Accumulation of Newly Synthesized F1 in Vivo in Arabidopsis Mitochondria Provides Evidence for Modular Assembly of the Plant F1Fo ATP Synthase

Li, Lie; Carrie, Chris; Nelson, Clark J.; Whelan, James; Millar, A. Harvey

doi:10.1074/jbc.m112.373506

Cited by 24 publications

(37 citation statements)

References 41 publications

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“…The different patterns of protein degradation rate and protein abundance observed may have obscured difference in populations of the same protein, present in insoluble/soluble fractions or in protein complexes of different sizes. We have observed this situation previously in directed analysis of OXPHOS complexes in membrane and soluble fractions (Li et al ., , ). In addition we were mindful of reports of insoluble protein bodies detected by microscopy in lon1 mitochondria (Suzuki et al ., ; Rigas et al ., ; Strauss et al ., ).…”

Section: Resultsmentioning

confidence: 99%

“…For example, within the members of the protein synthesis group, there is an apparent order in degradation rates with elongation factors being the fastest, small subunit proteins being intermediate and large subunit proteins being the slowest (Figure (b)). Protein degradation differences between subunits or subcomplexes can reflect the order of protein complex assembly pathways (Li et al ., ,b, ). The observed groups of degradation rates amongst proteins functioning in protein synthesis may support the hypothesis that small and large subunit proteins are assembled into the ribosome in a defined order (De Silva et al ., ).…”

Section: Discussionmentioning

confidence: 99%

“…We have used 15 N labelling to investigate mitochondrial protein degradation rates in Arabidopsis cell culture and the assembly of electron transport chain (ETC) complexes using BN‐PAGE separation of holoenzyme complexes and smaller subcomplexes undergoing assembly. Assembly intermediates have been shown to have different degradation rates to intact complexes due to the varying latency of proteins in subcomplexes during the assembly process (Li et al ., , ).…”

Section: Introductionmentioning

confidence: 99%

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Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Nelson

Fenske

et al. 2017

The Plant Journal

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SUMMARYMitochondrial Lon1 loss impairs oxidative phosphorylation complexes and TCA enzymes and causes accumulation of specific mitochondrial proteins. Analysis of over 400 mitochondrial protein degradation rates using 15 N labelling showed that 205 were significantly different between wild type (WT) and lon1-1. Those proteins included ribosomal proteins, electron transport chain subunits and TCA enzymes. For respiratory complexes I and V, decreased protein abundance correlated with higher degradation rate of subunits in total mitochondrial extracts. After blue native separation, however, the assembled complexes had slow degradation, while smaller subcomplexes displayed rapid degradation in lon1-1. In insoluble fractions, a number of TCA enzymes were more abundant but the proteins degraded slowly in lon1-1. In soluble protein fractions, TCA enzymes were less abundant but degraded more rapidly. These observations are consistent with the reported roles of Lon1 as a chaperone aiding the proper folding of newly synthesized/imported proteins to stabilise them and as a protease to degrade mitochondrial protein aggregates. HSP70, prohibitin and enzymes of photorespiration accumulated in lon1-1 and degraded slowly in all fractions, indicating an important role of Lon1 in their clearance from the proteome.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Nelson

Fenske

et al. 2017

The Plant Journal

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“…Several studies have assessed the utility of various metabolic labels ( 2 H, 13 C, and 15 N) for this purpose (Yang et al, 2010;Chen et al, 2011;Li et al, 2012b). More focused reports have refined our understanding of the dynamics in the assembly of mitochondrial electron transport complexes using these tools (Li et al, 2012a. Most recently, we performed a shotgun study of mitochondrial proteins from Arabidopsis (Arabidopsis thaliana) cell culture, measured K d values of 224 proteins, and assessed the turnover of several protein complexes by adaptation of this approach to assess larger scale liquid chromatography-tandem mass spectrometry (LC-MS/MS) data sets .…”

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Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

et al. 2014

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Protein turnover is a key component in cellular homeostasis; however, there is little quantitative information on degradation kinetics for individual plant proteins. We have used 15 N labeling of barley (Hordeum vulgare) plants and gas chromatography-mass spectrometry analysis of free amino acids and liquid chromatography-mass spectrometry analysis of proteins to track the enrichment of 15 N into the amino acid pools in barley leaves and then into tryptic peptides derived from newly synthesized proteins. Using information on the rate of growth of barley leaves combined with the rate of degradation of 14 N-labeled proteins, we calculate the turnover rates of 508 different proteins in barley and show that they vary by more than 100-fold. There was approximately a 9-h lag from label application until 15 N incorporation could be reliably quantified in extracted peptides. Using this information and assuming constant translation rates for proteins during the time course, we were able to quantify degradation rates for several proteins that exhibit half-lives on the order of hours. Our workflow, involving a stringent series of mass spectrometry filtering steps, demonstrates that 15 N labeling can be used for large-scale liquid chromatography-mass spectrometry studies of protein turnover in plants. We identify a series of abundant proteins in photosynthesis, photorespiration, and specific subunits of chlorophyll biosynthesis that turn over significantly more rapidly than the average protein involved in these processes. We also highlight a series of proteins that turn over as rapidly as the well-known D1 subunit of photosystem II. While these proteins need further verification for rapid degradation in vivo, they cluster in chlorophyll and thiamine biosynthesis.

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“…1). The stability of the ribosomal large subunit is reminiscent of the relatively more stable membrane arm of inner membrane protein complexes in plants, such as Complex I and V. 9,10 In contrast to these mitochondria ribosomal structural proteins, the three elongation factors studied had relatively fast degradation rates.…”

Section: Lon1 Stabilises the Mitochondrial Ribosome And Degrades Elonmentioning

confidence: 99%

Mitochondrial Lon1 has a role in homeostasis of the mitochondrial ribosome and pentatricopeptide repeat proteins in plants

Millar

Huang

2017

Plant Signaling & Behavior

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Lon is a highly conserved protein family in eukaryotes and eubacteria and its members all contain both a chaperone and a proteolytic domain that are important for Lon function. Loss of mitochondrial Lon1 leads to deleterious phenotypes in yeast and plants, and causes developmental disorders and aging-related diseases in humans. In Arabidopsis, we have recently reported the multiple roles of Lon1 in mitochondrial protein homeostasis through an evaluation of changes in protein degradation rates in the absence of Lon1.1 In this addendum, we extend our discussion to the roles of Lon1 in mitochondrial post-transcriptional regulation by considering the effects of its loss on ribosome proteins required for protein synthesis and mitochondrial PPR proteins required for RNA regulation. Mitochondria phosphorylate ADP to form ATP via respiratory oxidation of organic acids, the transfer of electrons to O 2 via the electron transport chain (ETC) and the cocommitted pumping of protons across the mitochondrial inner membrane. To maintain this energy production, protein quality control of a myriad of processes must be maintained inside mitochondria and this is predominately performed by the action of mitochondrial proteases including Lon1.2 Lon1 is a member of the highly conserved Lon protein family whose members all contain a chaperone and a proteolytic domain.3 Loss of mitochondrial Lon1 leads to reduction in growth rate in yeast and plants and causes developmental disorders and agingrelated diseases in humans. [4][5][6] In plants, Lon1 disruption causes changes in mitochondrial respiratory properties, lower protein abundances of oxidative phosphorylation protein complexes (such as Complex I and V), and increases in the abundance of prohibitins and heat shock proteins.5 Our recent analysis of a Lon1 loss-of-function line using a progressive 15 N labelling technique to measure protein turnover rates, suggested that Lon1 plays a chaperone role in aiding the proper folding of newly synthesized/imported proteins, particularly in the case of TCA enzymes, and complex I and V subunits.1 In this addendum, we will extend our discussion of the role of Lon1 by considering its effects on the homeostasis of mitochondrial ribosome proteins required for protein synthesis and on PPR proteins involved in RNA regulation. Lon1 stabilises the mitochondrial ribosome and degrades elongation factorsThe plant mitochondrion has its own ribosome to translate RNAs that are transcribed from the genes located in its genome. Like its yeast and mammal counterparts, the plant mitochondrial ribosome consists of »80 proteins that are resident in its large subunit, small subunit or act at the interface and are referred to as elongation factors. 7 In wild type Arabidopsis thaliana (cv Col-0) we quantified the degradation rates of 46 mitochondrial ribosomal proteins; 26 were subunits of the large subunit, 17 were subunits of the small subunit and 3 were elongation factors.1 Proteins in the large subunit were more stable than proteins in the small subunit.

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Accumulation of Newly Synthesized F1 in Vivo in Arabidopsis Mitochondria Provides Evidence for Modular Assembly of the Plant F1Fo ATP Synthase

Cited by 24 publications

References 41 publications

Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Changes in specific protein degradation rates in Arabidopsis thaliana reveal multiple roles of Lon1 in mitochondrial protein homeostasis

Proteins with High Turnover Rate in Barley Leaves Estimated by Proteome Analysis Combined with in Planta Isotope Labeling

Mitochondrial Lon1 has a role in homeostasis of the mitochondrial ribosome and pentatricopeptide repeat proteins in plants

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