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 The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information.

1987 ◽  
Vol 7 (11) ◽  
pp. 4038-4047 ◽  
Author(s):  
D M Bedwell ◽  
D J Klionsky ◽  
S D Emr
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Beta Subunit ◽  
Missense Mutations ◽  
Mature Protein ◽  
Mitochondrial Import ◽  
Gene Encoding ◽  
Mutant Proteins ◽  
F1 Atpase

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.

scholarly journals The yeast F1-ATPase beta subunit precursor contains functionally redundant mitochondrial protein import information

1987 ◽  
Vol 7 (11) ◽  
pp. 4038-4047
Author(s):  
D M Bedwell ◽  
D J Klionsky ◽  
S D Emr
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Beta Subunit ◽  
Missense Mutations ◽  
Mature Protein ◽  
Mitochondrial Import ◽  
Gene Encoding ◽  
Mutant Proteins ◽  
F1 Atpase

The NH2 terminus of the yeast F1-ATPase beta subunit precursor directs the import of this protein into mitochondria. To define the functionally important components of this import signal, oligonucleotide-directed mutagenesis was used to introduce a series of deletion and missense mutations into the gene encoding the F1-beta subunit precursor. Among these mutations were three nonoverlapping deletions, two within the 19-amino-acid presequence (delta 5-12 and delta 16-19) and one within the mature protein (delta 28-34). Characterization of the mitochondrial import properties of various mutant F1-beta subunit proteins containing different combinations of these deletions showed that import was blocked only when all three deletions were combined. Mutant proteins containing all possible single and pairwise combinations of these deletions were found to retain the ability to direct mitochondrial import of the F1-beta subunit. These data suggest that the F1-beta subunit contains redundant import information at its NH2 terminus. In fact, we found that deletion of the entire F1-beta subunit presequence did not prevent import, indicating that a functional mitochondrial import signal is present near the NH2 terminus of the mature protein. Furthermore, by analyzing mitochondrial import of the various mutant proteins in [rho-] yeast, we obtained evidence that different segments of the F1-beta subunit import signal may act in an additive or cooperative manner to optimize the import properties of this protein.

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 Shot-Gun Proteomic Analysis on Roots of Arabidopsis pldα1 Mutants Suggesting New Roles of PLDα1 in Mitochondrial Protein Import, Vesicular Trafficking and Glucosinolate Biosynthesis

2018 ◽  
Author(s):  
Tomáš Takáč ◽  
Olga Šamajová ◽  
Pavol Vadovič ◽  
Tibor Pechan ◽  
Jozef Šamaj
Keyword(s):  
Endoplasmic Reticulum ◽  
Stress Responses ◽  
Proteomic Analysis ◽  
Protein Import ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Import ◽  
Enzymatic Production ◽  
Glucosinolate Biosynthesis ◽  
Vacuolar Protein

Phospholipase Dα1 (PLDα1) belongs to phospholipases, a large phospholipid hydrolyzing protein family. PLDα1 has a substrate preference for phosphatidylcholine leading to enzymatic production of phosphatidic acid, a lipid second messenger with multiple cellular functions. PLDα1 itself is implicated in biotic and abiotic stress responses. We present here a shot-gun differential proteomic analysis on roots of two pldα1 mutants compared to the Col-0 wild type. Our data suggest new roles of PLDα1 in endomembrane transport, mitochondrial protein import and protein quality control and glucosinolate biosynthesis. Thus, we identified proteins involved in endocytosis, endoplasmic reticulum-Golgi transport and attachment sites of endoplasmic reticulum and plasma membrane (V-type proton ATPases, protein transport protein SEC13 homolog A, vesicle-associated protein 1-2, vacuolar protein sorting-associated protein 29, syntaxin-32, all upregulated in the mutants), mitochondrial import and electron transport chain (mitochondrial import inner membrane translocase subunits TIM23-2 and TIM13, mitochondrial NADH dehydrogenases, ATP synthases, cytochrome c oxidase subunit 6b-1, ADP,ATP carrier protein 2, downregulated in the mutants) and glucosinolate biosynthesis (3-isopropylmalate dehydrogenases 1, 2 and 3, methylthioalkylmalate synthase 1, cytochrome P450 83B1, Glutathione S-transferase F9, indole glucosinolate O-methyltransferase 1, adenylyl-sulfate kinase 1, all upregulated in mutants). Our results suggest broader biological roles of PLDα1 as anticipated so far.

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 MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import.

1992 ◽  
Vol 12 (1) ◽  
pp. 283-291 ◽  
Author(s):  
D P Atencio ◽  
M P Yaffe
Keyword(s):  
Protein Import ◽  
Elevated Temperatures ◽  
Mitochondrial Protein ◽  
Heat Shock Gene ◽  
Temperature Sensitive ◽  
Mitochondrial Protein Import ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Phenotypes ◽  
Cloned Gene ◽  
Yeast Homolog

The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.

scholarly journals ROMO1 is a constituent of the human presequence translocase required for YME1L protease import

2018 ◽  
Vol 218 (2) ◽  
pp. 598-614 ◽  
Author(s):  
Frank Richter ◽  
Sven Dennerlein ◽  
Miroslav Nikolov ◽  
Daniel C. Jans ◽  
Nataliia Naumenko ◽  
...  
Keyword(s):  
Membrane Structure ◽  
Protein Import ◽  
Protein Quality ◽  
Functional Characterization ◽  
Mitochondrial Protein ◽  
Mitochondrial Protein Import ◽  
Yeast Saccharomyces Cerevisiae ◽  
Inner Membrane ◽  
Inner Membrane Protein ◽  
General Protein

The mitochondrial presequence translocation machinery (TIM23 complex) is conserved between the yeast Saccharomyces cerevisiae and humans; however, functional characterization has been mainly performed in yeast. Here, we define the constituents of the human TIM23 complex using mass spectrometry and identified ROMO1 as a new translocase constituent with an exceptionally short half-life. Analyses of a ROMO1 knockout cell line revealed aberrant inner membrane structure and altered processing of the GTPase OPA1. We show that in the absence of ROMO1, mitochondria lose the inner membrane YME1L protease, which participates in OPA1 processing and ROMO1 turnover. While ROMO1 is dispensable for general protein import along the presequence pathway, we show that it participates in the dynamics of TIM21 during respiratory chain biogenesis and is specifically required for import of YME1L. This selective import defect can be linked to charge distribution in the unusually long targeting sequence of YME1L. Our analyses establish an unexpected link between mitochondrial protein import and inner membrane protein quality control.

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.

MAS5, a yeast homolog of DnaJ involved in mitochondrial protein import

1992 ◽  
Vol 12 (1) ◽  
pp. 283-291
Author(s):  
D P Atencio ◽  
M P Yaffe
Keyword(s):  
Protein Import ◽  
Elevated Temperatures ◽  
Mitochondrial Protein ◽  
Heat Shock Gene ◽  
Temperature Sensitive ◽  
Mitochondrial Protein Import ◽  
Yeast Saccharomyces Cerevisiae ◽  
Mutant Phenotypes ◽  
Cloned Gene ◽  
Yeast Homolog

The nuclear mas5 mutation causes temperature-sensitive growth and defects in mitochondrial protein import at the nonpermissive temperature in the yeast Saccharomyces cerevisiae. The MAS5 gene was isolated by complementation of the mutant phenotypes, and integrative transformation demonstrated that the complementing fragment encoded the authentic MAS5 gene. The deduced protein sequence of the cloned gene revealed a polypeptide of 410 amino acids which is homologous to Escherichia coli DnaJ and the yeast DnaJ log SCJ1. Northern (RNA blot) analysis revealed that MAS5 is a heat shock gene whose expression increases moderately at elevated temperatures. Cells with a deletion mutation in MAS5 grew slowly at 23 degrees C and were inviable at 37 degrees C, demonstrating that MAS5 is essential for growth at increased temperatures. The deletion mutant also displayed a modest import defect at 23 degrees C and a substantial import defect at 37 degrees C. These results indicate a role for a DnaJ cognate protein in mitochondrial protein import.

scholarly journals Role of the Mitochondrial Protein Import Machinery and Protein Processing in Heart Disease

2021 ◽  
Vol 8 ◽  
Author(s):  
Fujie Zhao ◽  
Ming-Hui Zou
Keyword(s):  
Heart Disease ◽  
Protein Import ◽  
Protein Quality ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Protein ◽  
Nuclear Genome ◽  
Major Protein ◽  
Intermembrane Space ◽  
Mitochondrial Protein Import ◽  
Mitochondrial Import

Mitochondria are essential organelles for cellular energy production, metabolic homeostasis, calcium homeostasis, cell proliferation, and apoptosis. About 99% of mammalian mitochondrial proteins are encoded by the nuclear genome, synthesized as precursors in the cytosol, and imported into mitochondria by mitochondrial protein import machinery. Mitochondrial protein import systems function not only as independent units for protein translocation, but also are deeply integrated into a functional network of mitochondrial bioenergetics, protein quality control, mitochondrial dynamics and morphology, and interaction with other organelles. Mitochondrial protein import deficiency is linked to various diseases, including cardiovascular disease. In this review, we describe an emerging class of protein or genetic variations of components of the mitochondrial import machinery involved in heart disease. The major protein import pathways, including the presequence pathway (TIM23 pathway), the carrier pathway (TIM22 pathway), and the mitochondrial intermembrane space import and assembly machinery, related translocases, proteinases, and chaperones, are discussed here. This review highlights the importance of mitochondrial import machinery in heart disease, which deserves considerable attention, and further studies are urgently needed. Ultimately, this knowledge may be critical for the development of therapeutic strategies in heart disease.

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