Coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes is assisted by CLPP2 in Arabidopsis thaliana

AbstractProtein homeostasis in eukaryotic organelles and their progenitor prokaryotes is regulated by a series of proteases including the caseinolytic protease (CLPP). CLPP has essential roles in chloroplast biogenesis and maintenance, but the significance of the plant mitochondrial CLPP remains unknown and factors that aid coordination of nuclear and mitochondrial encoded subunits for complex assembly in mitochondria await discovery. We generated knock-out lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis thaliana. Mutants had higher abundance of transcripts from mitochondrial genes encoding OXPHOS protein complexes, while transcripts for nuclear genes encoding other subunits of the same complexes showed no change in abundance. In contrast, the protein abundance of specific nuclear-encoded subunits in OXPHOS complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Protein complexes mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import, assembly and function of Complex I revealed that while function was retained, protein homeostasis was disrupted through decreased assembly, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.One sentence summaryCLPP contributes to the mitochondrial protein degradation network through supporting coordination and assembly of protein complexes encoded across mitochondrial and nuclear genomes.

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Arabidopsis thaliana G3BP Ortholog Rescues Mammalian Stress Granule Phenotype across Kingdoms

International Journal of Molecular Sciences ◽

10.3390/ijms22126287 ◽

2021 ◽

Vol 22 (12) ◽

pp. 6287

Author(s):

Hendrik Reuper ◽

Benjamin Götte ◽

Lucy Williams ◽

Timothy J. C. Tan ◽

Gerald M. McInerney ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Fusion Proteins ◽

Biological Function ◽

Protein Complexes ◽

Protein Family ◽

Knock Out ◽

Marker Proteins ◽

Interaction Partners ◽

Functional Analyses ◽

Rna Protein Complexes

Stress granules (SGs) are dynamic RNA–protein complexes localized in the cytoplasm that rapidly form under stress conditions and disperse when normal conditions are restored. The formation of SGs depends on the Ras-GAP SH3 domain-binding protein (G3BP). Formations, interactions and functions of plant and human SGs are strikingly similar, suggesting a conserved mechanism. However, functional analyses of plant G3BPs are missing. Thus, members of the Arabidopsis thaliana G3BP (AtG3BP) protein family were investigated in a complementation assay in a human G3BP knock-out cell line. It was shown that two out of seven AtG3BPs were able to complement the function of their human homolog. GFP-AtG3BP fusion proteins co-localized with human SG marker proteins Caprin-1 and eIF4G1 and restored SG formation in G3BP double KO cells. Interaction between AtG3BP-1 and -7 and known human G3BP interaction partners such as Caprin-1 and USP10 was also demonstrated by co-immunoprecipitation. In addition, an RG/RGG domain exchange from Arabidopsis G3BP into the human G3BP background showed the ability for complementation. In summary, our results support a conserved mechanism of SG function over the kingdoms, which will help to further elucidate the biological function of the Arabidopsis G3BP protein family.

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Skeletal Phenotypes Due to Abnormalities in Mitochondrial Protein Homeostasis and Import

International Journal of Molecular Sciences ◽

10.3390/ijms21218327 ◽

2020 ◽

Vol 21 (21) ◽

pp. 8327

Author(s):

Tian Zhao ◽

Caitlin Goedhart ◽

Gerald Pfeffer ◽

Steven C Greenway ◽

Matthew Lines ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Mitochondrial Disease ◽

Protein Import ◽

Mitochondrial Protein ◽

Membrane Lipid ◽

Disease Genes ◽

Protein Homeostasis ◽

Inner Mitochondrial Membrane ◽

Mitochondrial Functions ◽

Insight Into

Mitochondrial disease represents a collection of rare genetic disorders caused by mitochondrial dysfunction. These disorders can be quite complex and heterogeneous, and it is recognized that mitochondrial disease can affect any tissue at any age. The reasons for this variability are not well understood. In this review, we develop and expand a subset of mitochondrial diseases including predominantly skeletal phenotypes. Understanding how impairment ofdiverse mitochondrial functions leads to a skeletal phenotype will help diagnose and treat patients with mitochondrial disease and provide additional insight into the growing list of human pathologies associated with mitochondrial dysfunction. The underlying disease genes encode factors involved in various aspects of mitochondrial protein homeostasis, including proteases and chaperones, mitochondrial protein import machinery, mediators of inner mitochondrial membrane lipid homeostasis, and aminoacylation of mitochondrial tRNAs required for translation. We further discuss a complex of frequently associated phenotypes (short stature, cataracts, and cardiomyopathy) potentially explained by alterations to steroidogenesis, a process regulated by mitochondria. Together, these observations provide novel insight into the consequences of impaired mitochondrial protein homeostasis.

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Ups1p and Ups2p antagonistically regulate cardiolipin metabolism in mitochondria

The Journal of Cell Biology ◽

10.1083/jcb.200812018 ◽

2009 ◽

Vol 185 (6) ◽

pp. 1029-1045 ◽

Cited By ~ 117

Author(s):

Yasushi Tamura ◽

Toshiya Endo ◽

Miho Iijima ◽

Hiromi Sesaki

Keyword(s):

Fatty Acid ◽

Molecular Mechanism ◽

Protein Import ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Growth Defects ◽

Mitochondrial Inner Membrane ◽

Synthetic Growth

Cardiolipin, a unique phospholipid composed of four fatty acid chains, is located mainly in the mitochondrial inner membrane (IM). Cardiolipin is required for the integrity of several protein complexes in the IM, including the TIM23 translocase, a dynamic complex which mediates protein import into the mitochondria through interactions with the import motor presequence translocase–associated motor (PAM). In this study, we report that two homologous intermembrane space proteins, Ups1p and Ups2p, control cardiolipin metabolism and affect the assembly state of TIM23 and its association with PAM in an opposing manner. In ups1Δ mitochondria, cardiolipin levels were decreased, and the TIM23 translocase showed altered conformation and decreased association with PAM, leading to defects in mitochondrial protein import. Strikingly, loss of Ups2p restored normal cardiolipin levels and rescued TIM23 defects in ups1Δ mitochondria. Furthermore, we observed synthetic growth defects in ups mutants in combination with loss of Pam17p, which controls the integrity of PAM. Our findings provide a novel molecular mechanism for the regulation of cardiolipin metabolism.

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The Tim9p/10p and Tim8p/13p Complexes Bind to Specific Sites on Tim23p during Mitochondrial Protein Import

Molecular Biology of the Cell ◽

10.1091/mbc.e06-06-0546 ◽

2007 ◽

Vol 18 (2) ◽

pp. 475-486 ◽

Cited By ~ 37

Author(s):

Alison J. Davis ◽

Nathan N. Alder ◽

Robert E. Jensen ◽

Arthur E. Johnson

Keyword(s):

Protein Import ◽

Protein Complexes ◽

Mitochondrial Protein ◽

Cross Linking ◽

Intermembrane Space ◽

Mitochondrial Protein Import ◽

Mitochondrial Inner Membrane ◽

Specificity Of Binding ◽

Proper Orientation ◽

Tim Proteins

The import of polytopic membrane proteins into the mitochondrial inner membrane (IM) is facilitated by Tim9p/Tim10p and Tim8p/Tim13p protein complexes in the intermembrane space (IMS). These complexes are proposed to act as chaperones by transporting the hydrophobic IM proteins through the aqueous IMS and preventing their aggregation. To examine the nature of this interaction, Tim23p molecules containing a single photoreactive cross-linking probe were imported into mitochondria in the absence of an IM potential where they associated with small Tim complexes in the IMS. On photolysis and immunoprecipitation, a probe located at a particular Tim23p site (27 different locations were examined) was found to react covalently with, in most cases, only one of the small Tim proteins. Tim8p, Tim9p, Tim10p, and Tim13p were therefore positioned adjacent to specific sites in the Tim23p substrate before its integration into the IM. This specificity of binding to Tim23p strongly suggests that small Tim proteins do not function solely as general chaperones by minimizing the exposure of nonpolar Tim23p surfaces to the aqueous medium, but may also align a folded Tim23p substrate in the proper orientation for delivery and integration into the IM at the TIM22 translocon.

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Transcriptome analysis reveals rice MADS13 as an important repressor of the carpel development pathway in ovules

Journal of Experimental Botany ◽

10.1093/jxb/eraa460 ◽

2020 ◽

Author(s):

Michela Osnato ◽

Elia Lacchini ◽

Alessandro Pilatone ◽

Ludovico Dreni ◽

Andrea Grioni ◽

...

Keyword(s):

Transcription Factors ◽

Molecular Mechanisms ◽

Target Genes ◽

Protein Complexes ◽

Female Sterility ◽

Homeotic Genes ◽

Ovule Development ◽

Knock Out ◽

Regulatory Pathways ◽

Genes Encoding

Abstract In angiosperms, floral homeotic genes encoding MADS-domain transcription factors regulate the development of floral organs. Specifically, members of the SEPALLATA (SEP) and AGAMOUS (AG) subfamilies form higher-order protein complexes to control floral meristem determinacy and to specify the identity of female reproductive organs. In rice, the AG subfamily gene OsMADS13 is intimately involved in the determination of ovule identity, since knock-out mutant plants develop carpel-like structures in place of ovules, resulting in female sterility. Little is known about the regulatory pathways at the base of rice gynoecium development. To investigate molecular mechanisms acting downstream of OsMADS13, we obtained transcriptomes of immature inflorescences from wild-type and Osmads13 mutant plants. Among a total of 476 differentially expressed genes (DEGs), a substantial overlap with DEGs from the SEP-family Osmads1 mutant was found, suggesting that OsMADS1 and OsMADS13 may act on a common set of target genes. Expression studies and preliminary analyses of two up-regulated genes encoding Zinc-finger transcription factors indicated that our dataset represents a valuable resource for the identification of both OsMADS13 target genes and novel players in rice ovule development. Taken together, our study suggests that OsMADS13 is an important repressor of the carpel pathway during ovule development.

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

Genetics ◽

10.1093/genetics/165.1.35 ◽

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.

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Cytosolic phosphofructokinases are important for sugar homeostasis in leaves of Arabidopsis thaliana

Annals of Botany ◽

10.1093/aob/mcab122 ◽

2021 ◽

Author(s):

Laura Kathrine Perby ◽

Simon Richter ◽

Konrad Weber ◽

Alina Johanna Hieber ◽

Natalia Hess ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Double Mutant ◽

Normal Growth ◽

Growth Conditions ◽

Metabolic Phenotype ◽

Minor Role ◽

Knock Out ◽

Genes Encoding ◽

Fructose 1,6 Bisphosphate ◽

Sugar Homeostasis

Abstract Background and Aims ATP-dependent phosphofructokinases (PFKs) catalyse phosphorylation of the carbon-1 position of fructose-6-phosphate, to form fructose-1,6-bisphosphate. In the cytosol, this is considered a key step in channelling carbon into glycolysis. Arabidopsis thaliana has seven genes encoding PFK isoforms, two chloroplastic and five cytosolic. This study focusses on the four major cytosolic isoforms of PFK in vegetative tissues of A. thaliana. Methods We have isolated homozygous knock-out individual mutants (pfk1, pfk3, pfk6, pfk7) and two double mutants (pfk1/7 and pfk3/6) and characterized their growth and metabolic phenotypes. Key Results In contrast to single mutants and the double mutant pfk3/6 for the hypoxia-responsive isoforms, the double mutant pfk1/7 had reduced PFK activity and shows a clear visual and metabolic phenotype with reduced shoot growth, early flowering, and elevated hexose levels. This mutant also has an altered ratio of short/long aliphatic glucosinolates and an altered root-shoot distribution. Surprisingly, this mutant does not show any major changes in short-term carbon flux and in levels of hexose-phosphates. Conclusions We conclude that the two isoforms PFK1 and PFK7 are important for sugar homeostasis in leaf metabolism and apparently source/sink relations in Arabidopsis, while PFK3 and PFK6 only play a minor role under normal growth conditions.

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TOM9.2 Is a Calmodulin-Binding Protein Critical for TOM Complex Assembly but Not for Mitochondrial Protein Import in Arabidopsis thaliana

Molecular Plant ◽

10.1016/j.molp.2016.12.012 ◽

2017 ◽

Vol 10 (4) ◽

pp. 575-589 ◽

Cited By ~ 3

Author(s):

Nargis Parvin ◽

Chris Carrie ◽

Isabelle Pabst ◽

Antonia Läßer ◽

Debabrata Laha ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Protein Import ◽

Binding Protein ◽

Mitochondrial Protein ◽

Mitochondrial Protein Import ◽

Complex Assembly ◽

Calmodulin Binding ◽

Tom Complex

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Intramitochondrial proteostasis is directly coupled to α-synuclein and amyloid β1-42 pathologies

Journal of Biological Chemistry ◽

10.1074/jbc.ra119.011650 ◽

2020 ◽

Vol 295 (30) ◽

pp. 10138-10152 ◽

Cited By ~ 3

Author(s):

Janin Lautenschläger ◽

Sara Wagner-Valladolid ◽

Amberley D. Stephens ◽

Ana Fernández-Villegas ◽

Colin Hockings ◽

...

Keyword(s):

Mitochondrial Dysfunction ◽

Protein Import ◽

Protein Quality ◽

Neurodegenerative Disorder ◽

Mitochondrial Protein ◽

Amyloid Β ◽

Ubiquitin Proteasome System ◽

Protein Homeostasis ◽

Mitochondrial Inhibitors ◽

Ubiquitin Proteasome

Mitochondrial dysfunction has long been implicated in the neurodegenerative disorder Parkinson's disease (PD); however, it is unclear how mitochondrial impairment and α-synuclein pathology are coupled. Using specific mitochondrial inhibitors, EM analysis, and biochemical assays, we report here that intramitochondrial protein homeostasis plays a major role in α-synuclein aggregation. We found that interference with intramitochondrial proteases, such as HtrA2 and Lon protease, and mitochondrial protein import significantly aggravates α-synuclein seeding. In contrast, direct inhibition of mitochondrial complex I, an increase in intracellular calcium concentration, or formation of reactive oxygen species, all of which have been associated with mitochondrial stress, did not affect α-synuclein pathology. We further demonstrate that similar mechanisms are involved in amyloid-β 1-42 (Aβ42) aggregation. Our results suggest that, in addition to other protein quality control pathways, such as the ubiquitin–proteasome system, mitochondria per se can influence protein homeostasis of cytosolic aggregation-prone proteins. We propose that approaches that seek to maintain mitochondrial fitness, rather than target downstream mitochondrial dysfunction, may aid in the search for therapeutic strategies to manage PD and related neuropathologies.

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