisation ◽  
Structural Insights ◽  
Analogous Mechanism

Apoptotic stimuli activate and oligomerise the pro-apoptotic proteins Bak and Bax resulting in mitochondrial outer membrane permeabilisation and subsequent cell death. This activation can occur when certain BH3-only proteins directly interact with Bak and Bax. A recent crystal structure by Czabotar et al. (2013) revealed a novel conformational change for Bax upon activation by BH3-only peptides. Distinguishing characteristics of BH3-only proteins capable of directly activating Bax were also elucidated. Here we describe complementary studies on the related protein Bak. We identify specific BH3-only peptides capable of inducing Bak dimerisation and describe crystal structures that provide key insights into Bak activation and oligomerisation. These structures demonstrate that Bak undergoes similar conformational changes upon activation to those observed with Bax. Altogether our results confirm an analogous mechanism for activation and dimerization of Bak and Bax in response to BH3-only peptides.

scholarly journals The WD40 protein Caf4p is a component of the mitochondrial fission machinery and recruits Dnm1p to mitochondria

2005 ◽  
Vol 170 (2) ◽  
pp. 237-248 ◽  
Author(s):  
Erik E. Griffin ◽  
Johannes Graumann ◽  
David C. Chan
Keyword(s):  
Mitochondrial Dynamics ◽  
Mitochondrial Fission ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Related Protein ◽  
Wd40 Repeat ◽  
Mitochondrial Division ◽  
Late Step ◽  
Wd40 Domain ◽  
Molecular Adaptors

The mitochondrial division machinery regulates mitochondrial dynamics and consists of Fis1p, Mdv1p, and Dnm1p. Mitochondrial division relies on the recruitment of the dynamin-related protein Dnm1p to mitochondria. Dnm1p recruitment depends on the mitochondrial outer membrane protein Fis1p. Mdv1p interacts with Fis1p and Dnm1p, but is thought to act at a late step during fission because Mdv1p is dispensable for Dnm1p localization. We identify the WD40 repeat protein Caf4p as a Fis1p-associated protein that localizes to mitochondria in a Fis1p-dependent manner. Caf4p interacts with each component of the fission apparatus: with Fis1p and Mdv1p through its NH2-terminal half and with Dnm1p through its COOH-terminal WD40 domain. We demonstrate that mdv1Δ yeast contain residual mitochondrial fission due to the redundant activity of Caf4p. Moreover, recruitment of Dnm1p to mitochondria is disrupted in mdv1Δ caf4Δ yeast, demonstrating that Mdv1p and Caf4p are molecular adaptors that recruit Dnm1p to mitochondrial fission sites. Our studies support a revised model for assembly of the mitochondrial fission apparatus.

scholarly journals The Dynamin-Related Gtpase, Mgm1p, Is an Intermembrane Space Protein Required for Maintenance of Fusion Competent Mitochondria

2000 ◽  
Vol 151 (2) ◽  
pp. 341-352 ◽  
Author(s):  
Edith D. Wong ◽  
Jennifer A. Wagner ◽  
Steven W. Gorsich ◽  
J. Michael McCaffery ◽  
Janet M. Shaw ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Fission ◽  
Mitochondrial Fusion ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Morphology ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Direct Role ◽  
Membrane Fission ◽  
Mitochondrial Intermembrane Space

Mutations in the dynamin-related GTPase, Mgm1p, have been shown to cause mitochondrial aggregation and mitochondrial DNA loss in Saccharomyces cerevisiae cells, but Mgm1p's exact role in mitochondrial maintenance is unclear. To study the primary function of MGM1, we characterized new temperature sensitive MGM1 alleles. Examination of mitochondrial morphology in mgm1 cells indicates that fragmentation of mitochondrial reticuli is the primary phenotype associated with loss of MGM1 function, with secondary aggregation of mitochondrial fragments. This mgm1 phenotype is identical to that observed in cells with a conditional mutation in FZO1, which encodes a transmembrane GTPase required for mitochondrial fusion, raising the possibility that Mgm1p is also required for fusion. Consistent with this idea, mitochondrial fusion is blocked in mgm1 cells during mating, and deletion of DNM1, which encodes a dynamin-related GTPase required for mitochondrial fission, blocks mitochondrial fragmentation in mgm1 cells. However, in contrast to fzo1 cells, deletion of DNM1 in mgm1 cells restores mitochondrial fusion during mating. This last observation indicates that despite the phenotypic similarities observed between mgm1 and fzo1 cells, MGM1 does not play a direct role in mitochondrial fusion. Although Mgm1p was recently reported to localize to the mitochondrial outer membrane, our studies indicate that Mgm1p is localized to the mitochondrial intermembrane space. Based on our localization data and Mgm1p's structural homology to dynamin, we postulate that it functions in inner membrane remodeling events. In this context, the observed mgm1 phenotypes suggest that inner and outer membrane fission is coupled and that loss of MGM1 function may stimulate Dnm1p-dependent outer membrane fission, resulting in the formation of mitochondrial fragments that are structurally incompetent for fusion.

scholarly journals The Omp85 family of proteins is essential for outer membrane biogenesis in mitochondria and bacteria

2003 ◽  
Vol 164 (1) ◽  
pp. 19-24 ◽  
Author(s):  
Ian Gentle ◽  
Kipros Gabriel ◽  
Peter Beech ◽  
Ross Waller ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Mitochondrial Fission ◽  
Anion Channel ◽  
Mitochondrial Outer Membrane ◽  
Organelle Biogenesis ◽  
Voltage Dependent Anion Channel ◽  
Protein Insertion ◽  
Outer Membrane Biogenesis ◽  
Integral Proteins

Integral proteins in the outer membrane of mitochondria control all aspects of organelle biogenesis, being required for protein import, mitochondrial fission, and, in metazoans, mitochondrial aspects of programmed cell death. How these integral proteins are assembled in the outer membrane had been unclear. In bacteria, Omp85 is an essential component of the protein insertion machinery, and we show that members of the Omp85 protein family are also found in eukaryotes ranging from plants to humans. In eukaryotes, Omp85 is present in the mitochondrial outer membrane. The gene encoding Omp85 is essential for cell viability in yeast, and conditional omp85 mutants have defects that arise from compromised insertion of integral proteins like voltage-dependent anion channel (VDAC) and components of the translocase in the outer membrane of mitochondria (TOM) complex into the mitochondrial outer membrane.

scholarly journals Mdv1p Is a Wd Repeat Protein That Interacts with the Dynamin-Related Gtpase, Dnm1p, to Trigger Mitochondrial Division

2000 ◽  
Vol 151 (2) ◽  
pp. 353-366 ◽  
Author(s):  
Quinton Tieu ◽  
Jodi Nunnari
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Membrane ◽  
Mitochondrial Fission ◽  
Coiled Coil ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Targeting ◽  
Mitochondrial Membranes ◽  
Mitochondrial Division ◽  
Targeting Signal ◽  
Two Hybrid

Mitochondrial fission is mediated by the dynamin-related GTPase, Dnm1p, which assembles on the mitochondrial outer membrane into punctate structures associated with sites of membrane constriction and fission. We have identified additional nuclear genes required for mitochondrial fission, termed MDV (for mitochondrial division). MDV1 encodes a predicted soluble protein, containing a coiled-coil motif and seven COOH-terminal WD repeats. Genetic and two-hybrid analyses indicate that Mdv1p interacts with Dnm1p to mediate mitochondrial fission. In addition, Mdv1p colocalizes with Dnm1p in fission-mediating punctate structures on the mitochondrial outer membrane. Whereas localization of Mdv1p to these structures requires Dnm1p, localization of Mdv1p to mitochondrial membranes does not. This indicates that Mdv1p possesses a Dnm1p-independent mitochondrial targeting signal. Dnm1p-independent targeting of Mdv1p to mitochondria requires MDV2. Our data indicate that MDV2 also functions separately to regulate the assembly of Dnm1p into punctate structures. In contrast, Mdv1p is not required for the assembly of Dnm1p, but Dnm1p-containing punctate structures lacking Mdv1p are not able to complete division. Our studies suggest that mitochondrial fission is a multi-step process in which Mdv2p regulates the assembly of Dnm1p into punctate structures and together with Mdv1p functions later during fission to facilitate Dnm1p-dependent mitochondrial membrane constriction and/or division.

scholarly journals Identification of new channels by systematic analysis of the mitochondrial outer membrane

2017 ◽  
Vol 216 (11) ◽  
pp. 3485-3495 ◽  
Author(s):  
Vivien Krüger ◽  
Thomas Becker ◽  
Lars Becker ◽  
Malayko Montilla-Martinez ◽  
Lars Ellenrieder ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Protein Import ◽  
Molecular Level ◽  
Anion Channel ◽  
Helical Structure ◽  
Mitochondrial Outer Membrane ◽  
Related Protein ◽  
Voltage Dependent Anion Channel ◽  
Systematic Analysis ◽  
Voltage Dependent

The mitochondrial outer membrane is essential for communication between mitochondria and the rest of the cell and facilitates the transport of metabolites, ions, and proteins. All mitochondrial outer membrane channels known to date are β-barrel membrane proteins, including the abundant voltage-dependent anion channel and the cation-preferring protein-conducting channels Tom40, Sam50, and Mdm10. We analyzed outer membrane fractions of yeast mitochondria and identified four new channel activities: two anion-preferring channels and two cation-preferring channels. We characterized the cation-preferring channels at the molecular level. The mitochondrial import component Mim1 forms a channel that is predicted to have an α-helical structure for protein import. The short-chain dehydrogenase-related protein Ayr1 forms an NADPH-regulated channel. We conclude that the mitochondrial outer membrane contains a considerably larger variety of channel-forming proteins than assumed thus far. These findings challenge the traditional view of the outer membrane as an unspecific molecular sieve and indicate a higher degree of selectivity and regulation of metabolite fluxes at the mitochondrial boundary.

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