The protein import machine of the mitochondrial inner membrane

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

Molecular and Cellular Biology ◽

10.1128/mcb.13.12.7364 ◽

1993 ◽

Vol 13 (12) ◽

pp. 7364-7371 ◽

Cited By ~ 76

Author(s):

J Blom ◽

M Kübrich ◽

J Rassow ◽

W Voos ◽

P J Dekker ◽

...

Keyword(s):

Protein Import ◽

Early Stage ◽

Proteolytic Processing ◽

Early Step ◽

Direct Role ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Preprotein Translocation ◽

In Transit

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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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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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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Identification of MIM23, a putative component of the protein import machinery of the mitochondrial inner membrane

FEBS Letters ◽

10.1016/0014-5793(93)80921-g ◽

1993 ◽

Vol 330 (1) ◽

pp. 66-70 ◽

Cited By ~ 85

Author(s):

Peter J.T. Dekker ◽

Petra Keil ◽

Joachim Rassow ◽

Ammy C. Maarse ◽

Nikolaus Pfanner ◽

...

Keyword(s):

Protein Import ◽

Inner Membrane ◽

Mitochondrial Inner Membrane

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SMS1, a high-copy suppressor of the yeast mas6 mutant, encodes an essential inner membrane protein required for mitochondrial protein import.

Molecular Biology of the Cell ◽

10.1091/mbc.5.5.529 ◽

1994 ◽

Vol 5 (5) ◽

pp. 529-538 ◽

Cited By ~ 63

Author(s):

K R Ryan ◽

M M Menold ◽

S Garrett ◽

R E Jensen

Keyword(s):

Membrane Protein ◽

Protein Import ◽

Mitochondrial Protein ◽

Mitochondrial Protein Import ◽

Yeast Saccharomyces Cerevisiae ◽

Inner Membrane ◽

Mitochondrial Inner Membrane ◽

Inner Membrane Protein ◽

Membrane Spanning ◽

Membrane Spanning Domains

MAS6 encodes an essential inner membrane protein required for mitochondrial protein import in the yeast Saccharomyces cerevisiae (Emtage and Jensen, 1993). To identify new inner membrane import components, we isolated a high-copy suppressor (SMS1) of the mas6-1 mutant. SMS1 encodes a 16.5-kDa protein that contains several potential membrane-spanning domains. The Sms1 protein is homologous to the carboxyl-terminal domain of the Mas6 protein. Like Mas6p, Sms1p is located in the mitochondrial inner membrane and is an essential protein. Depletion of Sms1p from cells causes defects in the import of several mitochondrial precursor proteins, suggesting that Sms1p is a new inner membrane import component. Our observations raise the possibility that Sms1p and Mas6p act together to translocate proteins across the inner membrane.

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Suppression of a Defect in Mitochondrial Protein Import Identifies Cytosolic Proteins Required for Viability of Yeast Cells Lacking Mitochondrial DNA

Genetics ◽

10.1093/genetics/165.1.35 ◽

2003 ◽

Vol 165 (1) ◽

pp. 35-45

Author(s):

Cory D Dunn ◽

Robert E Jensen

Keyword(s):

Mitochondrial Dna ◽

Protein Import ◽

Mitochondrial Protein ◽

Yeast Cells ◽

Mitochondrial Protein Import ◽

Inner Membrane ◽

Cytosolic Proteins ◽

Mitochondrial Inner Membrane ◽

Genes Encoding ◽

Negative Phenotype

Abstract The TIM22 complex, required for the insertion of imported polytopic proteins into the mitochondrial inner membrane, contains the nonessential Tim18p subunit. To learn more about the function of Tim18p, we screened for high-copy suppressors of the inability of tim18Δ mutants to live without mitochondrial DNA (mtDNA). We identified several genes encoding cytosolic proteins, including CCT6, SSB1, ICY1, TIP41, and PBP1, which, when overproduced, rescue the mtDNA dependence of tim18Δ cells. Furthermore, these same plasmids rescue the petite-negative phenotype of cells lacking other components of the mitochondrial protein import machinery. Strikingly, disruption of the genes identified by the different suppressors produces cells that are unable to grow without mtDNA. We speculate that loss of mtDNA leads to a lowered inner membrane potential, and subtle changes in import efficiency can no longer be tolerated. Our results suggest that increased amounts of Cct6p, Ssb1p, Icy1p, Tip41p, and Pbp1p help overcome the problems resulting from a defect in protein import.

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Yeast mitochondria lacking the phosphate carrier/p32 are blocked in phosphate transport but can import preproteins after regeneration of a membrane potential.

Molecular and Cellular Biology ◽

10.1128/mcb.16.11.6524 ◽

1996 ◽

Vol 16 (11) ◽

pp. 6524-6531 ◽

Cited By ~ 24

Author(s):

V Zara ◽

K Dietmeier ◽

A Palmisano ◽

A Vozza ◽

J Rassow ◽

...

Keyword(s):

Membrane Potential ◽

Outer Membrane ◽

Protein Import ◽

Outer Membrane Proteins ◽

Phosphate Transport ◽

Inner Membrane ◽

Saccharomyces Cerevisiae Mitochondria ◽

Mitochondrial Inner Membrane ◽

Precursor Proteins ◽

Phosphate Carrier

Two different functions have been proposed for the phosphate carrier protein/p32 of Saccharomyces cerevisiae mitochondria: transport of phosphate and requirement for import of precursor proteins into mitochondria. We characterized a yeast mutant lacking the gene for the phosphate carrier/p32 and found both a block in the import of phosphate and a strong reduction in the import of preproteins transported to the mitochondrial inner membrane and matrix. Binding of preproteins to the surface of mutant mitochondria and import of outer membrane proteins were not inhibited, indicating that the inhibition of protein import occurred after the recognition step at the outer membrane. The membrane potential across the inner membrane of the mutant mitochondria was strongly reduced. Restoration of the membrane potential restored preprotein import but did not affect the block of phosphate transport of the mutant mitochondria. We conclude that the inhibition of protein import into mitochondria lacking the phosphate carrier/p32 is indirectly caused by a reduction of the mitochondrial membrane potential (delta(gamma)), and we propose a model that the reduction of delta(psi) is due to the defective phosphate import, suggesting that phosphate transport is the primary function of the phosphate carrier/p32.

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