scholarly journals Coassembly of Mgm1 isoforms requires cardiolipin and mediates mitochondrial inner membrane fusion

2009 ◽  
Vol 186 (6) ◽  
pp. 793-803 ◽  
Author(s):  
Rachel M. DeVay ◽  
Lenin Dominguez-Ramirez ◽  
Laura L. Lackner ◽  
Suzanne Hoppins ◽  
Henning Stahlberg ◽  
...  
Keyword(s):  
Membrane Fusion ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Related Protein ◽  
Mitochondrial Membranes ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
In Trans

Two dynamin-related protein (DRP) families are essential for fusion of the outer and inner mitochondrial membranes, Fzo1 (yeast)/Mfn1/Mfn2 (mammals) and Mgm1 (yeast)/Opa1 (mammals), respectively. Fzo1/Mfns possess two medial transmembrane domains, which place their critical GTPase and coiled-coil domains in the cytosol. In contrast, Mgm1/Opa1 are present in cells as long (l) isoforms that are anchored via the N terminus to the inner membrane, and short (s) isoforms were predicted to be soluble in the intermembrane space. We addressed the roles of Mgm1 isoforms and how DRPs function in membrane fusion. Our analysis indicates that in the absence of a membrane, l- and s-Mgm1 both exist as inactive GTPase monomers, but that together in trans they form a functional dimer in a cardiolipin-dependent manner that is the building block for higher-order assemblies.

scholarly journals Formation of Membrane-bound Ring Complexes by Prohibitins in Mitochondria

2005 ◽  
Vol 16 (1) ◽  
pp. 248-259 ◽  
Author(s):  
Takashi Tatsuta ◽  
Kirstin Model ◽  
Thomas Langer
Keyword(s):  
Cell Cycle Progression ◽  
Coiled Coil ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
High Molecular Weight Complex ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Membrane Bound ◽  
Ring Complexes ◽  
Carboxy Terminal

Prohibitins comprise a remarkably conserved protein family in eukaryotic cells with proposed functions in cell cycle progression, senescence, apoptosis, and the regulation of mitochondrial activities. Two prohibitin homologues, Phb1 and Phb2, assemble into a high molecular weight complex of ∼1.2 MDa in the mitochondrial inner membrane, but a nuclear localization of Phb1 and Phb2 also has been reported. Here, we have analyzed the biogenesis and structure of the prohibitin complex in Saccharomyces cerevisiae. Both Phb1 and Phb2 subunits are targeted to mitochondria by unconventional noncleavable targeting sequences at their amino terminal end. Membrane insertion involves binding of newly imported Phb1 to Tim8/13 complexes in the intermembrane space and is mediated by the TIM23-translocase. Assembly occurs via intermediate-sized complexes of ∼120 kDa containing both Phb1 and Phb2. Conserved carboxy-terminal coiled-coil regions in both subunits mediate the formation of large assemblies in the inner membrane. Single particle electron microscopy of purified prohibitin complexes identifies diverse ring-shaped structures with outer dimensions of ∼270 × 200 Å. Implications of these findings for proposed cellular activities of prohibitins are discussed.

scholarly journals The mitochondrial carrier pathway transports non-canonical substrates with an odd number of transmembrane segments

BMC Biology ◽  
2020 ◽  
Vol 18 (1) ◽  
Author(s):  
Heike Rampelt ◽  
Iva Sucec ◽  
Beate Bersch ◽  
Patrick Horten ◽  
Inge Perschil ◽  
...  
Keyword(s):  
Intermembrane Space ◽  
Inner Mitochondrial Membrane ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Transmembrane Segments ◽  
Mitochondrial Inner Membrane ◽  
N Terminus ◽  
The Matrix ◽  
Mitochondrial Carrier Family ◽  
Mitochondrial Intermembrane Space

Abstract Background The mitochondrial pyruvate carrier (MPC) plays a central role in energy metabolism by transporting pyruvate across the inner mitochondrial membrane. Its heterodimeric composition and homology to SWEET and semiSWEET transporters set the MPC apart from the canonical mitochondrial carrier family (named MCF or SLC25). The import of the canonical carriers is mediated by the carrier translocase of the inner membrane (TIM22) pathway and is dependent on their structure, which features an even number of transmembrane segments and both termini in the intermembrane space. The import pathway of MPC proteins has not been elucidated. The odd number of transmembrane segments and positioning of the N-terminus in the matrix argues against an import via the TIM22 carrier pathway but favors an import via the flexible presequence pathway. Results Here, we systematically analyzed the import pathways of Mpc2 and Mpc3 and report that, contrary to an expected import via the flexible presequence pathway, yeast MPC proteins with an odd number of transmembrane segments and matrix-exposed N-terminus are imported by the carrier pathway, using the receptor Tom70, small TIM chaperones, and the TIM22 complex. The TIM9·10 complex chaperones MPC proteins through the mitochondrial intermembrane space using conserved hydrophobic motifs that are also required for the interaction with canonical carrier proteins. Conclusions The carrier pathway can import paired and non-paired transmembrane helices and translocate N-termini to either side of the mitochondrial inner membrane, revealing an unexpected versatility of the mitochondrial import pathway for non-cleavable inner membrane proteins.

scholarly journals Ribosome-Associated Mba1 Escorts Cox2 from Insertion Machinery to Maturing Assembly Intermediates

2016 ◽  
Vol 36 (22) ◽  
pp. 2782-2793 ◽  
Author(s):  
Isotta Lorenzi ◽  
Silke Oeljeklaus ◽  
Christin Ronsör ◽  
Bettina Bareth ◽  
Bettina Warscheid ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Scaffold Protein ◽  
Intermembrane Space ◽  
Dependent Manner ◽  
Inner Membrane ◽  
Content Type ◽  
Mitochondrial Ribosomes ◽  
C Terminus ◽  
N Terminus ◽  
Conserved Core

The three conserved core subunits of the cytochrome c oxidase are encoded by mitochondria in close to all eukaryotes. The Cox2 subunit spans the inner membrane twice, exposing the N and C termini to the intermembrane space. For this, the N terminus is exported cotranslationally by Oxa1 and subsequently undergoes proteolytic maturation in Saccharomyces cerevisiae . Little is known about the translocation of the C terminus, but Cox18 has been identified to be a critical protein in this process. Here we find that the scaffold protein Cox20, which promotes processing of Cox2, is in complex with the ribosome receptor Mba1 and translating mitochondrial ribosomes in a Cox2-dependent manner. The Mba1-Cox20 complex accumulates when export of the C terminus of Cox2 is blocked by the loss of the Cox18 protein. While Cox20 engages with Cox18, Mba1 is no longer present at this stage. Our analyses indicate that Cox20 associates with nascent Cox2 and Mba1 to promote Cox2 maturation cotranslationally. We suggest that Mba1 stabilizes the Cox20-ribosome complex and supports the handover of Cox2 to the Cox18 tail export machinery.

scholarly journals Absence of cardiolipin from the mitochondrial inner membrane outer leaflet restricts Opa1-mediated fusion

2021 ◽  
Author(s):  
Yifan Ge ◽  
Sivakumar Boopathy ◽  
Tran H Nguyen ◽  
Camila Makhlouta Lugo ◽  
Luke H Chao
Keyword(s):  
Membrane Fusion ◽  
Lipid Bilayers ◽  
Short Form ◽  
Mitochondrial Protein ◽  
Membrane Properties ◽  
Intermembrane Space ◽  
Inner Membrane ◽  
Outer Leaflet ◽  
Mitochondrial Inner Membrane

Cardiolipin is a tetra-acylated di-phosphatidylglycerol lipid enriched in the matrix facing (inner) leaflet of the mitochondrial inner membrane. Cardiolipin plays an important role in regulating mitochondria function and dynamics. Yet, the mechanisms connecting cardiolipin distribution and mitochondrial protein function remain indirect. In our previous work, we established an in vitro system reconstituting mitochondrial inner membrane fusion mediated by Opa1. We found that the long form of Opa1 (l-Opa1) works together with the proteolytically processed short form (s-Opa1) to mediate fast and efficient membrane fusion. Here, we extend our reconstitution system to generate supported lipid bilayers with asymmetric CL distribution. Using this system, we find the presence of CL on the intermembrane space-facing (outer) leaflet is important for membrane tethering and fusion. We discuss how the presence of CL in this leaflet may influence protein and membrane properties, and future applications for this approach.

scholarly journals YME1L controls the accumulation of respiratory chain subunits and is required for apoptotic resistance, cristae morphogenesis, and cell proliferation

2012 ◽  
Vol 23 (6) ◽  
pp. 1010-1023 ◽  
Author(s):  
Lukas Stiburek ◽  
Jana Cesnekova ◽  
Olga Kostkova ◽  
Daniela Fornuskova ◽  
Kamila Vinsova ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Membrane Protein ◽  
Respiratory Chain ◽  
Integral Membrane Protein ◽  
Intermembrane Space ◽  
Wild Type ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
Human Orthologue ◽  
Excessive Accumulation

Mitochondrial ATPases associated with diverse cellular activities (AAA) proteases are involved in the quality control and processing of inner-membrane proteins. Here we investigate the cellular activities of YME1L, the human orthologue of the Yme1 subunit of the yeast i‑AAA complex, using stable short hairpin RNA knockdown and expression experiments. Human YME1L is shown to be an integral membrane protein that exposes its carboxy-terminus to the intermembrane space and exists in several complexes of 600–1100 kDa. The stable knockdown of YME1L in human embryonic kidney 293 cells led to impaired cell proliferation and apoptotic resistance, altered cristae morphology, diminished rotenone-sensitive respiration, and increased susceptibility to mitochondrial membrane protein carbonylation. Depletion of YME1L led to excessive accumulation of nonassembled respiratory chain subunits (Ndufb6, ND1, and Cox4) in the inner membrane. This was due to a lack of YME1L proteolytic activity, since the excessive accumulation of subunits was reversed by overexpression of wild-type YME1L but not a proteolytically inactive YME1L variant. Similarly, the expression of wild-type YME1L restored the lamellar cristae morphology of YME1L-deficient mitochondria. Our results demonstrate the importance of mitochondrial inner-membrane proteostasis to both mitochondrial and cellular function and integrity and reveal a novel role for YME1L in the proteolytic regulation of respiratory chain biogenesis.

scholarly journals The J-related Segment of Tim44 Is Essential for Cell Viability: A Mutant Tim44 Remains in the Mitochondrial Import Site, but Inefficiently Recruits mtHsp70 and Impairs Protein Translocation

1999 ◽  
Vol 145 (5) ◽  
pp. 961-972 ◽  
Author(s):  
Alessio Merlin ◽  
Wolfgang Voos ◽  
Ammy C. Maarse ◽  
Michiel Meijer ◽  
Nikolaus Pfanner ◽  
...  
Keyword(s):  
Protein Import ◽  
Protein Translocation ◽  
Adaptor Protein ◽  
Dependent Manner ◽  
Wild Type ◽  
Yeast Saccharomyces Cerevisiae ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
J Proteins

Tim44 is a protein of the mitochondrial inner membrane and serves as an adaptor protein for mtHsp70 that drives the import of preproteins in an ATP-dependent manner. In this study we have modified the interaction of Tim44 with mtHsp70 and characterized the consequences for protein translocation. By deletion of an 18-residue segment of Tim44 with limited similarity to J-proteins, the binding of Tim44 to mtHsp70 was weakened. We found that in the yeast Saccharomyces cerevisiae the deletion of this segment is lethal. To investigate the role of the 18-residue segment, we expressed Tim44Δ18 in addition to the endogenous wild-type Tim44. Tim44Δ18 is correctly targeted to mitochondria and assembles in the inner membrane import site. The coexpression of Tim44Δ18 together with wild-type Tim44, however, does not stimulate protein import, but reduces its efficiency. In particular, the promotion of unfolding of preproteins during translocation is inhibited. mtHsp70 is still able to bind to Tim44Δ18 in an ATP-regulated manner, but the efficiency of interaction is reduced. These results suggest that the J-related segment of Tim44 is needed for productive interaction with mtHsp70. The efficient cooperation of mtHsp70 with Tim44 facilitates the translocation of loosely folded preproteins and plays a crucial role in the import of preproteins which contain a tightly folded domain.

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