The essential yeast protein MIM44 (encoded by MPI1) is involved in an early step of preprotein translocation across the mitochondrial inner membrane.

The essential yeast gene MPI1 encodes a mitochondrial membrane protein that is possibly involved in protein import into the organelle (A. C. Maarse, J. Blom, L. A. Grivell, and M. Meijer, EMBO J. 11:3619-3628, 1992). For this report, we determined the submitochondrial location of the MPI1 gene product and investigated whether it plays a direct role in the translocation of preproteins. By fractionation of mitochondria, the mature protein of 44 kDa was localized to the mitochondrial inner membrane and therefore termed MIM44. Import of the precursor of MIM44 required a membrane potential across the inner membrane and involved proteolytic processing of the precursor. A preprotein in transit across the mitochondrial membranes was cross-linked to MIM44, whereas preproteins arrested on the mitochondrial surface or fully imported proteins were not cross-linked. When preproteins were arrested at two distinct stages of translocation across the inner membrane, only preproteins at an early stage of translocation could be cross-linked to MIM44. Moreover, solubilized MIM44 was found to interact with in vitro-synthesized preproteins. We conclude that MIM44 is a component of the mitochondrial inner membrane import machinery and interacts with preproteins in an early step of translocation.

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MAS6 encodes an essential inner membrane component of the yeast mitochondrial protein import pathway

The Journal of Cell Biology ◽

10.1083/jcb.122.5.1003 ◽

1993 ◽

Vol 122 (5) ◽

pp. 1003-1012 ◽

Cited By ~ 104

Author(s):

JL Emtage ◽

RE Jensen

Keyword(s):

Protein Import ◽

Early Stage ◽

Osmotic Shock ◽

Alkali Treatment ◽

Mitochondrial Protein ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

Mitochondrial Inner Membrane

To identify new components that mediate mitochondrial protein import, we analyzed mas6, an import mutant in the yeast Saccharomyces cerevisiae. mas6 mutants are temperature sensitive for viability, and accumulate mitochondrial precursor proteins at the restrictive temperature. We show that mas6 does not correspond to any of the presently identified import mutants, and we find that mitochondria isolated from mas6 mutants are defective at an early stage of the mitochondrial protein import pathway. MAS6 encodes a 23-kD protein that contains several potential membrane spanning domains, and yeast strains disrupted for MAS6 are inviable at all temperatures and on all carbon sources. The Mas6 protein is located in the mitochondrial inner membrane and cannot be extracted from the membrane by alkali treatment. Antibodies to the Mas6 protein inhibit import into isolated mitochondria, but only when the outer membrane has been disrupted by osmotic shock. Mas6p therefore represents an essential import component located in the mitochondrial inner membrane.

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Quaternary Structure of the Mitochondrial TIM23 Complex Reveals Dynamic Association between Tim23p and Other Subunits

Molecular Biology of the Cell ◽

10.1091/mbc.e07-07-0669 ◽

2008 ◽

Vol 19 (1) ◽

pp. 159-170 ◽

Cited By ~ 42

Author(s):

Nathan N. Alder ◽

Jennifer Sutherland ◽

Ashley I. Buhring ◽

Robert E. Jensen ◽

Arthur E. Johnson

Keyword(s):

Structural Changes ◽

Protein Import ◽

Quaternary Structure ◽

Physiological State ◽

Transmembrane Segment ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Hydrophilic Region ◽

Chemical Cross Linking

Tim23p is an essential channel-forming component of the multisubunit TIM23 complex of the mitochondrial inner membrane that mediates protein import. Radiolabeled Tim23p monocysteine mutants were imported in vitro, incorporated into functional TIM23 complexes, and subjected to chemical cross-linking. Three regions of proximity between Tim23p and other subunits of the TIM23 complex were identified: Tim17p and the first transmembrane segment of Tim23p; Tim50p and the C-terminal end of the Tim23p hydrophilic region; and the entire hydrophilic domains of Tim23p molecules. These regions of proximity reversibly change in response to changes in membrane potential across the inner membrane and also when a translocating substrate is trapped in the TIM23 complex. These structural changes reveal that the macromolecular arrangement within the TIM23 complex is dynamic and varies with the physiological state of the mitochondrion.

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Protein import into mitochondria: the requirement for external ATP is precursor-specific whereas intramitochondrial ATP is universally needed for translocation into the matrix.

Molecular Biology of the Cell ◽

10.1091/mbc.5.4.465 ◽

1994 ◽

Vol 5 (4) ◽

pp. 465-474 ◽

Cited By ~ 81

Author(s):

C Wachter ◽

G Schatz ◽

B S Glick

Keyword(s):

Protein Import ◽

Protein Translocation ◽

Mitochondrial Protein ◽

Inner Membrane ◽

In Vitro System ◽

Mitochondrial Inner Membrane ◽

Precursor Proteins ◽

The Matrix

ATP is needed for the import of precursor proteins into mitochondria. However, the role of ATP and its site of action have been unclear. We have now investigated the ATP requirements for protein import into the mitochondrial matrix. These experiments employed an in vitro system that allowed ATP levels to be manipulated both inside and outside the mitochondrial inner membrane. Our results indicate that there are two distinct ATP requirements for mitochondrial protein import. ATP in the matrix is always needed for complete import of precursor proteins into this compartment, even when the precursors are presented to mitochondria in an unfolded conformation. In contrast, the requirement for external ATP is precursor-specific; depletion of external ATP strongly inhibits import of some precursors but has little or no effect with other precursors. A requirement for external ATP can often be overcome by denaturing the precursor with urea. We suggest that external ATP promotes the release of precursors from cytosolic chaperones, whereas matrix ATP drives protein translocation across the inner membrane.

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Characterization of Mmp37p, aSaccharomyces cerevisiaeMitochondrial Matrix Protein with a Role in Mitochondrial Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.e06-04-0366 ◽

2006 ◽

Vol 17 (9) ◽

pp. 4051-4062 ◽

Cited By ~ 47

Author(s):

Michelle R. Gallas ◽

Mary K. Dienhart ◽

Rosemary A. Stuart ◽

Roy M. Long

Keyword(s):

Matrix Protein ◽

Protein Import ◽

Mitochondrial Protein ◽

Temperature Sensitive ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Close Proximity ◽

The Matrix ◽

Targeting Signal

Many mitochondrial proteins are encoded by nuclear genes and after translation in the cytoplasm are imported via translocases in the outer and inner membranes, the TOM and TIM complexes, respectively. Here, we report the characterization of the mitochondrial protein, Mmp37p (YGR046w) and demonstrate its involvement in the process of protein import into mitochondria. Haploid cells deleted of MMP37 are viable but display a temperature-sensitive growth phenotype and are inviable in the absence of mitochondrial DNA. Mmp37p is located in the mitochondrial matrix where it is peripherally associated with the inner membrane. We show that Mmp37p has a role in the translocation of proteins across the mitochondrial inner membrane via the TIM23-PAM complex and further demonstrate that substrates containing a tightly folded domain in close proximity to their mitochondrial targeting sequences display a particular dependency on Mmp37p for mitochondrial import. Prior unfolding of the preprotein, or extension of the region between the targeting signal and the tightly folded domain, relieves their dependency for Mmp37p. Furthermore, evidence is presented to show that Mmp37 may affect the assembly state of the TIM23 complex. On the basis of these findings, we hypothesize that the presence of Mmp37p enhances the early stages of the TIM23 matrix import pathway to ensure engagement of incoming preproteins with the mtHsp70p/PAM complex, a step that is necessary to drive the unfolding and complete translocation of the preprotein into the matrix.

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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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Identification of the essential yeast protein MIM17, an integral mitochondrial inner membrane protein involved in protein import

FEBS Letters ◽

10.1016/0014-5793(94)00669-5 ◽

1994 ◽

Vol 349 (2) ◽

pp. 215-221 ◽

Cited By ~ 72

Author(s):

Ammy C. Maarse ◽

Jolanda Blom ◽

Petra Keil ◽

Nikolaus Pfanner ◽

Michiel Meijer

Keyword(s):

Membrane Protein ◽

Protein Import ◽

Yeast Protein ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Inner Membrane Protein

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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

The Journal of Cell Biology ◽

10.1083/jcb.145.5.961 ◽

1999 ◽

Vol 145 (5) ◽

pp. 961-972 ◽

Cited By ~ 36

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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Targeting and assembly of the oxoglutarate carrier: general principles for biogenesis of carrier proteins of the mitochondrial inner membrane

Biochemical Journal ◽

10.1042/bj3330151 ◽

1998 ◽

Vol 333 (1) ◽

pp. 151-158 ◽

Cited By ~ 42

Author(s):

Annamaria PALMISANO ◽

Vincenzo ZARA ◽

Angelika HÖNLINGER ◽

Angelo VOZZA ◽

Peter J. T. DEKKER ◽

...

Keyword(s):

High Efficiency ◽

Dependent Manner ◽

Yeast Mitochondria ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Native Electrophoresis ◽

Surface Receptor ◽

Inner Membrane Protein ◽

Oxoglutarate Carrier

We have studied the targeting and assembly of the 2-oxoglutarate carrier (OGC), an integral inner-membrane protein of mitochondria. The precursor of OGC, synthesized without a cleavable presequence, is transported into mitochondria in an ATP- and membrane potential-dependent manner. Import of the mammalian OGC occurs efficiently into both mammalian and yeast mitochondria. Targeting of OGC reveals a clear dependence on the mitochondrial surface receptor Tom70 (the 70 kDa subunit of the translocase of the outer mitochondrial membrane), whereas a cleavable preprotein depends on Tom20 (the 20 kDa subunit), supporting a model of specificity differences of the receptors and the existence of distinct targeting pathways to mitochondria. The assembly of minute amounts of OGC imported in vitro to the dimeric form can be monitored by blue native electrophoresis of digitonin-lysed mitochondria. The assembly of mammalian OGC and fungal ADP/ATP carrier occurs with high efficiency in both mammalian and yeast mitochondria. These findings indicate a dynamic behaviour of the carrier dimers in the mitochondrial inner membrane and suggest a high conservation of the assembly reactions from mammals to fungi.

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