Detection of Cysteine Redox States in Mitochondrial Proteins in Intact Mammalian Cells

2017 ◽  
pp. 105-138 ◽  
Author(s):  
Markus Habich ◽  
Jan Riemer
Keyword(s):  
Mammalian Cells ◽  
Mitochondrial Proteins ◽  
Redox States

scholarly journals CLUH interactome reveals an association to SPAG5 and a proximity to the translation of mitochondrial protein

2021 ◽  
Author(s):  
Mickaële Hemono ◽  
Alexandre Haller ◽  
Johana Chicher ◽  
Anne-Marie Duchêne ◽  
Richard Patryk Ngondo
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Mitochondrial Protein ◽  
Embryonic Stem ◽  
Nuclear Genome ◽  
Mrna Translation ◽  
Mitochondrial Proteins ◽  
Molecular Function ◽  
First Time

Mitochondria require thousands of proteins to fulfil their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal a stable RNA-independent interaction of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. Additionally, our data highlight the importance of CLUH TPR domain for its interactions with both proteins and mRNAs. Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by highlighting its association to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

scholarly journals The two mammalian mitochondrial stress proteins, grp 75 and hsp 58, transiently interact with newly synthesized mitochondrial proteins.

1991 ◽  
Vol 2 (2) ◽  
pp. 165-179 ◽  
Author(s):  
L A Mizzen ◽  
A N Kabiling ◽  
W J Welch
Keyword(s):  
Mammalian Cells ◽  
Stress Proteins ◽  
Mitochondrial Proteins ◽  
Amino Acid Analogue ◽  
Hsp 70 ◽  
Protein Maturation ◽  
Mitochondrial Stress ◽  
Chase Period ◽  
Acid Analogue ◽  
Glucose Regulated Protein

In mammalian cells, two of the so-called heat shock (hsp) or stress proteins are components of the mitochondria. One of these, hsp 58, is a member of the bacterial GroEL family, whereas the other, glucose-regulated protein (grp) 75, represents a member of the hsp 70 family of stress proteins. Owing to previous studies implicating a role for both the hsp 70 and GroEL families in facilitating protein maturation events, we used the method of native immunoprecipitation to examine whether hsp 58 and grp 75 might interact with other proteins of the mitochondria. In cells pulse-labeled with [35S]-methionine, a significant number of newly synthesized mitochondrial proteins co-precipitated with either hsp 58 or grp 75. Such interactions appeared transient. For example, providing the pulse-labeled cells a subsequent chase period in the absence of radiolabel resulted in a reduction of co-precipitating proteins. If the pulse-chase labeling experiments were performed in the presence of an amino acid analogue, somewhat different results were obtained. Specifically, although many of the newly synthesized and analogue-containing proteins again were observed to co-precipitate with grp 75, the interactions did not appear transient, but instead were stable. Under steady-state labeling conditions, we also observed a portion of hsp 58 and grp 75 in an apparent complex with one another. On addition of ATP, the complex was dissociated. Accompanying this dissociation was the concomitant autophosphorylation of grp 75. On the basis of these observations, as well as previous studies examining the structure/function of the hsp 70 and GroEL proteins, we suspect that both hsp 58 and grp 75 interact with and facilitate the folding and assembly of proteins as they enter into the mitochondria.

scholarly journals Mitochondrial proteome of mouse oocytes and cisplatin-induced shifts in protein profile

2021 ◽  
Author(s):  
Na Zhang ◽  
An-di Sun ◽  
Si-man Sun ◽  
Rui Yang ◽  
Yan-yan Shi ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Protein Profile ◽  
Chemotherapeutic Agents ◽  
Mitochondrial Proteins ◽  
Cellular Respiration ◽  
Inherited Disorders ◽  
Mitochondrial Proteome ◽  
Mouse Oocytes ◽  
Atp Metabolism ◽  
Functional Components

AbstractMitochondria are essential organelles that provide energy for mammalian cells and participate in multiple functions, such as signal transduction, cellular differentiation, and regulation of apoptosis. Compared with the mitochondria in somatic cells, oocyte mitochondria have an additional level of importance since they are required for germ cell maturation, dysfunction in which can lead to severe inherited disorders. Thus, a systematic proteomic profile of oocyte mitochondria is urgently needed to support the basic and clinical research, but the acquisition of such a profile has been hindered by the rarity of oocyte samples and technical challenges associated with capturing mitochondrial proteins from live oocytes. Here, in this work, using proximity labeling proteomics, we established a mitochondria-specific ascorbate peroxidase (APEX2) reaction in live GV-stage mouse oocytes and identified a total of 158 proteins in oocyte mitochondria. This proteome includes intrinsic mitochondrial structural and functional components involved in processes associated with “cellular respiration”, “ATP metabolism”, “mitochondrial transport”, etc. In addition, mitochondrial proteome capture after oocyte exposure to the antitumor chemotherapeutic cisplatin revealed differential changes in the abundance of several oocyte-specific mitochondrial proteins. Our study provides the first description of a mammalian oocyte mitochondrial proteome of which we are aware, and further illustrates the dynamic shifts in protein abundance associated with chemotherapeutic agents.

scholarly journals The interactome of CLUH reveals its association to SPAG5 and its co-translational proximity to mitochondrial proteins

BMC Biology ◽  
2022 ◽  
Vol 20 (1) ◽  
Author(s):  
Mickaële Hémono ◽  
Alexandre Haller ◽  
Johana Chicher ◽  
Anne-Marie Duchêne ◽  
Richard Patryk Ngondo
Keyword(s):  
Mammalian Cells ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Embryonic Stem ◽  
Nuclear Genome ◽  
Mrna Translation ◽  
Mitochondrial Proteins ◽  
Molecular Function ◽  
First Time ◽  
Tpr Domain

Abstract Background Mitochondria require thousands of proteins to fulfill their essential function in energy production and other fundamental biological processes. These proteins are mostly encoded by the nuclear genome, translated in the cytoplasm before being imported into the organelle. RNA binding proteins (RBPs) are central players in the regulation of this process by affecting mRNA translation, stability, or localization. CLUH is an RBP recognizing specifically mRNAs coding for mitochondrial proteins, but its precise molecular function and interacting partners remain undiscovered in mammals. Results Here we reveal for the first time CLUH interactome in mammalian cells. Using both co-IP and BioID proximity-labeling approaches, we identify novel molecular partners interacting stably or transiently with CLUH in HCT116 cells and mouse embryonic stem cells. We reveal stable RNA-independent interactions of CLUH with itself and with SPAG5 in cytosolic granular structures. More importantly, we uncover an unexpected proximity of CLUH to mitochondrial proteins and their cognate mRNAs in the cytosol. We show that this interaction occurs during the process of active translation and is dependent on CLUH TPR domain. Conclusions Overall, through the analysis of CLUH interactome, our study sheds a new light on CLUH molecular function by revealing new partners and by highlighting its link to the translation and subcellular localization of some mRNAs coding for mitochondrial proteins.

scholarly journals Cryo-EM Structure of the Human Mitochondrial Translocase TIM22 Complex

2019 ◽  
Author(s):  
Liangbo Qi ◽  
Qiang Wang ◽  
Zeyuan Guan ◽  
Yan Wu ◽  
Jianbo Cao ◽  
...  
Keyword(s):  
Molecular Basis ◽  
Mammalian Cells ◽  
Transmembrane Protein ◽  
Complex Function ◽  
Mitochondrial Proteins ◽  
Molar Ratio ◽  
Intermembrane Space ◽  
Transmembrane Helices ◽  
The Core ◽  
Vital Functions

AbstractMitochondria play vital functions in cellular metabolism, homeostasis, and apoptosis1-3. Most of the mitochondrial proteins are synthesized as precursors in the cytosol and imported into mitochondria for folding or maturation4,5. The translocase TIM22 complex is responsible for the import of multiple hydrophobic carrier proteins that are then folded in the inner membrane of mitochondria6-8. In mammalian cells, the TIM22 complex consists of at least six components, Tim22, Tim29, AGK, and three Tim chaperones (Tim9, Tim10a and Tim10b)9-14. Here, we report the cryo-EM structure of the human translocase TIM22 complex at an overall resolution of 3.7 angstrom. The core subunit, Tim22, contains four transmembrane helices, forming a partial pore that is open to the lipid bilayer. Tim29 is a single transmembrane protein that provides an N-terminal helix to stabilize Tim22 and a C-terminal intermembrane space (IMS) domain to connect AGK and two TIM chaperone hexamers to maintain complex integrity. One TIM hexamer comprises Tim9 and Tim10a in a 3:3 molar ratio, and the other consists of two Tim9 units, three Tim10a units, and one Tim10b unit. The latter hexamer faces the intramembrane region of Tim22, likely providing the dock to load the precursors to the partial pore of Tim22. Our structure serves as a molecular basis for the mechanistic understanding of TIM22 complex function.

Effect of Chloramphenicol on Enzyme Synthesis and Growth of Mammalian Cells

1972 ◽  
Vol 50 (2) ◽  
pp. 200-209 ◽  
Author(s):  
I. M. Fettes ◽  
D. Haldar ◽  
K. B. Freeman
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Oxidase Activity ◽  
Mammalian Cells ◽  
Nadh Dehydrogenase ◽  
Enzyme Synthesis ◽  
Mitochondrial Proteins ◽  
Mitochondrial Enzymes ◽  
L Cells ◽  
Two Generations

D-threo-Chloramphenicol and its sulfamoyl analogue Tevenel, but not L-chloramphenicol, prevent the formation of a functional cytochrome c oxidase in L cells. The syntheses of several mitochondrial enzymes, malate dehydrogenase, succinate dehydrogenase, NADH dehydrogenase, ATPase, and cytochromes b, c, and c1, were not inhibited. Cycloheximide also inhibited the formation of cytochrome c oxidase. After removal of D-chloramphenicol or Tevenel, cytochrome oxidase activity returned at a rate equivalent to its normal synthesis. L cells continued to grow for up to five generations in the presence of Tevenel at 100 μg/ml but only for 1.5 to two generations with D-chloramphenicol at the same concentration. D-Chloramphenicol specifically inhibited the synthesis of insoluble mitochondrial proteins in ascites cells.

Ultrastructural Alterations in Hela Cells Induced by Spider Venom

1967 ◽  
Vol 25 ◽  
pp. 20-21
Author(s):  
Dale E. McClendon ◽  
Paul N. Morgan ◽  
Bernard L. Soloff
Keyword(s):  
Growth Medium ◽  
Mammalian Cells ◽  
Carcinoma Cells ◽  
Venom Protein ◽  
Sterile Saline ◽  
Carbon Coated ◽  
Available Information ◽  
Loxosceles Reclusa ◽  
Essential Growth ◽  
Solution Cultures

It has been observed that minute amounts of venom from the brown recluse spider, Loxosceles reclusa, are capable of producing cytotoxic changes in cultures of certain mammalian cells (Morgan and Felton, 1965). Since there is little available information concerning the effect of venoms on susceptible cells, we have attempted to characterize, at the electron microscope level, the cytotoxic changes produced by the venom of this spider.Cultures of human epithelial carcinoma cells, strain HeLa, were initiated on sterile, carbon coated coverslips contained in Leighton tubes. Each culture was seeded with approximately 1x105 cells contained in 1.5 ml of a modified Eagle's minimum essential growth medium prepared in Hank's balanced salt solution. Cultures were incubated at 36° C. for three days prior to the addition of venom. The venom was collected from female brown recluse spiders and diluted in sterile saline. Protein determinations on the venom-were made according to the spectrophotometric method of Waddell (1956). Approximately 10 μg venom protein per ml of fresh medium was added to each culture after discarding the old growth medium. Control cultures were treated similarly, except that no venom was added. All cultures were reincubated at 36° C.

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