Alteration of a Mitochondrial Outer Membrane Signal Anchor Sequence That Permits Its Insertion into the Inner Membrane

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

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A signal-anchor sequence selective for the mitochondrial outer membrane.

The Journal of Cell Biology ◽

10.1083/jcb.119.6.1451 ◽

1992 ◽

Vol 119 (6) ◽

pp. 1451-1457 ◽

Cited By ~ 83

Author(s):

H M McBride ◽

D G Millar ◽

J M Li ◽

G C Shore

Keyword(s):

Amino Acids ◽

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Hybrid Protein ◽

Transmembrane Segment ◽

Structural Domains ◽

Signal Anchor ◽

Anchor Sequence ◽

Rate Limiting

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

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Role of mitochondrial inner membrane organizing system in protein biogenesis of the mitochondrial outer membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e12-04-0295 ◽

2012 ◽

Vol 23 (20) ◽

pp. 3948-3956 ◽

Cited By ~ 82

Author(s):

Maria Bohnert ◽

Lena-Sophie Wenz ◽

Ralf M. Zerbes ◽

Susanne E. Horvath ◽

David A. Stroud ◽

...

Keyword(s):

Outer Membrane ◽

Early Stage ◽

Outer Membrane Proteins ◽

Mitochondrial Outer Membrane ◽

Large Core ◽

Large Protein ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Protein Biogenesis

Mitochondria contain two membranes, the outer membrane and the inner membrane with folded cristae. The mitochondrial inner membrane organizing system (MINOS) is a large protein complex required for maintaining inner membrane architecture. MINOS interacts with both preprotein transport machineries of the outer membrane, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It is unknown, however, whether MINOS plays a role in the biogenesis of outer membrane proteins. We have dissected the interaction of MINOS with TOM and SAM and report that MINOS binds to both translocases independently. MINOS binds to the SAM complex via the conserved polypeptide transport–associated domain of Sam50. Mitochondria lacking mitofilin, the large core subunit of MINOS, are impaired in the biogenesis of β-barrel proteins of the outer membrane, whereas mutant mitochondria lacking any of the other five MINOS subunits import β-barrel proteins in a manner similar to wild-type mitochondria. We show that mitofilin is required at an early stage of β-barrel biogenesis that includes the initial translocation through the TOM complex. We conclude that MINOS interacts with TOM and SAM independently and that the core subunit mitofilin is involved in biogenesis of outer membrane β-barrel proteins.

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Influence of the mitochondrial outer membrane and the binding of creatine kinase to the mitochondrial inner membrane on the compartmentation of adenine nucleotides in the intermembrane space of rat heart mitochondria

Biochimica et Biophysica Acta (BBA) - Bioenergetics ◽

10.1016/0005-2728(93)90073-o ◽

1993 ◽

Vol 1140 (3) ◽

pp. 327-334 ◽

Cited By ~ 32

Author(s):

Frank N. Gellerich ◽

Zaza A. Khuchua ◽

Andrey V. Kuznetsov

Keyword(s):

Creatine Kinase ◽

Outer Membrane ◽

Rat Heart ◽

Adenine Nucleotides ◽

Heart Mitochondria ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Rat Heart Mitochondria

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Role of the intermembrane-space domain of the preprotein receptor Tom22 in protein import into mitochondria.

Molecular and Cellular Biology ◽

10.1128/mcb.16.8.4035 ◽

1996 ◽

Vol 16 (8) ◽

pp. 4035-4042 ◽

Cited By ~ 55

Author(s):

D A Court ◽

F E Nargang ◽

H Steiner ◽

R S Hodges ◽

W Neupert ◽

...

Keyword(s):

Outer Membrane ◽

Protein Import ◽

Protein Translocation ◽

Specific Binding ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Inner Membrane ◽

Terminal Domain ◽

Tom Complex

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix and inner membrane were imported less efficiently. The reduced import was not due to impaired interaction of presequences with their specific binding site on the trans side of the outer membrane. Rather, the IMS domain of Tom22 appears to slightly enhance the efficiency of the transfer of these preproteins to the import machinery of the inner membrane.

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Biogenesis of Mitochondrial Metabolite Carriers

Biomolecules ◽

10.3390/biom10071008 ◽

2020 ◽

Vol 10 (7) ◽

pp. 1008 ◽

Cited By ~ 5

Author(s):

Patrick Horten ◽

Lilia Colina-Tenorio ◽

Heike Rampelt

Keyword(s):

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Inner Membrane ◽

Chaperone Function ◽

Mitochondrial Inner Membrane ◽

Metabolic Reactions ◽

Almost All ◽

Related Proteins ◽

Shed Light

Metabolite carriers of the mitochondrial inner membrane are crucial for cellular physiology since mitochondria contribute essential metabolic reactions and synthesize the majority of the cellular ATP. Like almost all mitochondrial proteins, carriers have to be imported into mitochondria from the cytosol. Carrier precursors utilize a specialized translocation pathway dedicated to the biogenesis of carriers and related proteins, the carrier translocase of the inner membrane (TIM22) pathway. After recognition and import through the mitochondrial outer membrane via the translocase of the outer membrane (TOM) complex, carrier precursors are ushered through the intermembrane space by hexameric TIM chaperones and ultimately integrated into the inner membrane by the TIM22 carrier translocase. Recent advances have shed light on the mechanisms of TOM translocase and TIM chaperone function, uncovered an unexpected versatility of the machineries, and revealed novel components and functional crosstalk of the human TIM22 translocase.

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Mgm1p, a Dynamin-related GTPase, Is Essential for Fusion of the Mitochondrial Outer Membrane

Molecular Biology of the Cell ◽

10.1091/mbc.e02-12-0788 ◽

2003 ◽

Vol 14 (6) ◽

pp. 2342-2356 ◽

Cited By ~ 178

Author(s):

Hiromi Sesaki ◽

Sheryl M. Southard ◽

Michael P. Yaffe ◽

Robert E. Jensen

Keyword(s):

Outer Membrane ◽

Direct Evidence ◽

Outer Membrane Proteins ◽

Mitochondrial Fusion ◽

Mitochondrial Outer Membrane ◽

Wild Type ◽

Membrane Structures ◽

Gtpase Domain ◽

Inner Membrane ◽

Mitochondrial Inner Membrane

In Saccharomyces cerevisiae, mitochondrial fusion requires at least two outer membrane proteins, Fzo1p and Ugo1p. We provide direct evidence that the dynamin-related Mgm1 protein is also required for mitochondrial fusion. Like fzo1 and ugo1 mutants, cells disrupted for the MGM1 gene contain numerous mitochondrial fragments instead of the few long, tubular organelles seen in wild-type cells. Fragmentation of mitochondria in mgm1 mutants is rescued by disrupting DNM1, a gene required for mitochondrial division. In zygotes formed by mating mgm1 mutants, mitochondria do not fuse and mix their contents. Introducing mutations in the GTPase domain of Mgm1p completely block mitochondrial fusion. Furthermore, we show that mgm1 mutants fail to fuse both their mitochondrial outer and inner membranes. Electron microscopy demonstrates that although mgm1 mutants display aberrant mitochondrial inner membrane cristae, mgm1 dnm1 double mutants restore normal inner membrane structures. However, mgm1 dnm1 mutants remain defective in mitochondrial fusion, indicating that mitochondrial fusion requires Mgm1p regardless of the morphology of mitochondria. Finally, we find that Mgm1p, Fzo1p, and Ugo1p physically interact in the mitochondrial outer membrane. Our results raise the possibility that Mgm1p regulates fusion of the mitochondrial outer membrane through its interactions with Fzo1p and Ugo1p.

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Mitochondria Use Different Mechanisms for Transport of Multispanning Membrane Proteins through the Intermembrane Space

Molecular and Cellular Biology ◽

10.1128/mcb.23.21.7818-7828.2003 ◽

2003 ◽

Vol 23 (21) ◽

pp. 7818-7828 ◽

Cited By ~ 52

Author(s):

Ann E. Frazier ◽

Agnieszka Chacinska ◽

Kaye N. Truscott ◽

Bernard Guiard ◽

Nikolaus Pfanner ◽

...

Keyword(s):

Outer Membrane ◽

Heat Shock Protein 70 ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Carrier Proteins ◽

Inner Membrane ◽

Tom Complex ◽

Mitochondrial Inner Membrane ◽

Integral Proteins ◽

Matrix Heat

ABSTRACT The mitochondrial inner membrane contains numerous multispanning integral proteins. The precursors of these hydrophobic proteins are synthesized in the cytosol and therefore have to cross the mitochondrial outer membrane and intermembrane space to reach the inner membrane. While the import pathways of noncleavable multispanning proteins, such as the metabolite carriers, have been characterized in detail by the generation of translocation intermediates, little is known about the mechanism by which cleavable preproteins of multispanning proteins, such as Oxa1, are transferred from the outer membrane to the inner membrane. We have identified a translocation intermediate of the Oxa1 preprotein in the translocase of the outer membrane (TOM) and found that there are differences from the import mechanisms of carrier proteins. The intermembrane space domain of the receptor Tom22 supports the stabilization of the Oxa1 intermediate. Transfer of the Oxa1 preprotein to the inner membrane is not affected by inactivation of the soluble TIM complexes. Both the inner membrane potential and matrix heat shock protein 70 are essential to release the preprotein from the TOM complex, suggesting a close functional cooperation of the TOM complex and the presequence translocase of the inner membrane. We conclude that mitochondria employ different mechanisms for translocation of multispanning proteins across the aqueous intermembrane space.

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