Mitochondrial preproteins en route from the outer membrane to the inner membrane are exposed to the intermembrane space

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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Multiple pathways for mitochondrial protein traffic

Biological Chemistry ◽

10.1515/bc.2009.087 ◽

2009 ◽

Vol 390 (8) ◽

Cited By ~ 130

Author(s):

Toshiya Endo ◽

Koji Yamano

Keyword(s):

Outer Membrane ◽

Protein Trafficking ◽

Protein Import ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

Protein Traffic

Abstract Mitochondria are two-membrane bounded organelles consisting of 1000–2000 different proteins, most of which are synthesized in the cytosol and subsequently imported into mitochondria. The imported proteins are further sorted to one of the four compartments, the outer membrane, intermembrane space, inner membrane, and matrix, mostly following one of the five major pathways. Mitochondrial protein import and sorting are mediated by the translocator complexes in the membranes and chaperones in the aqueous compartments operating along the import pathways. Here, we summarize the expanding knowledge on the roles of translocators, chaperones, and related components in the multiple pathways for mitochondrial protein trafficking.

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Discrimination of distinct proteinases at the four structural levels of rat liver mitochondria

Biochemical Journal ◽

10.1042/bj2330283 ◽

1986 ◽

Vol 233 (1) ◽

pp. 283-286 ◽

Cited By ~ 3

Author(s):

M C Duque-Magalhães ◽

P Régnier

Keyword(s):

Outer Membrane ◽

Rat Liver ◽

Degradation Products ◽

Liver Mitochondria ◽

Rat Liver Mitochondria ◽

Intermembrane Space ◽

Peptidase Activity ◽

Inner Membrane ◽

Mitochondrial Fractions ◽

The Matrix

Rat liver mitochondrial fractions corresponding to four morphological structures (matrix, inner membrane, intermembrane space and outer membrane) contain proteinases that cleave casein components at different rates. Proteinases of the intermembrane space preferentially cleave kappa-casein, whereas the proteinases of the outer membrane, inner membrane and matrix fractions degrade alpha S1-casein more rapidly. Electrophoretic separation of the degradation products of alpha S1-casein and kappa-casein in polyacrylamide gels shows that different polypeptides are produced when the substrate is degraded by the matrix, by both membranes and by the intermembrane-space fraction. Some of the degradation products resulting from incubation of the caseins with the mitochondrial fractions are probably the result of digestion by contaminating lysosomal proteinase(s). The matrix has a high peptidase activity, since glucagon, a small peptide, is very rapidly degraded by this fraction. These observations strongly suggest that distinct proteinases, with different specificities, are associated respectively with the intermembrane space and with both membrane fractions.

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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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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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The role of the Tim8p–Tim13p complex in a conserved import pathway for mitochondrial polytopic inner membrane proteins

The Journal of Cell Biology ◽

10.1083/jcb.200205124 ◽

2002 ◽

Vol 158 (6) ◽

pp. 1017-1027 ◽

Cited By ~ 97

Author(s):

Sean P. Curran ◽

Danielle Leuenberger ◽

Einhard Schmidt ◽

Carla M. Koehler

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Intermembrane Space ◽

Hydrophobic Membrane ◽

Inner Membrane ◽

Membrane Complex ◽

Mitochondrial Carrier Family ◽

Pass Through ◽

Membrane Spanning ◽

Membrane Spanning Domains

Tim23p is imported via the TIM (translocase of inner membrane)22 pathway for mitochondrial inner membrane proteins. In contrast to precursors with an NH2-terminal targeting presequence that are imported in a linear NH2-terminal manner, we show that Tim23p crosses the outer membrane as a loop before inserting into the inner membrane. The Tim8p–Tim13p complex facilitates translocation across the intermembrane space by binding to the membrane spanning domains as shown by Tim23p peptide scans with the purified Tim8p–Tim13p complex and crosslinking studies with Tim23p fusion constructs. The interaction between Tim23p and the Tim8p–Tim13p complex is not dependent on zinc, and the purified Tim8p–Tim13p complex does not coordinate zinc in the conserved twin CX3C motif. Instead, the cysteine residues seemingly form intramolecular disulfide linkages. Given that proteins of the mitochondrial carrier family also pass through the TOM (translocase of outer membrane) complex as a loop, our study suggests that this translocation mechanism may be conserved. Thus, polytopic inner membrane proteins, which lack an NH2-terminal targeting sequence, pass through the TOM complex as a loop followed by binding of the small Tim proteins to the hydrophobic membrane spanning domains.

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p166 links membrane and intramitochondrial modules of the trypanosomal tripartite attachment complex

10.1101/2021.12.16.473092 ◽

2021 ◽

Author(s):

Bernd Schimanski ◽

Salome Aeschlimann ◽

Sandro Käser ◽

Maria Gomez-Fabra Gala ◽

Nora Vögtle ◽

...

Keyword(s):

Outer Membrane ◽

Transmembrane Domain ◽

Proper Function ◽

Intermembrane Space ◽

Kinetoplast Dna ◽

Mitochondrial Membranes ◽

Inner Membrane ◽

Large N ◽

Associated Proteins ◽

Single Mitochondrion

The protist parasite Trypanosoma brucei has a single mitochondrion with a single unit genome termed kinetoplast DNA (kDNA). Faithfull segregation of replicated kDNA is ensured by a complicated structure termed tripartite attachment complex (TAC). The TAC physically links the basal body of the flagellum with the kDNA spanning the two mitochondrial membranes. Here, we characterized p166 as the only TAC subunit that is anchored in the inner membrane. Its C-terminal transmembrane domain separates the protein into a large N-terminal region that interacts with the kDNA-localized TAC102 and a 34 aa C-tail that binds to the intermembrane space-exposed loop of the integral outer membrane protein TAC60. Thus, in contrast to the outer membrane TAC region which requires four essential subunits for proper function a single inner membrane TAC subunit is sufficient to bridge the distance from the OM to the kDNA. Surprisingly, non-functional p166 lacking the C-terminal 34 aa still localizes to the TAC region. This suggests the existence of nonessential TAC-associated proteins in the OM. These proteins can loosely bind to non-functional p166 lacking the C-terminal 34 aa and keep it at the TAC but their binding would not be strong enough to withstand the mechanical force upon kDNA segregation.

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Sam50-Mic19-Mic60 axis Determines Mitochondrial Cristae Architecture by Mediating Mitochondrial Outer and Inner Membrane Contact

10.1101/345959 ◽

2018 ◽

Author(s):

Junhui Tang ◽

Kuan Zhang ◽

Jun Dong ◽

Chaojun Yan ◽

Shi Chen ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Morphology ◽

Intermembrane Space ◽

Inner Membrane ◽

Atp Production ◽

Mitochondrial Intermembrane Space ◽

Membrane Contact ◽

Inner Membrane Protein

ABSTRACTMitochondrial cristae are critical for efficient oxidative phosphorylation, however, how cristae architecture is precisely organized remains largely unknown. Here, we discovered that Mic19, a core component of MICOS (mitochondrial contact site and cristae organizing system) complex, can be cleaved at N-terminal by mitochondrial protease OMA1. Mic19 directly interacts with mitochondrial outer-membrane protein Sam50 (the key subunit of SAM complex) and inner-membrane protein Mic60 (the key component of MICOS complex) to form Sam50-Mic19-Mic60 axis, which dominantly connects SAM and MICOS complexes to assemble MIB (mitochondrial intermembrane space bridging) supercomplex for mediating mitochondrial outer- and inner-membrane contact. OMA1-mediated Mic19 cleavage causes Sam50-Mic19-Mic60 axis disruption, which separates SAM and MICOS and leads to MIB disassembly. Disrupted Sam50-Mic19-Mic60 axis, even in the presence of SAM and MICOS complexes, causes the abnormal mitochondrial morphology, loss of mitochondrial cristae junctions, abnormal cristae distribution and reduced ATP production. Importantly, Sam50 displays punctate distribution at mitochondrial outer membrane, and acts as an anchoring point to guide the formation of mitochondrial cristae junctions. Therefore, we propose a model that Sam50-Mic19-Mic60 axis mediated SAM-MICOS complexes integration determines mitochondrial cristae architecture.

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Mitochondrial inner-membrane protease Yme1 degrades outer-membrane proteins Tom22 and Om45

The Journal of Cell Biology ◽

10.1083/jcb.201702125 ◽

2017 ◽

Vol 217 (1) ◽

pp. 139-149 ◽

Cited By ~ 16

Author(s):

Xi Wu ◽

Lanlan Li ◽

Hui Jiang

Keyword(s):

Outer Membrane ◽

Outer Membrane Proteins ◽

Ubiquitin Proteasome System ◽

Mitochondrial Outer Membrane ◽

Intermembrane Space ◽

Inner Membrane ◽

Mitochondrial Proteome ◽

Mitochondrial Inner Membrane ◽

Ubiquitin Proteasome

Mitochondria are double-membraned organelles playing essential metabolic and signaling functions. The mitochondrial proteome is under surveillance by two proteolysis systems: the ubiquitin–proteasome system degrades mitochondrial outer-membrane (MOM) proteins, and the AAA proteases maintain the proteostasis of intramitochondrial compartments. We previously identified a Doa1–Cdc48-Ufd1-Npl4 complex that retrogradely translocates ubiquitinated MOM proteins to the cytoplasm for degradation. In this study, we report the unexpected identification of MOM proteins whose degradation requires the Yme1-Mgr1-Mgr3 i-AAA protease complex in mitochondrial inner membrane. Through immunoprecipitation and in vivo site-specific photo–cross-linking experiments, we show that both Yme1 adapters Mgr1 and Mgr3 recognize the intermembrane space (IMS) domains of the MOM substrates and facilitate their recruitment to Yme1 for proteolysis. We also provide evidence that the cytoplasmic domain of substrate can be dislocated into IMS by the ATPase activity of Yme1. Our findings indicate a proteolysis pathway monitoring MOM proteins from the IMS side and suggest that the MOM proteome is surveilled by mitochondrial and cytoplasmic quality control machineries in parallel.

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