scholarly journals Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis

1989 ◽  
Vol 9 (3) ◽  
pp. 1014-1025
Author(s):  
D M Bedwell ◽  
S A Strobel ◽  
K Yun ◽  
G D Jongeward ◽  
S D Emr
Keyword(s):  
Protein Import ◽  
Critical Role ◽  
Mitochondrial Protein ◽  
Point Mutations ◽  
Beta Subunit ◽  
Yeast Cells ◽  
Signal Function ◽  
Subunit Gene ◽  
Mitochondrial Protein Import ◽  
Beta Subunit Gene

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and import.

scholarly journals Sequence and structural requirements of a mitochondrial protein import signal defined by saturation cassette mutagenesis.

1989 ◽  
Vol 9 (3) ◽  
pp. 1014-1025 ◽  
Author(s):  
D M Bedwell ◽  
S A Strobel ◽  
K Yun ◽  
G D Jongeward ◽  
S D Emr
Keyword(s):  
Protein Import ◽  
Critical Role ◽  
Mitochondrial Protein ◽  
Point Mutations ◽  
Beta Subunit ◽  
Yeast Cells ◽  
Signal Function ◽  
Subunit Gene ◽  
Mitochondrial Protein Import ◽  
Beta Subunit Gene

The Saccharomyces cerevisiae F1-ATPase beta subunit precursor contains redundant mitochondrial protein import information at its NH2 terminus (D. M. Bedwell, D. J. Klionsky, and S. D. Emr, Mol. Cell. Biol. 7:4038-4047, 1987). To define the critical sequence and structural features contained within this topogenic signal, one of the redundant regions (representing a minimal targeting sequence) was subjected to saturation cassette mutagenesis. Each of 97 different mutant oligonucleotide isolates containing single (32 isolates), double (45 isolates), or triple (20 isolates) point mutations was inserted in front of a beta-subunit gene lacking the coding sequence for its normal import signal (codons 1 through 34 were deleted). The phenotypic and biochemical consequences of these mutations were then evaluated in a yeast strain deleted for its normal beta-subunit gene (delta atp2). Consistent with the lack of an obvious consensus sequence for mitochondrial protein import signals, many mutations occurring throughout the minimal targeting sequence did not significantly affect its import competence. However, some mutations did result in severe import defects. In these mutants, beta-subunit precursor accumulated in the cytoplasm, and the yeast cells exhibited a respiration defective phenotype. Although point mutations have previously been identified that block mitochondrial protein import in vitro, a subset of the mutations reported here represents the first single missense mutations that have been demonstrated to significantly block mitochondrial protein import in vivo. The previous lack of such mutations in the beta-subunit precursor apparently relates to the presence of redundant import information in this import signal. Together, our mutants define a set of constraints that appear to be critical for normal activity of this (and possibly other) import signals. These include the following: (i) mutant signals that exhibit a hydrophobic moment greater than 5.5 for the predicted amphiphilic alpha-helical conformation of this sequence direct near normal levels of beta-subunit import (ii) at least two basic residues are necessary for efficient signal function, (iii) acidic amino acids actively interfere with import competence, and (iv) helix-destabilizing residues also interfere with signal function. These experimental observations provide support for mitochondrial protein import models in which both the structure and charge of the import signal play a critical role in directing mitochondrial protein targeting and 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 The amino terminus of the F1-ATPase beta-subunit precursor functions as an intramolecular chaperone to facilitate mitochondrial protein import.

1997 ◽  
Vol 17 (12) ◽  
pp. 7169-7177 ◽  
Author(s):  
P Hájek ◽  
J Y Koh ◽  
L Jones ◽  
D M Bedwell
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Beta Subunit ◽  
Amino Terminus ◽  
Missense Mutations ◽  
Primary Defect ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Import ◽  
Yeast Saccharomyces Cerevisiae ◽  
F1 Atpase

Mitochondrial import signals have been shown to function in many steps of mitochondrial protein import. Previous studies have shown that the F1-ATPase beta-subunit precursor (pre-F1beta) of the yeast Saccharomyces cerevisiae contains an extended, functionally redundant mitochondrial import signal at its amino terminus. However, the full significance of this functionally redundant targeting sequence has not been determined. We now report that the extended pre-F1beta signal acts to maintain the precursor in an import-competent conformation prior to import, in addition to its previously characterized roles in mitochondrial targeting and translocation. We found that this extended signal is required for the efficient posttranslational mitochondrial import of pre-F1beta both in vivo and in vitro. To determine whether the pre-F1beta signal directly influences precursor conformation, fusion proteins that contain wild-type and mutant forms of the pre-F1beta import signal attached to the model passenger protein dihydrofolate reductase (DHFR) were constructed. Deletions that reduced the import signal to a minimal functional unit decreased both the half-time of precursor folding and the efficiency of mitochondrial import. To confirm that the reduced mitochondrial import associated with this truncated signal was due to a defect in its ability to maintain DHFR in a loosely folded conformation, we introduced structurally destabilizing missense mutations into the DHFR passenger to block precursor folding independently of the import signal. We found that the truncated signal imported this destabilized form of DHFR as efficiently as the intact targeting signal, indicating that the primary defect associated with the minimal signal is an inability to maintain the precursor in a loosely folded conformation. Our results suggest that the loss of this intramolecular chaperone function leads to defects in the early stages of the import process.

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.

A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle

1991 ◽  
Vol 11 (5) ◽  
pp. 2647-2655 ◽  
Author(s):  
B J Smith ◽  
M P Yaffe
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Cell Cycle Progression ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Heat Shock Gene ◽  
Temperature Sensitive ◽  
Mitochondrial Protein Import ◽  
Cycle Progression

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

scholarly journals A mutation in the yeast heat-shock factor gene causes temperature-sensitive defects in both mitochondrial protein import and the cell cycle.

1991 ◽  
Vol 11 (5) ◽  
pp. 2647-2655 ◽  
Author(s):  
B J Smith ◽  
M P Yaffe
Keyword(s):  
Cell Cycle ◽  
Heat Shock ◽  
Cell Cycle Progression ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Yeast Cells ◽  
Heat Shock Gene ◽  
Temperature Sensitive ◽  
Mitochondrial Protein Import ◽  
Cycle Progression

Yeast cells containing the recessive mas3 mutation display temperature-sensitive defects in both mitochondrial protein import and the cell division cycle. The import defect is characterized by two pools of mitochondrial precursors and a dramatically slower rate of posttranslational import. The effect of mas3 on cell cycle progression occurs within one cell cycle at the nonpermissive temperature and retards progression through the G2 stage. The mas3 mutation maps to the gene encoding yeast heat-shock transcription factor (HSF), and expression of wild-type HSF complements the temperature-sensitive defects. The mas3 lesion has no apparent effect on protein secretion. In mas3 cells, induction of a major heat-shock gene, SSA1, is defective at 37 degrees C. The properties of the mas3 mutant cells indicate that HSF mediates the response to stress of two basic cellular processes: mitochondrial protein import and cell cycle progression.

scholarly journals Identification of Tam41 maintaining integrity of the TIM23 protein translocator complex in mitochondria

2006 ◽  
Vol 174 (5) ◽  
pp. 631-637 ◽  
Author(s):  
Yasushi Tamura ◽  
Yoshihiro Harada ◽  
Koji Yamano ◽  
Kazuaki Watanabe ◽  
Daigo Ishikawa ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Molecular Size ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Inner Mitochondrial Membrane ◽  
Mitochondrial Protein Import ◽  
The Matrix

Newly synthesized mitochondrial proteins are imported into mitochondria with the aid of protein translocator complexes in the outer and inner mitochondrial membranes. We report the identification of yeast Tam41, a new member of mitochondrial protein translocator systems. Tam41 is a peripheral inner mitochondrial membrane protein facing the matrix. Disruption of the TAM41 gene led to temperature-sensitive growth of yeast cells and resulted in defects in protein import via the TIM23 translocator complex at elevated temperature both in vivo and in vitro. Although Tam41 is not a constituent of the TIM23 complex, depletion of Tam41 led to a decreased molecular size of the TIM23 complex and partial aggregation of Pam18 and -16. Import of Pam16 into mitochondria without Tam41 was retarded, and the imported Pam16 formed aggregates in vitro. These results suggest that Tam41 facilitates mitochondrial protein import by maintaining the functional integrity of the TIM23 protein translocator complex from the matrix side of the inner membrane.

scholarly journals Role of Mitochondrial Protein Import in Age-Related Neurodegenerative and Cardiovascular Diseases

Cells ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 3528
Author(s):  
Andrey Bogorodskiy ◽  
Ivan Okhrimenko ◽  
Dmitrii Burkatovskii ◽  
Philipp Jakobs ◽  
Ivan Maslov ◽  
...  
Keyword(s):  
Cardiovascular Diseases ◽  
Cell Survival ◽  
Protein Import ◽  
Fundamental System ◽  
State Of The Art ◽  
Critical Role ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Age Related

Mitochondria play a critical role in providing energy, maintaining cellular metabolism, and regulating cell survival and death. To carry out these crucial functions, mitochondria employ more than 1500 proteins, distributed between two membranes and two aqueous compartments. An extensive network of dedicated proteins is engaged in importing and sorting these nuclear-encoded proteins into their designated mitochondrial compartments. Defects in this fundamental system are related to a variety of pathologies, particularly engaging the most energy-demanding tissues. In this review, we summarize the state-of-the-art knowledge about the mitochondrial protein import machinery and describe the known interrelation of its failure with age-related neurodegenerative and cardiovascular diseases.

scholarly journals Cryo-EM reveals the dynamic interplay between mitochondrial Hsp90 and SdhB folding intermediates

2020 ◽  
Author(s):  
Yanxin Liu ◽  
Daniel Elnatan ◽  
Ming Sun ◽  
Alexander G. Myasnikov ◽  
David A. Agard
Keyword(s):  
Protein Import ◽  
Atp Hydrolysis ◽  
Mitochondrial Dynamics ◽  
Critical Role ◽  
Mitochondrial Protein ◽  
Client Protein ◽  
Mitochondrial Protein Import ◽  
Folding Intermediates ◽  
B Subunit ◽  
Client Proteins

AbstractTRAP1 is a mitochondrion specific Hsp90, a ubiquitous chaperone family that mediates the folding and maturation of hundreds of “client” proteins. Through the interaction with client proteins, TRAP1 regulates mitochondrial protein homeostasis, oxidative phosphorylation/glycolysis balance, and plays a critical role in mitochondrial dynamics and disease. However, the molecular mechanism of client protein recognition and remodeling by TRAP1 remains elusive. Here we established the succinate dehydrogenase B subunit (SdhB) from mitochondrial complex II as a client protein for TRAP1 amenable to detailed biochemical and structural investigation. SdhB accelerates the rate of TRAP1 dimer closure and ATP hydrolysis by 5-fold. Cryo-EM structures of the TRAP1:SdhB complex show TRAP1 stabilizes SdhB folding intermediates by trapping an SdhB segment in the TRAP1 lumen. Unexpectedly, client protein binding induces an asymmetric to symmetric transition in the TRAP1 closed state. Our results highlight a client binding mechanism conserved throughout Hsp90s that transcends the need for cochaperones and provide molecular insights into how TRAP1 modulates protein folding within mitochondria. Our structures also suggest a potential role for TRAP1 in Fe-S cluster biogenesis and mitochondrial protein import and will guide small molecule development for therapeutic intervention in specific TRAP1 client interactions.

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