scholarly journals Mitochondrial Protein Import Motor: Differential Role of Tim44 in the Recruitment of Pam17 and J-Complex to the Presequence Translocase

2008 ◽  
Vol 19 (6) ◽  
pp. 2642-2649 ◽  
Author(s):  
Dana P. Hutu ◽  
Bernard Guiard ◽  
Agnieszka Chacinska ◽  
Dorothea Becker ◽  
Nikolaus Pfanner ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Adaptor Protein ◽  
Inner Membrane ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Yeast Mutants ◽  
Matrix Heat

The presequence translocase of the mitochondrial inner membrane (TIM23 complex) mediates the import of preproteins with amino-terminal presequences. To drive matrix translocation the TIM23 complex recruits the presequence translocase-associated motor (PAM) with the matrix heat shock protein 70 (mtHsp70) as central subunit. Activity and localization of mtHsp70 are regulated by four membrane-associated cochaperones: the adaptor protein Tim44, the stimulatory J-complex Pam18/Pam16, and Pam17. It has been proposed that Tim44 serves as molecular platform to localize mtHsp70 and the J-complex at the TIM23 complex, but it is unknown how Pam17 interacts with the translocase. We generated conditional tim44 yeast mutants and selected a mutant allele, which differentially affects the association of PAM modules with TIM23. In tim44-804 mitochondria, the interaction of the J-complex with the TIM23 complex is impaired, whereas unexpectedly the binding of Pam17 is increased. Pam17 interacts with the channel protein Tim23, revealing a new interaction site between TIM23 and PAM. Thus, the motor PAM is composed of functional modules that bind to different sites of the translocase. We suggest that Tim44 is not simply a scaffold for binding of motor subunits but plays a differential role in the recruitment of PAM modules to the inner membrane translocase.

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

1994 ◽  
Vol 5 (4) ◽  
pp. 465-474 ◽  
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.

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

2006 ◽  
Vol 17 (9) ◽  
pp. 4051-4062 ◽  
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.

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.

scholarly journals Tim23p Contains Separate and Distinct Signals for Targeting to Mitochondria and Insertion into the Inner Membrane

1998 ◽  
Vol 9 (9) ◽  
pp. 2577-2593 ◽  
Author(s):  
Alison J. Davis ◽  
Kathleen R. Ryan ◽  
Robert E. Jensen
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Amino Terminal ◽  
Transmembrane Segments ◽  
Charged Amino Acids ◽  
The Matrix ◽  
Positively Charged

The Tim23 protein is an essential inner membrane (IM) component of the yeast mitochondrial protein import pathway. Tim23p does not carry an amino-terminal presequence; therefore, the targeting information resides within the mature protein. Tim23p is anchored in the IM via four transmembrane segments and has two positively charged loops facing the matrix. To identify the import signal for Tim23p, we have constructed several altered versions of the Tim23 protein and examined their function and import in yeast cells, as well as their import into isolated mitochondria. We replaced the positively charged amino acids in one or both loops with alanine residues and found that the positive charges are not required for import into mitochondria, but at least one positively charged loop is required for insertion into the IM. Furthermore, we find that the signal to target Tim23p to mitochondria is carried in at least two of the hydrophobic transmembrane segments. Our results suggest that Tim23p contains separate import signals: hydrophobic segments for targeting Tim23p to mitochondria, and positively charged loops for insertion into the IM. We therefore propose that Tim23p is imported into mitochondria in at least two distinct steps.

scholarly journals Characterization of the Mitochondrial Inner Membrane Translocase Complex: the Tim23p Hydrophobic Domain Interacts with Tim17p but Not with Other Tim23p Molecules

1998 ◽  
Vol 18 (1) ◽  
pp. 178-187 ◽  
Author(s):  
Kathleen R. Ryan ◽  
Roxanne S. Leung ◽  
Robert E. Jensen
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Direct Interaction ◽  
Full Length ◽  
Yeast Cells ◽  
Inner Membrane ◽  
Hydrophobic Domain ◽  
Amino Terminal ◽  
Mitochondrial Inner Membrane ◽  
Inner Membrane Protein

ABSTRACT Tim23p is a mitochondrial inner membrane protein essential for the import of proteins from the cytosol. Tim23p contains an amino-terminal hydrophilic segment and a carboxyl-terminal hydrophobic domain (Tim23Cp). To study the functions and interactions of the two parts of Tim23p separately, we constructed tim23N, encoding only the hydrophilic region of Tim23p, and tim23C, encoding only the hydrophobic domain of Tim23p. Only the Tim23C protein is imported into mitochondria, indicating that the mitochondrial targeting information in Tim23p resides in its membrane spans or intervening loops. Tim23Cp, however, cannot substitute for full-length Tim23p, suggesting that the hydrophilic portion of Tim23p also performs an essential function in mitochondrial protein import. We found that overexpression of Tim23Cp is toxic to yeast cells that carry the tim23-1 mutation. Excess Tim23Cp causes Tim23-1p to disappear, leavingtim23-1 cells without a full-length version of the Tim23 protein. If Tim17p, another inner membrane import component, is overexpressed along with Tim23Cp, the toxicity of Tim23Cp is largely reversed and the Tim23-1 protein no longer disappears. In coimmunoprecipitations from solubilized mitochondria, Tim17p associates with the Tim23C protein. In addition, we show that Tim23p and Tim17p can be chemically cross-linked to each other in intact mitochondria. We conclude that the hydrophobic domain encoded by tim23Ctargets Tim23p to the mitochondria and mediates the direct interaction between Tim23p and Tim17p. In contrast, Tim23Cp cannot be coimmunoprecipitated with Tim23p, raising the possibility that the hydrophobic domain of Tim23p does not interact with other Tim23 molecules.

scholarly journals Mitochondrial protein import: biochemical and genetic evidence for interaction of matrix hsp70 and the inner membrane protein MIM44.

1994 ◽  
Vol 127 (6) ◽  
pp. 1547-1556 ◽  
Author(s):  
J Rassow ◽  
A C Maarse ◽  
E Krainer ◽  
M Kübrich ◽  
H Müller ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Binding Proteins ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Growth Defect ◽  
Inner Membrane ◽  
The Matrix ◽  
Inner Membrane Protein ◽  
Polypeptide Chains ◽  
Matrix Heat

The import of preproteins into mitochondria involves translocation of the polypeptide chains through putative channels in the outer and inner membranes. Preprotein-binding proteins are needed to drive the unidirectional translocation of the precursor polypeptides. Two of these preprotein-binding proteins are the peripheral inner membrane protein MIM44 and the matrix heat shock protein hsp70. We report here that MIM44 is mainly exposed on the matrix side, and a fraction of mt-hsp70 is reversibly bound to the inner membrane. Mt-hsp70 binds to MIM44 in a 1:1 ratio, suggesting that mt-hsp70 is localizing to the membrane via its interaction with MIM44. Formation of the complex requires a functional ATPase domain of mt-hsp70. Addition of Mg-ATP leads to dissociation of the complex. Overexpression of mt-hsp70 rescues the protein import defect of mutants in MIM44; conversely, overexpression of MIM44 rescues protein import defects of mt-hsp70 mutants. In addition, yeast strains with conditional mutations in both MIM44 and mt-hsp70 are barely viable, showing a synthetic growth defect compared to strains carrying single mutations. We propose that MIM44 and mt-hsp70 cooperate in translocation of preproteins. By binding to MIM44, mt-hsp70 is recruited at the protein import sites of the inner membrane, and preproteins arriving at MIM44 may be directly handed over to mt-hsp70.

How to get to the other side of the mitochondrial inner membrane – the protein import motor

2020 ◽  
Vol 401 (6-7) ◽  
pp. 723-736 ◽  
Author(s):  
Dejana Mokranjac
Keyword(s):  
Protein Import ◽  
Atp Hydrolysis ◽  
Mitochondrial Protein ◽  
The Other ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Matrix ◽  
Biogenesis Of Mitochondria ◽  
Translocation Pathway

AbstractBiogenesis of mitochondria relies on import of more than 1000 different proteins from the cytosol. Approximately 70% of these proteins follow the presequence pathway – they are synthesized with cleavable N-terminal extensions called presequences and reach the final place of their function within the organelle with the help of the TOM and TIM23 complexes in the outer and inner membranes, respectively. The translocation of proteins along the presequence pathway is powered by the import motor of the TIM23 complex. The import motor of the TIM23 complex is localized at the matrix face of the inner membrane and is likely the most complicated Hsp70-based system identified to date. How it converts the energy of ATP hydrolysis into unidirectional translocation of proteins into mitochondria remains one of the biggest mysteries of this translocation pathway. Here, the knowns and the unknowns of the mitochondrial protein import motor are discussed.

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

1993 ◽  
Vol 122 (5) ◽  
pp. 1003-1012 ◽  
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.

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

1994 ◽  
Vol 5 (5) ◽  
pp. 529-538 ◽  
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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