scholarly journals The non-canonical mitochondrial inner membrane presequence translocase of trypanosomatids contains two essential rhomboid-like proteins

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Anke Harsman ◽  
Silke Oeljeklaus ◽  
Christoph Wenger ◽  
Jonathan L. Huot ◽  
Bettina Warscheid ◽  
...  
Keyword(s):  
Pore Formation ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Carrier ◽  
Mitochondrial Inner Membrane ◽  
Mitochondrial Carrier Protein ◽  
Compositional Variations

Abstract Mitochondrial protein import is essential for all eukaryotes. Here we show that the early diverging eukaryote Trypanosoma brucei has a non-canonical inner membrane (IM) protein translocation machinery. Besides TbTim17, the single member of the Tim17/22/23 family in trypanosomes, the presequence translocase contains nine subunits that co-purify in reciprocal immunoprecipitations and with a presequence-containing substrate that is trapped in the translocation channel. Two of the newly discovered subunits are rhomboid-like proteins, which are essential for growth and mitochondrial protein import. Rhomboid-like proteins were proposed to form the protein translocation pore of the ER-associated degradation system, suggesting that they may contribute to pore formation in the presequence translocase of T. brucei. Pulldown of import-arrested mitochondrial carrier protein shows that the carrier translocase shares eight subunits with the presequence translocase. This indicates that T. brucei may have a single IM translocase that with compositional variations mediates import of presequence-containing and carrier proteins.

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.

scholarly journals Tim23–Tim50 pair coordinates functions of translocators and motor proteins in mitochondrial protein import

2009 ◽  
Vol 184 (1) ◽  
pp. 129-141 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Takuya Shiota ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
...  
Keyword(s):  
Motor Proteins ◽  
Protein Import ◽  
Protein Translocation ◽  
Mitochondrial Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
The Matrix ◽  
General Protein ◽  
Mitochondrial Hsp70

Mitochondrial protein traffic requires coordinated operation of protein translocator complexes in the mitochondrial membrane. The TIM23 complex translocates and inserts proteins into the mitochondrial inner membrane. Here we analyze the intermembrane space (IMS) domains of Tim23 and Tim50, which are essential subunits of the TIM23 complex, in these functions. We find that interactions of Tim23 and Tim50 in the IMS facilitate transfer of precursor proteins from the TOM40 complex, a general protein translocator in the outer membrane, to the TIM23 complex. Tim23–Tim50 interactions also facilitate a late step of protein translocation across the inner membrane by promoting motor functions of mitochondrial Hsp70 in the matrix. Therefore, the Tim23–Tim50 pair coordinates the actions of the TOM40 and TIM23 complexes together with motor proteins for mitochondrial protein import.

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

Genetics ◽  
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.

scholarly journals Tim23, a Protein Import Component of the Mitochondrial Inner Membrane, Is Required for Normal Activity of the Multiple Conductance Channel, MCC

1997 ◽  
Vol 137 (2) ◽  
pp. 377-386 ◽  
Author(s):  
Timothy A. Lohret ◽  
Robert E. Jensen ◽  
Kathleen W. Kinnally
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Normal Activity ◽  
Mitochondrial Outer Membrane ◽  
Wild Type ◽  
Membrane Channel ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Inner Membrane ◽  
The Relationship

We previously showed that the conductance of a mitochondrial inner membrane channel, called MCC, was specifically blocked by peptides corresponding to mitochondrial import signals. To determine if MCC plays a role in protein import, we examined the relationship between MCC and Tim23p, a component of the protein import complex of the mitochondrial inner membrane. We find that antibodies against Tim23p, previously shown to inhibit mitochondrial protein import, inhibit MCC activity. We also find that MCC activity is altered in mitochondria isolated from yeast carrying the tim23-1 mutation. In contrast to wild-type MCC, we find that the conductance of MCC from the tim23-1 mutant is not significantly blocked by mitochondrial presequence peptides. Tim23 antibodies and the tim23-1 mutation do not, however, alter the activity of PSC, a presequence-peptide sensitive channel in the mitochondrial outer membrane. Our results show that Tim23p is required for normal MCC activity and raise the possibility that precursors are translocated across the inner membrane through the pore of MCC.

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 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 Mutant huntingtin disrupts mitochondrial proteostasis by interacting with TIM23

2019 ◽  
Vol 116 (33) ◽  
pp. 16593-16602 ◽  
Author(s):  
Svitlana Yablonska ◽  
Vinitha Ganesan ◽  
Lisa M. Ferrando ◽  
JinHo Kim ◽  
Anna Pyzel ◽  
...  
Keyword(s):  
Cell Lines ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Biological Significance ◽  
Intermembrane Space ◽  
Mutant Huntingtin ◽  
Mitochondrial Protein Import ◽  
Inner Membrane ◽  
Mitochondrial Proteome ◽  
Brain Tissues

Mutant huntingtin (mHTT), the causative protein in Huntington’s disease (HD), associates with the translocase of mitochondrial inner membrane 23 (TIM23) complex, resulting in inhibition of synaptic mitochondrial protein import first detected in presymptomatic HD mice. The early timing of this event suggests that it is a relevant and direct pathophysiologic consequence of mHTT expression. We show that, of the 4 TIM23 complex proteins, mHTT specifically binds to the TIM23 subunit and that full-length wild-type huntingtin (wtHTT) and mHTT reside in the mitochondrial intermembrane space. We investigated differences in mitochondrial proteome between wtHTT and mHTT cells and found numerous proteomic disparities between mHTT and wtHTT mitochondria. We validated these data by quantitative immunoblotting in striatal cell lines and human HD brain tissue. The level of soluble matrix mitochondrial proteins imported through the TIM23 complex is lower in mHTT-expressing cell lines and brain tissues of HD patients compared with controls. In mHTT-expressing cell lines, membrane-bound TIM23-imported proteins have lower intramitochondrial levels, whereas inner membrane multispan proteins that are imported via the TIM22 pathway and proteins integrated into the outer membrane generally remain unchanged. In summary, we show that, in mitochondria, huntingtin is located in the intermembrane space, that mHTT binds with high-affinity to TIM23, and that mitochondria from mHTT-expressing cells and brain tissues of HD patients have reduced levels of nuclearly encoded proteins imported through TIM23. These data demonstrate the mechanism and biological significance of mHTT-mediated inhibition of mitochondrial protein import, a mechanism likely broadly relevant to other neurodegenerative diseases.

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