The mitochondrial LYR protein SDHAF1 is required for succinate dehydrogenase activity in Arabidopsis.

Succinate dehydrogenase (SDH, complex II), which plays an essential role in mitochondrial respiration and tricarboxylic acid metabolism, requires the assembly of eight nuclear-encoded subunits and the insertion of various cofactors. Here, we report on the characterization of an Arabidopsis thaliana leucine-tyrosine-arginine (LYR) protein family member SDHAF1, (At2g39725) is a factor required for SDH activity. SDHAF1 is located in mitochondria and can fully complement the yeast SDHAF1 deletion strain. Knockdown of SDHAF1 using RNA interference resulted in a decrease in seedling hypocotyl elongation and reduced SDH activity. Proteomic analyses revealed a decreased abundance of various SDH subunits and assembly factors. Protein interaction assays revealed that SDHAF1 can interact exclusively with the Fe-S cluster-containing subunit SDH2 and HSCB, a cochaperone involved in Fe-S cluster complex recruitment. Therefore, we propose that in Arabidopsis, SDHAF1 plays a role in the biogenesis of SDH2 to form the functional complex II, which is essential for mitochondrial respiration and metabolism.

Knockdown of Succinate Dehydrogenase Assembly Factor 2 Induces Reactive Oxygen Species-Mediated Auxin Hypersensitivity Causing pH-Dependent Root Elongation.

Metabolism, auxin signaling and reactive oxygen species (ROS) all contribute to plant growth, and each is linked to plant mitochondria and the process of respiration. Knockdown of mitochondrial succinate dehydrogenase assembly factor 2 (SDHAF2) in Arabidopsis thaliana lowered succinate dehydrogenase activity and led to pH-inducible root inhibition when the growth medium pH was poised at different points between 7.0 and 5.0, but this phenomenon was not observed in wildtype (WT). Roots of sdhaf2 mutants showed high accumulation of succinate, depletion of citrate and malate and up-regulation of ROS-related and stress-inducible genes at pH 5.5. A change of oxidative status in sdhaf2 roots at low pH was also evidenced by low ROS staining in root tips and altered root sensitivity to H2O2. sdhaf2 had low auxin activity in root tips via DR5-GUS staining but displayed increased indole-3-acetic acid (IAA, auxin) abundance and IAA hypersensitivity, which is most likely caused by the change in ROS levels. On this basis, we conclude that knockdown of SDHAF2 induces pH-related root elongation and auxin hyperaccumulation and hypersensitivity, mediated by altered ROS homeostasis. This observation extends the existing evidence of associations between mitochondrial function and auxin by establishing a cascade of cellular events that link them through ROS formation, metabolism and root growth at different pH values.

Loss of conserved mitochondrial CLPP and its functions lead to different phenotypes in plants and other organisms.

Caseinolytic protease (CLPP) is an energy-dependent serine-type protease that plays a role in protein quality control. The CLPP gene is highly conserved across kingdoms and the protein is present in both bacteria and eukaryote organelles like mitochondria across a wide phylogenetic range. This pedigree has all the hallmarks of CLPP being an essential gene. However, in plants, disruption of mitochondrial CLPP has no impact on its growth, reminiscent of its nonessential role in some model fungi. Deletion of mitochondrial CLPP improves health and increased life span in the filamentous fungus, Podospora anserina, while loss of human mitochondrial CLPP leads to infertility and hearing loss. Recently it was revealed that both plant and human CLPP share a similar role in maintenance of the N-module of respiratory complex I. In addition, plant mitochondrial CLPP also coordinates the homeostasis of other mitochondrial protein complexes encoded by genes across mitochondrial and nuclear genomes. Understanding the contextual role of mitochondrial CLPP across kingdoms may help to understand these diverse sets of clpp phenotypes and the widespread conservation of CLPP genes.

Mitochondrial CLPP2 Assists Coordination and Homeostasis of Respiratory Complexes.

Protein 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 knockout lines of the single gene for the mitochondrial CLP protease subunit, CLPP2, in Arabidopsis (Arabidopsis thaliana). Mutants showed a higher abundance of transcripts from mitochondrial genes encoding oxidative phosphorylation protein complexes, whereas nuclear genes encoding other subunits of the same complexes showed no change in transcript abundance. By contrast, the protein abundance of specific nuclear-encoded subunits in oxidative phosphorylation complexes I and V increased in CLPP2 knockouts, without accumulation of mitochondrial-encoded counterparts in the same complex. Complexes with subunits mainly or entirely encoded in the nucleus were unaffected. Analysis of protein import and function of complex I revealed that while function was retained, protein homeostasis was disrupted, leading to accumulation of soluble subcomplexes of nuclear-encoded subunits. Therefore, CLPP2 contributes to the mitochondrial protein degradation network through supporting coordination and homeostasis of protein complexes encoded across mitochondrial and nuclear genomes.

Protein turnover rates in plant mitochondria.

Plant mitochondria operate as the powerhouses for cellular energy production by using the combined functions of both imported and organelle-synthesised proteins. Homeostasis of mitochondrial proteins is controlled by both synthesis and degradation processes which together define protein turnover in this organelle. Better understanding of plant mitochondrial protein turnover will provide information on protein quality control inside these organelles and its importance for proper function and regulation of mitochondrial machinery. This review discusses methods used for measurement of turnover rates of plant mitochondrial proteins and presents our current understanding of these rates for key mitochondrial proteins and protein complexes. The assembly and maintenance of mitochondrial OXPHOS complexes, in particular Complexes I and V, will be discussed in detail based on the evidence for differential protein turnover rates of the same protein subunits in different mitochondrial fractions. The impact of the loss of specific plant mitochondrial proteases on proteolysis events and rates of mitochondrial protein turnover will be highlighted. The challenges and future directions for investigation of plant mitochondrial protein turnover are also discussed.

The peptidases involved in plant mitochondrial protein import.

The endosymbiotic origin of the mitochondrion and the subsequent transfer of its genome to the host nucleus has resulted in intricate mechanisms of regulating mitochondrial biogenesis and protein content. The majority of mitochondrial proteins are nuclear encoded and synthesized in the cytosol, thus requiring specialized and dedicated machinery for the correct targeting import and sorting of its proteome. Most proteins targeted to the mitochondria utilize N-terminal targeting signals called presequences that are cleaved upon import. This cleavage is carried out by a variety of peptidases, generating free peptides that can be detrimental to organellar and cellular activity. Research over the last few decades has elucidated a range of mitochondrial peptidases that are involved in the initial removal of the targeting signal and its sequential degradation, allowing for the recovery of single amino acids. The significance of these processing pathways goes beyond presequence degradation after protein import, whereby the deletion of processing peptidases induces plant stress responses, compromises mitochondrial respiratory capability, and alters overall plant growth and development. Here, we review the multitude of plant mitochondrial peptidases that are known to be involved in protein import and processing of targeting signals to detail how their activities can affect organellar protein homeostasis and overall plant growth.

A Mitochondrial LYR Protein Is Required for Complex I Assembly.

Complex I biogenesis requires the expression of both nuclear and mitochondrial genes, the import of proteins, cofactor biosynthesis, and the assembly of at least 49 individual subunits. Assembly factors interact with subunits of Complex I but are not part of the final holocomplex. We show that in Arabidopsis (Arabidopsis thaliana), a mitochondrial matrix protein (EMB1793, At1g76060), which we term COMPLEX I ASSEMBLY FACTOR 1 (CIAF1), contains a LYR domain and is required for Complex I assembly. T-DNA insertion mutants of CIAF1 lack Complex I and the Supercomplex I+III. Biochemical characterization shows that the assembly of Complex I is stalled at 650 and 800 kD intermediates in mitochondria isolated from ciaf1 mutant lines.I. Yeast-two-hybrid interaction and complementation assays indicate that CIAF1 specifically interacts with the 23-kD TYKY-1 matrix domain subunit of Complex I and likely plays a role in Fe-S insertion into this subunit. These data show that CIAF1 plays an essential role in assembling the peripheral matrix arm Complex I subunits into the Complex I holoenzyme.

Mitochondrial Pyruvate Dehydrogenase Contributes to Auxin-Regulated Organ Development.

Pyruvate dehydrogenase is the first enzyme (E1) of the PDH complex (PDC). This multienzyme complex contains E1, E2, and E3 components and controls the entry of carbon into the mitochondrial tricarboxylic acid cycle to enable cellular energy production. The E1 component of the PDC is composed of an E1α catalytic subunit and an E1ß regulatory subunit. In Arabidopsis (Arabidopsis thaliana), there are two mitochondrial E1α homologs encoded by IAA-CONJUGATE-RESISTANT 4 (IAR4) and IAR4-LIKE (IAR4L), and one mitochondrial E1ß homolog. Although IAR4 was reported to be involved in auxin conjugate sensitivity and auxin homeostasis in root development, its precise role remains unknown. Here, we provide experimental evidence that mitochondrial PDC E1 contributes to polar auxin transport during organ development. We performed genetic screens for factors involved in cotyledon development and identified an uncharacterized mutant, macchi-bou 1 (mab1). MAB1 encodes a mitochondrial PDC E1ß subunit that can form both a homodimer and a heterodimer with IAR4. The mab1 mutation impaired MAB1 homodimerization, reduced the abundance of IAR4 and IAR4L, weakened PDC enzymatic activity, and diminished mitochondrial respiration. A metabolomics analysis showed significant changes in metabolites including amino acids in mab1 and, in particular, identified an accumulation of Ala. These results suggest that MAB1 is a component of the Arabidopsis mitochondrial PDC E1. Furthermore, in mab1 mutants and seedlings where the TCA cycle was pharmacologically blocked, we found reduced abundance of the PIN-FORMED (PIN) auxin efflux carriers, possibly due to impaired PIN recycling and enhanced PIN degradation in vacuoles. Therefore, we suggest that mab1 induces defective polar auxin transport via metabolic abnormalities.

Mitochondrial complex II of plants: subunit composition, assembly, and function in respiration and signaling.

Complex II [succinate dehydrogenase (succinate-ubiquinone oxidoreductase); EC 1.3.5.1; SDH] is the only enzyme shared by both the electron transport chain and the tricarboxylic acid (TCA) cycle in mitochondria. Complex II in plants is considered unusual because of its accessory subunits (SDH5-SDH8), in addition to the catalytic subunits of SDH found in all eukaryotes (SDH1-SDH4). Here, we review compositional and phylogenetic analysis and biochemical dissection studies to both clarify the presence and propose a role for these subunits. We also consider the wider functional and phylogenetic evidence for SDH assembly factors and the reports from plants on the control of SDH1 flavination and SDH1-SDH2 interaction. Plant complex II has been shown to influence stomatal opening, the plant defense response and reactive oxygen species-dependent stress responses. Signaling molecules such as salicyclic acid (SA) and nitric oxide (NO) are also reported to interact with the ubiquinone (UQ) binding site of SDH, influencing signaling transduction in plants. Future directions for SDH research in plants and the specific roles of its different subunits and assembly factors are suggested, including the potential for reverse electron transport to explain the succinate-dependent production of reactive oxygen species in plants and new avenues to explore the evolution of plant mitochondrial complex II and its utility.

An Assembly Factor Promotes Assembly of Flavinated SDH1 into the Succinate Dehydrogenase Complex.

Succinate dehydrogenase (Complex II; SDH) plays an important role in mitochondrial respiratory metabolism. The SDH complex consists of four core subunits and multiple cofactors, which must be assembled correctly to ensure enzyme function. To date, only an assembly factor (SDHAF2) required for FAD insertion into subunit SDH1 has been identified in plants. Here, we report the identification of Arabidopsis (Arabidopsis thaliana) At5g67490 as a second SDH assembly factor. Knockout of At5g67490 (sdhaf4) did not cause any phenotypic variation in seedlings but resulted in a decrease in both SDH activity and the succinate-dependent respiration rate as well as increased accumulation of succinate. Mass spectrometry analyses revealed stable levels of FAD-SDH1 in sdhaf4, together with increased levels of the FAD-SDH1 assembly factor, SDHAF2, and reduced levels of SDH2 compared with the wild type. Loss of SDHAF4 in sdhaf4 inhibited the formation of the SDH1/SDH2 intermediate, leading to the accumulation of soluble SDH1 in the mitochondrial matrix and reduced levels of SDH1 in the membrane. The increased levels of SDHAF2 suggest that the stabilization of soluble FAD-SDH1 depends on SDHAF2 availability. We conclude that SDHAF4 acts on FAD-SDH1 and promotes its assembly with SDH2, thereby stabilizing SDH2 and enabling its full assembly with SDH3/SDH4 to form the SDH complex.

Temperature-dependent metabolic adaptation of Triticum aestivum seedlings to anoxia.

Wheat (Triticum aestivum) is considered anoxia intolerant but it shows variance in anoxia responses between genotypes and environmental treatments. We firstly examined 4 day old seedlings of five wheat genotypes in response to anoxia at 15 °C and 28 °C by assessing growth rate, tissue damage and changes in metabolite abundances. Significant genotypic variations in anoxia tolerance were observed, especially at 28 °C. Wheat seedlings grown at 15 °C appeared to be more anoxia tolerant and showed less genotypic variation than those at 28 °C. To minimize seedling size variations and define the temperature effects, we grew two contrasting genotypes at 15 °C for 3.5 d and adapted to 4 different temperatures for 0.5 d before exposing them to anoxia at each adapted temperature. Genotypic variation in abundance of anoxia induced metabolites occurred at 24 °C and 28 °C but not at 15 °C and 20 °C. Tissue- and temperature-dependent metabolic adaptations to anoxia were revealed. In roots, the ability to maintain sugar/sugar-phosphate and TCA cycle metabolite levels and the accumulation of amino acids when temperature was below 24 °C correlated with anoxia tolerance. Temperatures between 20 °C-24 °C are critical for metabolic adaptation and suggest that further assessment of waterlogging/flooding tolerance of wheat seedlings should consider the temperature-dependence of tolerance in evaluations.

Salicylic Acid-Dependent Plant Stress Signaling via Mitochondrial Succinate Dehydrogenase.

Mitochondria are known for their role in ATP production and generation of reactive oxygen species, but little is known about the mechanism of their early involvement in plant stress signaling. The role of mitochondrial succinate dehydrogenase (SDH) in salicylic acid (SA) signaling was analyzed using two mutants: disrupted in stress response1 (dsr1), which is a point mutation in SDH1 identified in a loss of SA signaling screen, and a knockdown mutant (sdhaf2) for SDH assembly factor 2 that is required for FAD insertion into SDH1. Both mutants showed strongly decreased SA-inducible stress promoter responses and low SDH maximum capacity compared to wild type, while dsr1 also showed low succinate affinity, low catalytic efficiency, and increased resistance to SDH competitive inhibitors. The SA-induced promoter responses could be partially rescued in sdhaf2, but not in dsr1, by supplementing the plant growth media with succinate. Kinetic characterization showed that low concentrations of either SA or ubiquinone binding site inhibitors increased SDH activity and induced mitochondrial H2O2 production. Both dsr1 and sdhaf2 showed lower rates of SA-dependent H2O2 production in vitro in line with their low SA-dependent stress signaling responses in vivo. This provides quantitative and kinetic evidence that SA acts at or near the ubiquinone binding site of SDH to stimulate activity and contributes to plant stress signaling by increased rates of mitochondrial H2O2 production, leading to part of the SA-dependent transcriptional response in plant cells.

Protein Degradation Rate in Arabidopsis thaliana Leaf Growth and Development.

We applied 15N labeling approaches to leaves of the Arabidopsis thaliana rosette to characterize their protein degradation rate and understand its determinants. The progressive labeling of new peptides with 15N and measuring the decrease in the abundance of >60,000 existing peptides over time allowed us to define the degradation rate of 1228 proteins in vivo. We show that Arabidopsis protein half-lives vary from several hours to several months based on the exponential constant of the decay rate for each protein. This rate was calculated from the relative isotope abundance of each peptide and the fold change in protein abundance during growth. Protein complex membership and specific protein domains were found to be strong predictors of degradation rate, while N-end amino acid, hydrophobicity, or aggregation propensity of proteins were not. We discovered rapidly degrading subunits in a variety of protein complexes in plastids and identified the set of plant proteins whose degradation rate changed in different leaves of the rosette and correlated with leaf growth rate. From this information, we have calculated the protein turnover energy costs in different leaves and their key determinants within the proteome.

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

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.

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

Mitochondrial 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.

Retrograde signalling caused by heritable mitochondrial dysfunction is partially mediated by ANAC017 and improves plant performance.

Mitochondria are crucial for plant viability and are able to communicate information on their functional status to the cellular nucleus via retrograde signalling, thereby affecting gene expression. It is currently unclear if retrograde signalling in response to constitutive mitochondrial biogenesis defects is mediated by the same pathways as those triggered during acute mitochondrial dysfunction. Furthermore, it is unknown if retrograde signalling can effectively improve plant performance when mitochondrial function is constitutively impaired. Here we show that retrograde signalling in mutants defective in mitochondrial proteins RNA polymerase rpotmp or prohibitin atphb3 can be suppressed by knocking out the transcription factor ANAC017. Genome-wide RNA-seq expression analysis revealed that ANAC017 is almost solely responsible for the most dramatic transcriptional changes common to rpotmp and atphb3 mutants, regulating classical marker genes such as alternative oxidase 1a (AOX1a) and also previously-uncharacterised DUF295 genes that appear to be new retrograde markers. In contrast, ANAC017 does not regulate intra-mitochondrial gene expression or transcriptional changes unique to either rpotmp or atphb3 genotype, suggesting the existence of currently unknown signalling cascades. The data show that ANAC017 function extends beyond common retrograde transcriptional responses and affects downstream protein abundance and enzyme activity of alternative oxidase, as well as steady-state energy metabolism in atphb3 plants. Furthermore, detailed growth analysis revealed that ANAC017-dependent retrograde signalling provides benefits for growth and productivity in plants with mitochondrial defects. In conclusion, ANAC017 plays a key role in both biogenic and operational mitochondrial retrograde signalling, and improves plant performance when mitochondrial function is constitutively impaired.

The Roles of Mitochondrial Reactive Oxygen Species in Cellular Signaling and Stress Response in Plants.

Mitochondria produce ATP via respiratory oxidation of organic acids and transfer of electrons to O2 via the mitochondrial electron transport chain. This process produces reactive oxygen species (ROS) at various rates that can impact respiratory and cellular function, affecting a variety of signaling processes in the cell. Roles in redox signaling, retrograde signaling, plant hormone action, programmed cell death, and defense against pathogens have been attributed to ROS generated in plant mitochondria (mtROS). The shortcomings of the black box-idea of mtROS are discussed in the context of mechanistic considerations and the measurement of mtROS The overall aim of this update is to better define our current understanding of mtROS and appraise their potential influence on cellular function in plants. Furthermore, directions for future research are provided, along with suggestions to increase reliability of mtROS measurements.

Inactivation of Mitochondrial Complex I Induces the Expression of a Twin Cysteine Protein that Targets and Affects Cytosolic, Chloroplastidic and Mitochondrial Function.

At12Cys-1 (At5g64400) and At12Cys-2 (At5g09570) are two closely related isogenes that encode small, twin cysteine proteins, typically located in mitochondria. At12Cys-2 transcript is induced in a variety of mutants with disrupted mitochondrial proteins, but an increase in At12Cys protein is only detected in mutants with reduced mitochondrial complex I abundance. Induction of At12Cys protein in mutants that lack mitochondrial complex I is accompanied by At12Cys protein located in mitochondria, chloroplasts, and the cytosol. Biochemical analyses revealed that even single gene deletions, i.e., At12cys-1 or At12cys-2, have an effect on mitochondrial and chloroplast functions. However, only double mutants, i.e., At12cys-1:At12cys-2, affect the abundance of protein and mRNA transcripts encoding translation elongation factors as well as rRNA abundance. Blue native PAGE showed that At12Cys co-migrated with mitochondrial supercomplex I + III. Likewise, deletion of both At12cys-1 and At12cys-2 genes, but not single gene deletions, results in enhanced tolerance to drought and light stress and increased anti-oxidant capacity. The induction and multiple localization of At12Cys upon a reduction in complex I abundance provides a mechanism to specifically signal mitochondrial dysfunction to the cytosol and then beyond to other organelles in the cell.

Activity assay for plant mitochondrial enzymes.

Mitochondria are sites for respiration to produce chemical energy via oxidative phosphorylation. Their primary role has been viewed as the oxidation of organic acids via the tricarboxylic acid (TCA) cycle and the synthesis of ATP coupled to the transfer of electrons to O2. TCA cycle enzymes are essential for plant carbon metabolism and provide the reductant for the electron transport chain (ETC) enzymes that in turn drives ATP synthesis. The activity of individual enzymes will determine the flux of metabolism and thus the downstream consequences for respiration rate. Measurements of activities of mitochondrial enzymes, such as components of TCA cycle and the ETC, can provide insight into regulation of mitochondrial function. The activities of these enzymes vary between developmental stages, in different tissues, and in response to environmental conditions. In this chapter, methods for enzymatic assay of TCA cycle enzymes and a number of the ETC complex enzymes are described in detail.

INTERMEDIATE CLEAVAGE PEPTIDASE55 Modifies Enzyme Amino Termini and Alters Protein Stability in Arabidopsis Mitochondria.

Precursor proteins containing mitochondrial peptide signals are cleaved after import by a mitochondrial processing peptidase. In yeast (Saccharomyces cerevisiae) and human (Homo sapiens), intermediate cleavage peptidase55 (ICP55) plays a role in stabilizing mitochondrial proteins by the removal of single amino acids from mitochondrial processing peptidase-processed proteins. We have investigated the role of a metallopeptidase (At1g09300) from Arabidopsis (Arabidopsis thaliana) that has sequence similarity to yeast ICP55. We identified this protein in mitochondria by mass spectrometry and have studied its function in a transfer DNA insertion line (icp55). Monitoring of amino-terminal peptides showed that Arabidopsis ICP55 was responsible for the removal of single amino acids, and its action explained the -3 arginine processing motif of a number of mitochondrial proteins. ICP55 also removed single amino acids from mitochondrial proteins known to be cleaved at nonconserved arginine sites, a subset of mitochondrial proteins specific to plants. Faster mitochondrial protein degradation rates not only for ICP55 cleaved protein but also for some non-ICP55 cleaved proteins were observed in Arabidopsis mitochondrial samples isolated from icp55 than from the wild type, indicating that a complicated protease degradation network has been affected. The lower protein stability of isolated mitochondria and the lack of processing of target proteins in icp55 were complemented by transformation with the full-length ICP55. Analysis of in vitro degradation rates and protein turnover rates in vivo of specific proteins indicated that serine hydroxymethyltransferase was affected in icp55. The maturation of serine hydroxymethyltransferase by ICP55 is unusual, as it involves breaking an amino-terminal diserine that is not known as an ICP55 substrate in other organisms and that is typically considered a sequence that stabilizes rather than destabilizes a protein.