Clark J. Nelson

Tandem Phosphorylation of Ser-911 and Thr-912 at the C Terminus of Yeast Plasma Membrane H+-ATPase Leads to Glucose-dependent Activation

In recent years there has been growing interest in the post-translational regulation of P-type ATPases by protein kinase-mediated phosphorylation. Pma1 H+-ATPase, which is responsible for H+-dependent nutrient uptake in yeast (Saccharomyces cerevisiae), is one such example, displaying a rapid 5–10-fold increase in activity when carbon-starved cells are exposed to glucose. Activation has been linked to Ser/Thr phosphorylation in the C-terminal tail of the ATPase, but the specific phosphorylation sites have not previously been mapped. The present study has used nanoflow high pressure liquid chromatography coupled with electrospray electron transfer dissociation tandem mass spectrometry to identify Ser-911 and Thr-912 as two major phosphorylation sites that are clearly related to glucose activation. In carbon-starved cells with low Pma1 activity, peptide 896–918, which was derived from the C terminus upon Lys-C proteolysis, was found to be singly phosphorylated at Thr-912, whereas in glucose-metabolizing cells with high ATPase activity, the same peptide was doubly phosphorylated at Ser-911 and Thr-912. Reciprocal 14N/15N metabolic labeling of cells was used to measure the relative phosphorylation levels at the two sites. The addition of glucose to carbon-starved cells led to a 3-fold reduction in the singly phosphorylated form and an 11-fold increase in the doubly phosphorylated form. These results point to a mechanism in which the stepwise phosphorylation of two tandemly positioned residues near the C terminus mediates glucose-dependent activation of the H+-ATPase.

A Quantitative Analysis of Arabidopsis Plasma Membrane Using Trypsin-catalyzed 18O Labeling

Typical mass spectrometry-based protein lists from purified fractions are confounded by the absence of tools for evaluating contaminants. In this report, we compare the results of a standard survey experiment using an ion trap mass spectrometer with those obtained using dual isotope labeling and a Q-TOF mass spectrometer to quantify the degree of enrichment of proteins in purified subcellular fractions of Arabidopsis plasma membrane. Incorporation of a stable isotope, either H218O or H216O, during trypsinization allowed relative quantification of the degree of enrichment of proteins within membranes after phase partitioning with polyethylene glycol/dextran mixtures. The ratios allowed the quantification of 174 membrane-associated proteins with 70 showing plasma membrane enrichment equal to or greater than ATP-dependent proton pumps, canonical plasma membrane proteins. Enriched proteins included several hallmark plasma membrane proteins, such as H+-ATPases, aquaporins, receptor-like kinases, and various transporters, as well as a number of proteins with unknown functions. Most importantly, a comparison of the datasets from a sequencing “survey” analysis using the ion trap mass spectrometer with that from the quantitative dual isotope labeling ratio method indicates that as many as one-fourth of the putative survey identifications are biological contaminants rather than bona fide plasma membrane proteins.

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

Proteins turn over at different rates in plant tissues, and these have been quantified using stable isotope labeling of nitrogen and peptide mass spectrometry of leaf tissue from hydroponically grown barley. 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 15N 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 15N 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 14N-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 15N 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 15N 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.

Determining Degradation and Synthesis Rates of Arabidopsis Proteins Using the Kinetics of Progressive 15N Labeling of Two-dimensional Gel-separated Protein Spots

The growth and development of plant tissues is associated with an ordered succession of cellular processes that are reflected in the appearance and disappearance of proteins. The control of the kinetics of protein turnover is central to how plants can rapidly and specifically alter protein abundance and thus molecular function in response to environmental or developmental cues. However, the processes of turnover are largely hidden during periods of apparent steady-state protein abundance, and even when proteins accumulate it is unclear whether enhanced synthesis or decreased degradation is responsible. We have used a 15N labeling strategy with inorganic nitrogen sources coupled to a two-dimensional fluorescence difference gel electrophoresis and mass spectrometry analysis of two-dimensional IEF/SDS-PAGE gel spots to define the rate of protein synthesis (KS) and degradation (KD) of Arabidopsis cell culture proteins. Through analysis of MALDI-TOF/TOF mass spectra from 120 protein spots, we were able to quantify KS and KD for 84 proteins across six functional groups and observe over 65-fold variation in protein degradation rates. KS and KD correlate with functional roles of the proteins in the cell and the time in the cell culture cycle. This approach is based on progressive 15N labeling that is innocuous for the plant cells and, because it can be used to target analysis of proteins through the use of specific gel spots, it has broad applicability.

Protein degradation rate in Arabidopsis thaliana leaf growth and development

The degradation rate of 1228 Arabidopsis proteins was measured, their variation assessed, and the data used to calculate the protein turnover energy costs in different leaves of the rosette. 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.

Analysis of the hibernation cycle using LC-MS-based metabolomics in ground squirrel liver

A hallmark of hibernation in mammals is metabolic flexibility, which is typified by reversible bouts of metabolic depression (torpor) and the seasonal shift from predominantly carbohydrate to lipid metabolism from summer to winter. To provide new insight into the control and consequences of hibernation, we used LC/MS-based metabolomics to measure differences in small molecules in ground squirrel liver in five activity states: summer, entering torpor, late torpor, arousing from torpor, and interbout arousal. There were significant alterations both seasonally and within torpor-arousal cycles in enzyme cofactor metabolism, amino acid catabolism, and purine and pyrimidine metabolism, with observed metabolites reduced during torpor and increased upon arousal. Multiple lipids also changed, including 1-oleoyllysophosphatidylcholine, cholesterol sulfate, and sphingosine, which tended to be lowest during torpor, and hexadecanedioic acid, which accumulated during a torpor bout. The results reveal the dramatic alterations that occur in several classes of metabolites, highlighting the value of metabolomic analyses in deciphering the hibernation phenotype.

Degradation Rate of Mitochondrial Proteins in Arabidopsis thaliana Cells

The turnover of the proteomes of organelles in plant cells are known to be governed by both whole cell and organelle-specific processes. However, the rate and specificity of this protein turnover has not been explored in depth to understand how it affects different organellar processes. Here we have used progressive ¹⁵N labeling of Arabidopsis cells, and focused on the turnover rate of proteins in mitochondria. We provide estimates of degradation rate (K(d)) for 224 mitochondrial proteins, showing a range of over 50-fold in K(d). Protein complexes, most notably the respiratory chain complexes, had K(d) values that were generally coordinated and we have interpreted these measurements to outline how protein K(d) differs within protein complexes and between functional categories. The fastest turnover rates were reported for DNA/RNA metabolism enzymes, chaperones, and proteases.

Subcomplexes of Ancestral Respiratory Complex I Subunits Rapidly Turn Over in Vivo as Productive Assembly Intermediates in Arabidopsis

Background: Plant complex I contains γ-CA subunits whose role is unclear. Results: 15N labeling and import show CI assembly via a rapidly turning over γ-CA subcomplex. Conclusion: γ-CAs form an ancient pathway for CI assembly. Significance: The assembly pathway of complex I in plants is different from that in animals and more closely represents the ancestral enzyme. Subcomplexes of mitochondrial respiratory complex I (CI; EC 1.6.5.3) are shown to turn over in vivo, and we propose a role in an ancestral assembly pathway. By progressively labeling Arabidopsis cell cultures with 15N and isolating mitochondria, we have identified CI subcomplexes through differences in 15N incorporation into their protein subunits. The 200-kDa subcomplex, containing the ancestral γ-carbonic anhydrase (γ-CA), γ-carbonic anhydrase-like, and 20.9-kDa subunits, had a significantly higher turnover rate than intact CI or CI+CIII2. In vitro import of precursors for these CI subunits demonstrated rapid generation of subcomplexes and revealed that their specific abundance varied when different ancestral subunits were imported. Time course studies of precursor import showed the further assembly of these subcomplexes into CI and CI+CIII2, indicating that the subcomplexes are productive intermediates of assembly. The strong transient incorporation of new subunits into the 200-kDa subcomplex in a γ-CA mutant is consistent with this subcomplex being a key initiator of CI assembly in plants. This evidence alongside the pattern of coincident occurrence of genes encoding these particular proteins broadly in eukaryotes, except for opisthokonts, provides a framework for the evolutionary conservation of these accessory subunits and evidence of their function in ancestral CI assembly.

Quantitative analysis of protein turnover in plants

Proteins are constantly being synthesised and degraded as plant cells age and as plants grow, develop and adapt the proteome. Given that plants develop through a series of events from germination to fruiting and even undertake whole organ senescence, an understanding of protein turnover as a fundamental part of this process in plants is essential. Both synthesis and degradation processes are spatially separated in a cell across its compartmented structure. The majority of protein synthesis occurs in the cytosol, while synthesis of specific components occurs inside plastids and mitochondria. Degradation of proteins occurs in both the cytosol, through the action of the plant proteasome, and in organelles and lytic structures through different protease classes. Tracking the specific synthesis and degradation rate of individual proteins can be undertaken using stable isotope feeding and the ability of peptide MS to track labelled peptide fractions over time. Mathematical modelling can be used to follow the isotope signature of newly synthesised protein as it accumulates and natural abundance proteins as they are lost through degradation. Different technical and biological constraints govern the potential for the use of 13C, 15N, 2H and 18O for these experiments in complete labelling and partial labelling strategies. Future development of quantitative protein turnover analysis will involve analysis of protein populations in complexes and subcellular compartments, assessing the effect of PTMs and integrating turnover studies into wider system biology study of plants.

Global analysis of circulating metabolites in hibernating ground squirrels

Hibernation in mammals involves major alterations in nutrition and metabolism that would be expected to affect levels of circulating molecules. To gain insight into these changes we conducted a non-targeted LC-MS based metabolomic analysis of plasma using hibernating ground squirrels in late torpor (LT, T(b)~5 °C) or during an interbout arousal period (IBA, T(b)~5 °C) and non-hibernating squirrels in spring (T(b)~37 °C). Several metabolites varied and allowed differentiation between hibernators and spring squirrels, and between torpid and euthermic squirrels. Methionine and the short-chain carnitine esters of propionate and butyryate/isobutyrate were reduced in LT compared with the euthermic groups. Pantothenic acid and several lysophosphatidylcholines were elevated in LT relative to the euthermic groups, whereas lysophosphatidylethanolamines were elevated during IBA compared to LT and spring animals. Two regulatory lipids varied among the groups: sphingosine 1-phosphate was lower in LT vs. euthermic groups, whereas cholesterol sulfate was elevated in IBA compared to spring squirrels. Levels of long-chain fatty acids (LCFA) and total NEFA tended to be elevated in hibernators relative to spring squirrels. Three long-chain acylcarnitines were reduced in LT relative to IBA; free carnitine was also lower in LT vs. IBA. Our results identified several biochemical changes not previously observed in the seasonal hibernation cycle, including some that may provide insight into the metabolic limitations of mammalian torpor.