scholarly journals Import of a major mitochondrial enzyme depends on synergy between two distinct helices of its presequence

2016 ◽  
Vol 473 (18) ◽  
pp. 2813-2829 ◽  
Author(s):  
Ester Kalef-Ezra ◽  
Dimitra Kotzamani ◽  
Ioannis Zaganas ◽  
Nitsa Katrakili ◽  
Andreas Plaitakis ◽  
...  
Keyword(s):  
Matrix Proteins ◽  
Mitochondrial Proteins ◽  
Mitochondrial Enzyme ◽  
Yeast Mitochondria ◽  
Mitochondrial Targeting ◽  
Mitochondrial Import ◽  
Transport Efficiency ◽  
Isolated Mitochondria ◽  
Intervening Sequences ◽  
Positively Charged

Mammalian glutamate dehydrogenase (GDH), a nuclear-encoded enzyme central to cellular metabolism, is among the most abundant mitochondrial proteins (constituting up to 10% of matrix proteins). To attain such high levels, GDH depends on very efficient mitochondrial targeting that, for human isoenzymes hGDH1 and hGDH2, is mediated by an unusually long cleavable presequence (N53). Here, we studied the mitochondrial transport of these proteins using isolated yeast mitochondria and human cell lines. We found that both hGDHs were very rapidly imported and processed in isolated mitochondria, with their presequences (N53) alone being capable of directing non-mitochondrial proteins into mitochondria. These presequences were predicted to form two α helices (α1: N 1–10; α2: N 16–32) separated by loops. Selective deletion of the α1 helix abolished the mitochondrial import of hGDHs. While the α1 helix alone had a very weak hGDH mitochondrial import capacity, it could direct efficiently non-mitochondrial proteins into mitochondria. In contrast, the α2 helix had no autonomous mitochondrial-targeting capacity. A peptide consisting of α1 and α2 helices without intervening sequences had GDH transport efficiency comparable with that of N53. Mutagenesis of the cleavage site blocked the intra-mitochondrial processing of hGDHs, but did not affect their mitochondrial import. Replacement of all three positively charged N-terminal residues (Arg3, Lys7 and Arg13) by Ala abolished import. We conclude that the synergistic interaction of helices α1 and α2 is crucial for the highly efficient import of hGDHs into mitochondria.

scholarly journals The nucleus is a quality control center for non-imported mitochondrial proteins

2020 ◽  
Author(s):  
Viplendra P.S. Shakya ◽  
William A. Barbeau ◽  
Tianyao Xiao ◽  
Christina S. Knutson ◽  
Adam L. Hughes
Keyword(s):  
Quality Control ◽  
Steady State ◽  
Nuclear Protein ◽  
Protein Quality ◽  
Mitochondrial Proteins ◽  
Mitochondrial Targeting ◽  
Mitochondrial Import ◽  
Control Center ◽  
Mitochondrial Impairment ◽  
Precursor Proteins

AbstractMitochondrial import deficiency causes cellular stress due to the accumulation of non-imported mitochondrial precursor proteins. Despite the burden mis-localized mitochondrial precursors place on cells, our understanding of the systems that dispose of these proteins is incomplete. Here, we catalog the location and steady-state abundance of mitochondrial precursor proteins during mitochondrial impairment in S. cerevisiae. We find that a number of non-imported mitochondrial proteins localize to the nucleus, where they are eliminated by proteasome-based nuclear protein quality control. Recognition of mitochondrial precursors by the nuclear quality control machinery requires the presence of an N-terminal mitochondrial targeting sequence (MTS), and impaired breakdown of precursors leads to their buildup in nuclear-associated foci. These results identify the nucleus as a key destination for the disposal of non-imported mitochondrial precursors.

scholarly journals A nuclear-based quality control pathway for non-imported mitochondrial proteins

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Viplendra PS Shakya ◽  
William A Barbeau ◽  
Tianyao Xiao ◽  
Christina S Knutson ◽  
Max H Schuler ◽  
...  
Keyword(s):  
Quality Control ◽  
Steady State ◽  
Mitochondrial Proteins ◽  
Mitochondrial Targeting ◽  
Mitochondrial Import ◽  
Cellular Toxicity ◽  
Mitochondrial Impairment ◽  
Induced Stress ◽  
Precursor Proteins ◽  
Combined Action

Mitochondrial import deficiency causes cellular toxicity due to the accumulation of non-imported mitochondrial precursor proteins, termed mitoprotein-induced stress. Despite the burden mis-localized mitochondrial precursors place on cells, our understanding of the systems that dispose of these proteins is incomplete. Here, we cataloged the location and steady-state abundance of mitochondrial precursor proteins during mitochondrial impairment in S. cerevisiae. We found that a number of non-imported mitochondrial proteins localize to the nucleus, where they are subjected to proteasome-dependent degradation through a process we term nuclear-associated mitoprotein degradation (mitoNUC). Recognition and destruction of mitochondrial precursors by the mitoNUC pathway requires the presence of an N-terminal mitochondrial targeting sequence (MTS) and is mediated by combined action of the E3 ubiquitin ligases San1, Ubr1, and Doa10. Impaired breakdown of precursors leads to alternative sequestration in nuclear-associated foci. These results identify the nucleus as an important destination for the disposal of non-imported mitochondrial precursors.

scholarly journals The synthesis of murine ferrochelatase in vitro and in vivo

1988 ◽  
Vol 254 (3) ◽  
pp. 799-803 ◽  
Author(s):  
S R Karr ◽  
H A Dailey
Keyword(s):  
Membrane Protein ◽  
Biosynthetic Pathway ◽  
Integral Membrane Protein ◽  
Mitochondrial Proteins ◽  
Isolated Mitochondria ◽  
Mitochondrial Inner Membrane ◽  
Carbonyl Cyanide ◽  
A Cell

Ferrochelatase (protohaem ferro-lyase, EC 4.99.1.1), the terminal enzyme of the haem-biosynthetic pathway, is an integral membrane protein of the mitochondrial inner membrane. When murine erythroleukaemia cells are labelled in vivo with [35S]methionine, lysed, and the extract is immunoprecipitated with rabbit anti-(mouse ferrochelatase) antibody, a protein of Mr 40,000 is isolated. However, when isolated mouse RNA is translated in a cell-free reticulocyte extract, a protein of Mr 43,000 is isolated. Incubation of this Mr 43,000 protein with isolated mitochondria resulted in processing of the Mr 43,000 precursor to the Mr 40,000 mature-sized protein. Addition of carbonyl cyanide m-chlorophenylhydrazone and/or phenanthroline inhibits this processing. These data indicate that ferrochelatase, like most mitochondrial proteins, is synthesized in the cytoplasm as a larger precursor and is then translocated and processed to a mature-sized protein in an energy-required step.

Oxidative phosphorylation and respiratory control in mitochondria from Aspergillus oryzae

1969 ◽  
Vol 15 (8) ◽  
pp. 975-977 ◽  
Author(s):  
K. Watson ◽  
W. Paton ◽  
J. E. Smith
Keyword(s):  
Bovine Serum Albumin ◽  
Oxidative Phosphorylation ◽  
Aspergillus Oryzae ◽  
Reaction Medium ◽  
Respiratory Control ◽  
Adenosine Diphosphate ◽  
Yeast Mitochondria ◽  
Active Oxidation ◽  
Isolated Mitochondria ◽  
Ph Range

Mitochondria isolated from Aspergillus oryzae exhibited respiratory control with a range of substrates. Bovine serum albumin was required in the reaction medium to observe adenosine diphosphate (ADP) controlled respiration. The mitochondria carried out active oxidation and phosphorylation with citrate as substrate in the pH range 6–7 and showed a slight optimum for oxidative phosphorylation at pH 6.5. The respiratory properties of the isolated mitochondria were similar to those reported for A. niger and yeast mitochondria.

scholarly journals Tom20 Mediates Localization of mRNAs to Mitochondria in a Translation-Dependent Manner

2009 ◽  
Vol 30 (1) ◽  
pp. 284-294 ◽  
Author(s):  
Erez Eliyahu ◽  
Lilach Pnueli ◽  
Daniel Melamed ◽  
Tanja Scherrer ◽  
André P. Gerber ◽  
...  
Keyword(s):  
Large Scale ◽  
Dna Microarrays ◽  
Protein Transport ◽  
Yeast Cells ◽  
Mitochondrial Proteins ◽  
Mitochondrial Outer Membrane ◽  
Dependent Manner ◽  
Mitochondrial Targeting ◽  
Double Knockout ◽  
Mitochondrial Fractions

ABSTRACT mRNAs encoding mitochondrial proteins are enriched in the vicinity of mitochondria, presumably to facilitate protein transport. A possible mechanism for enrichment may involve interaction of the translocase of the mitochondrial outer membrane (TOM) complex with the precursor protein while it is translated, thereby leading to association of polysomal mRNAs with mitochondria. To test this hypothesis, we isolated mitochondrial fractions from yeast cells lacking the major import receptor, Tom20, and compared their mRNA repertoire to that of wild-type cells by DNA microarrays. Most mRNAs encoding mitochondrial proteins were less associated with mitochondria, yet the extent of decrease varied among genes. Analysis of several mRNAs revealed that optimal association of Tom20 target mRNAs requires both translating ribosomes and features within the encoded mitochondrial targeting signal. Recently, Puf3p was implicated in the association of mRNAs with mitochondria through interaction with untranslated regions. We therefore constructed a tom20Δ puf3Δ double-knockout strain, which demonstrated growth defects under conditions where fully functional mitochondria are required. Mislocalization effects for few tested mRNAs appeared stronger in the double knockout than in the tom20Δ strain. Taken together, our data reveal a large-scale mRNA association mode that involves interaction of Tom20p with the translated mitochondrial targeting sequence and may be assisted by Puf3p.

scholarly journals The GET pathway safeguards against non-imported mitochondrial protein stress

2020 ◽  
Author(s):  
Tianyao Xiao ◽  
Viplendra P.S. Shakya ◽  
Adam L. Hughes
Keyword(s):  
Endoplasmic Reticulum ◽  
Mitochondrial Protein ◽  
Transmembrane Proteins ◽  
Mitochondrial Proteins ◽  
Mitochondrial Import ◽  
Cellular Toxicity ◽  
Precursor Proteins ◽  
Dependent Protein ◽  
Er Targeting ◽  
Get Complex

SUMMARYDeficiencies in mitochondrial import cause the toxic accumulation of non-imported mitochondrial precursor proteins. Numerous fates for non-imported mitochondrial precursors have been identified, including proteasomal destruction, deposition into protein aggregates, and mis-targeting to other organelles. Amongst organelles, the endoplasmic reticulum (ER) has emerged as a key destination for non-imported mitochondrial proteins, but how ER-targeting of these proteins is achieved remains unclear. Here, we show that the guided entry of tail-anchored proteins (GET) complex is required for ER-targeting of endogenous mitochondrial multi-transmembrane proteins. Without a functional GET pathway, non-imported mitochondrial proteins destined for the ER are alternatively sequestered into Hsp42-dependent protein foci. The ER targeting of non-imported mitochondrial proteins by the GET complex prevents cellular toxicity and facilitates re-import of mitochondrial proteins from the ER via the recently identified ER-SURF pathway. Overall, this study outlines an important and unconventional role for the GET complex in mitigating stress associated with non-imported mitochondrial proteins.

scholarly journals Causes and consequences of mitochondrial proteome size-variation in animals

2019 ◽  
Author(s):  
Viraj Muthye ◽  
Dennis Lavrov
Keyword(s):  
Functional Significance ◽  
Size Variation ◽  
Mitochondrial Proteins ◽  
Mitochondrial Targeting ◽  
Factors Affecting ◽  
Mitochondrial Proteome ◽  
Mitochondrial Functions ◽  
Size Evolution ◽  
Targeting Signals ◽  
Gain And Loss

AbstractDespite a conserved set of core mitochondrial functions, animal mitochondrial proteomes show a large variation in size. In this study, we analyzed the putative mechanisms behind and functional significance of this variation using experimentally-verified mt-proteomes of four bilaterian animals and two non-animal outgroups. We found that, of several factors affecting mitochondrial proteome size, evolution of novel mitochondrial proteins in mammals and loss of ancestral proteins in protostomes were the main contributors. Interestingly, gain and loss of conventional mitochondrial targeting signals was not a significant factor in the proteome size evolution.

scholarly journals The intermembrane space protein Mix23 is a novel stress-induced mitochondrial import factor

2020 ◽  
Vol 295 (43) ◽  
pp. 14686-14697 ◽  
Author(s):  
Eva Zöller ◽  
Janina Laborenz ◽  
Lena Krämer ◽  
Felix Boos ◽  
Markus Räschle ◽  
...  
Keyword(s):  
Protein Import ◽  
Mitochondrial Protein ◽  
Mitochondrial Proteins ◽  
Temperature Sensitive ◽  
Intermembrane Space ◽  
Mitochondrial Import ◽  
Induced Stress ◽  
Evolutionarily Conserved ◽  
Precursor Proteins ◽  
Biogenesis Of Mitochondria

The biogenesis of mitochondria requires the import of hundreds of precursor proteins. These proteins are transported post-translationally with the help of chaperones, meaning that the overproduction of mitochondrial proteins or the limited availability of chaperones can lead to the accumulation of cytosolic precursor proteins. This imposes a severe challenge to cytosolic proteostasis and triggers a specific transcription program called the mitoprotein-induced stress response, which activates the proteasome system. This coincides with the repression of mitochondrial proteins, including many proteins of the intermembrane space. In contrast, herein we report that the so-far-uncharacterized intermembrane space protein Mix23 is considerably up-regulated when mitochondrial import is perturbed. Mix23 is evolutionarily conserved and a homolog of the human protein CCDC58. We found that, like the subunits of the proteasome, Mix23 is under control of the transcription factor Rpn4. It is imported into mitochondria by the mitochondrial disulfide relay. Mix23 is critical for the efficient import of proteins into the mitochondrial matrix, particularly if the function of the translocase of the inner membrane 23 is compromised such as in temperature-sensitive mutants of Tim17. Our observations identify Mix23 as a novel regulator or stabilizer of the mitochondrial protein import machinery that is specifically up-regulated upon mitoprotein-induced stress conditions.

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