Asparagine metabolism and nitrogen distribution during protein degradation in sugar-starved maize root tips

Excised maize (Zea mays L.) root tips were used to monitor the effects of prolonged glucose starvation on nitrogen metabolism. Following root-tip excision, sugar content was rapidly exhausted, and protein content declined to 40 and 8% of its initial value after 96 and 192 h, respectively. During starvation the contents of free amino acids changed. Amino acids that belonged to the same synthetic family showed a similar pattern of changes, indicating that their content, during starvation, is controlled mainly at the level of their common biosynthetic steps. Asparagine, which is a good marker of protein and amino-acid degradation under stress conditions, accumulated considerably until 45 h of starvation and accounted for 50% of the nitrogen released by protein degradation at that time. After 45 h of starvation, nitrogen ceased to be stored in asparagine and was excreted from the cell, first as ammonia until 90–100 h and then, when starvation had become irreversible, as amino acids and aminated compounds. The study of asparagine metabolism and nitrogen-assimilation pathways throughout starvation showed that: (i) asparagine synthesis occurred via asparagine synthetase (EC 6.3.1.1) rather than asparagine aminotransferase (EC 2.6.1.14) or the -cyanoalanine pathway, and asparagine degradation occurred via asparaginase (EC 3.5.1.1); and (ii) the enzymic activities related to nitrogen reduction and assimilation and amino-acid synthesis decreased continuously, whereas glutamate dehydrogenase (EC 1.4.1.2–4) activities increased during the reversible period of starvation. Considered together, metabolite analysis and enzymic-activity measurements showed that starvation may be divided into three phases: (i) the acclimation phase (0 to 30–35 h) in which the root tips adapt to transient sugar deprivation and partly store the nitrogen released by protein degradation, (ii) the survival phase (30–35 to 90–100 h) in which the root tips expel the nitrogen released by protein degradation and starvation may be reversed by sugar addition and (iii) the cell-disorganization phase (beyond 100 h) in which all metabolites and enzymic activities decrease and the root tips die.Abbreviations AlaAT alanine aminotransferase - AspAT aspartate aminotransferase - AS asparagine synthetase - Asnase asparaginase - AsnAT asparagine aminotransferase - -CS -cyanoalanine synthase - GDH glutamate dehydrogenase - Glnase glutaminase - GOGAT glutamate synthase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Characterization of the hexose transport system in maize root tips

Sugar-depleted excised maize (Zea mays L.) root tips were used to study the kinetics and the specificity of hexose uptake. It was found that difficulties induced by bulk diffusion and penetration barriers did not exist with root tips. Several lines of evidence indicate the existence of a complex set of uptake systems for hexoses showing an overall biphasic dependence on external sugar concentrations. The results suggest that the high and the low affinity components might be located on the same carrier. One uptake system was specific for fructose, but the high affinity component was repressed by high concentrations of external glucose. A second system was specific for glucose and its analogs (2-deoxy-d-glucose and 3-O-methyl-d-glucose), and a third one, more complex, had a high affinity for glucose and its analogs but could transport fructose when glucose was not present in the external solution. A simple method is proposed to determine the inhibitor constants in competition experiments.     

Regulated phosphorylation of 40S ribosomal protein S6 in root tips of maize

Ribosomal protein S6 (RPS6) is located in the mRNA binding site of the 40S subunit of cytosolic ribosomes. Two maize (Zea mays) rps6 genes were identified that encode polypeptides (30 kD, 11.4 pI) with strong primary amino acid sequence and predicted secondary structure similarity to RPS6 of other eukaryotes. Maize RPS6 was analyzed by the use of two-dimensional gel electrophoresis systems, in vivo labeling with [(32)P]P(i) and immunological detection. Nine RPS6 isoforms were resolved in a two-dimensional basic-urea/sodium dodecyl sulfate-polyacrylamide gel electrophoresis system. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry performed on trypsin-digested isoforms identified four serine (Ser) and one threonine (Thr) residue in the carboxy-terminal region as phosphorylation sites (RRS(238)KLS(241)AAAKAS(247)AAT(250)S(251)A-COOH). Heterogeneity in RPS6 phosphorylation was a consequence of the presence of zero to five phosphorylated residues. Phosphorylated isoforms fell into two groups characterized by (a) sequential phosphorylation of Ser-238 and Ser-241 and (b) the absence of phospho-Ser-238 and presence of phospho-Ser-241. The accumulation of hyper-phosphorylated isoforms with phospho-Ser-238 was reduced in response to oxygen deprivation and heat shock, whereas accumulation of these isoforms was elevated by cold stress. Salt and osmotic stress had no reproducible effect on RPS6 phosphorylation. The reduction in hyper-phosphorylated isoforms under oxygen deprivation was blocked by okadaic acid, a Ser/Thr phosphatase inhibitor. By contrast, the recovery of hyper-phosphorylated isoforms upon re-oxygenation was blocked by LY-294002, an inhibitor of phosphatidylinositol 3-kinases. Thus, differential activity of phosphatase(s) and kinase(s) determine complex heterogeneity in RPS6 phosphorylation.     

Regulation of protein tyrosine kinase signaling by substrate degradation during brain development

Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.     

Regulation of maize catalase by changing rates of synthesis and degradation.

Rates of catalase synthesis and degradation were measured in the scutellum of the germinating maize seedling by the technique of Price, Sterling, Tarantola, Hartley & Rechcigl [J. Biol. Chem. (1963) 237, 3468-3475] by using the porphyrinogenic drug 2-allyl-2-isopropylacetamide to inhibit catalase synthesis. Results indicate that developmental changes in catalase activity are determined by changes in the rate of enzyme degradation as well as by changes in the rates of its synthesis.     

A metabolic study of the regulation of proteolysis by sugars in maize root tips: effects of glycerol and dihydroxyacetone

Sugars, the main growth substrates of plants, act as physiological signals in the complex regulatory network of sugar metabolism. To investigate the function of different glycolytic steps in sugar sensing and signaling we compared the effects of carbon starvation with those of glucose, glycerol and dihydroxyacetone on carbon metabolism, proteolysis, and protease expression in excised maize (Zea mays L.) root tips. Respiration, soluble proteins, protein turnover and proteolytic activities were monitored as a function of time, along with in vitro and in vivo analysis of a variety of metabolites (sugars, amino and organic acids, phosphoesters, adenine nucleotides...) using ¹³C, ³¹P and ¹H NMR spectroscopy. Our results indicate that, in maize root tips, endopeptidase activities and protease expression are induced in response to a decrease in carbon supply to the upper part of the glycolytic pathway, i.e. at the hexokinase step. Proteolysis would be controlled downstream glycolysis, probably at the level of the respiratory substrate supply to mitochondria. Electronic Supplementary Material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Substrate cycles in the central metabolism of maize root tips under hypoxia

Substrate cycles, also called "futile" cycles, are ubiquitous and lead to a net consumption of ATP which, in the normoxic maize root, have been estimated at about 50% of the total ATP produced [Alonso, A.P., Vigeolas, H., Raymond, P., Rolin, D., Dieuaide-Noubhani, M., 2005. A new substrate cycle in plants. Evidence for a high glucose-phosphate-to-glucose turnover from in vivo steady-state and pulse-labeling experiments with [(13)C] glucose and [(14)C] glucose. Plant Physiol. 138, 2220-2232]. To evaluate their role we studied the substrate cycles of maize root tips under an oxygen limitation of respiration (3% O(2)). Short-time labeling experiments with [U-(14)C]-Glc were performed to quantify the fluxes through sucrose and starch cycles of synthesis and degradation. Steady-state labeling with [1-(13)C]-Glc followed by (1)H NMR and (13)C NMR analysis of sugars and free alanine was used to quantify fluxes in the central metabolic pathways, including the Glc-P/Glc cycle and the fructose-P/triose-P cycle of glycolysis. Comparison with results previously obtained in normoxia [Alonso et al., as mentioned above] showed that 3% O(2) induced fermentation and reduced respiration, which led to a lesser amount of ATP produced. The rates of Glc consumption, glycolytic flux and all substrate cycles were lower, but the proportion of ATP consumed in the substrate cycles remained unchanged. These findings suggest that substrate cycles are not a luxury but an integral part of the organization of the plant central metabolism.     

Hypoxia enhances phosphorylation of eukaryotic initiation factor 4A in maize root tips.

We have identified two isoforms of initiation factor 4A (eIF-4A) in maize root tips, with distinct isoelectric points and similar molecular mass (approximately 50 kDa). Both isoforms of maize eIF-4A cross-react with antibodies raised against wheat germ eIF-4A, and one of the maize proteins (higher pI isoform) comigrates with purified wheat germ eIF-4A on two-dimensional gels. The two maize eIF-4As were indistinguishable by comparative peptide fingerprint analysis, which also showed a very strong similarity between eIF-4A in maize roots and wheat germ. Maize eIF-4As copurify with eIF-4F and eIF-(iso)4F on a 7-methyl-GTP-Sepharose affinity column, indicating that they are part of the 5'-cap-binding complex. Two-dimensional gel electrophoresis and immunoblotting of proteins from 32P-labeled maize root tips revealed that the lower pI isoform of eIF-4A is phosphorylated. Two-dimensional phosphopeptide maps of trypsin-digested eIF-4A contained one principal phosphorylated fragment; phosphoamino acid analysis indicated phosphorylation of threonine. In oxygenated maize root tips, the ratio of phosphorylated to nonphosphorylated eIF-4A is approximately 0.2. This ratio increases to approximately 1 within 20 min following the onset of hypoxia, due to interconversion between the two maize eIF-4A isoforms. The hypoxia-induced phosphorylation of eIF-4A is discussed with respect to metabolic responses, and the translational control of gene expression, in hypoxic plant tissues.     

Regulation of C1-Ten protein tyrosine phosphatase by p62/SQSTM1-mediated sequestration and degradation

C1-Ten is a member of the tensin family of focal adhesion molecules but recent studies suggest it plays a more active role in many biological processes because of its potential association with diabetes and cancers. However, relatively little is known about the regulation of C1-Ten, such as changes in its protein level or cellular localization. The cellular localization of C1-Ten is unique because it is expressed in cytoplasmic puncta but nothing is known about these puncta. Here, we show that p62 sequestrates C1-Ten into puncta, making C1-Ten diffuse into the cytoplasm upon p62 depletion. More importantly, p62-mediated C1-Ten sequestration promoted C1-Ten ubiquitination and proteasomal degradation. p62-mediated protein reduction was specific to C1-Ten, and not other tensins such as tensin1 and tensin3. Thus, our results link cellular localization of C1-Ten to an off-switch site for C1-Ten. Additionally, p62 expression increased but C1-Ten protein decreased during muscle differentiation, supporting a role for p62 as a physiological regulator of C1-Ten.     

Regulation of stress-activated protein kinase signaling pathways by protein phosphatases.

Stress-activated protein kinase (SAPK) signaling plays essential roles in eliciting adequate cellular responses to stresses and proinflammatory cytokines. SAPK pathways are composed of three successive protein kinase reactions. The phosphorylation of SAPK signaling components on Ser/Thr or Thr/Tyr residues suggests the involvement of various protein phosphatases in the negative regulation of these systems. Accumulating evidence indicates that three families of protein phosphatases, namely the Ser/Thr phosphatases, the Tyr phosphatases and the dual specificity Ser/Thr/Tyr phosphatases regulate these pathways, each mediating a distinct function. Differences in substrate specificities and regulatory mechanisms for these phosphatases form the molecular basis for the complex regulation of SAPK signaling. Here we describe the properties of the protein phosphatases responsible for the regulation of SAPK signaling pathways.     

Intracellular localization of the multiple forms of ATP-ase activity in maize root tips

Summary The multiple forms of ATP-ase activity in maize root tips have been examined by separation and staining using polyacrylamide gel electrophoresis. The band pattern obtained using a whole homogenate supernatant fraction was compared with the pattern obtained from various cell organelle fractions. The kinetic properties of the ATP-ase activity in various fractions were also examined. The results support the view that ATP-ase activity exists in a number of molecular forms in the same tissue and that these forms may show specific intracellular localizations.     

Prohibitins regulate membrane protein degradation by the m-AAA protease in mitochondria

Prohibitins comprise a protein family in eukaryotic cells with potential roles in senescence and tumor suppression. Phb1p and Phb2p, members of the prohibitin family in Saccharomyces cerevisiae, have been implicated in the regulation of the replicative life span of the cells and in the maintenance of mitochondrial morphology. The functional activities of these proteins, however, have not been elucidated. We demonstrate here that prohibitins regulate the turnover of membrane proteins by the m-AAA protease, a conserved ATP-dependent protease in the inner membrane of mitochondria. The m-AAA protease is composed of the homologous subunits Yta10p (Afg3p) and Yta12p (Rca1p). Deletion of PHB1 or PHB2 impairs growth of Deltayta10 or Deltayta12 cells but does not affect cell growth in the presence of the m-AAA protease. A prohibitin complex with a native molecular mass of approximately 2 MDa containing Phb1p and Phb2p forms a supercomplex with the m-AAA protease. Proteolysis of nonassembled inner membrane proteins by the m-AAA protease is accelerated in mitochondria lacking Phb1p or Phb2p, indicating a negative regulatory effect of prohibitins on m-AAA protease activity. These results functionally link members of two conserved protein families in eukaryotes to the degradation of membrane proteins in mitochondria.     

Alzheimer's amyloid precursor protein produced by recombinant baculovirus expression. Proteolytic processing and protease inhibitory properties

The baculovirus expression system was used to generate recombinant Alzheimer's amyloid precursor (AAP) proteins. Recombinant baculoviruses were constructed, designed to express full-length 695-, 751-, and 770-amino acid forms. Recombinant baculoviruses designed for constitutive secretion were engineered by placing a termination codon between the beta-protein domain and cytoplasmic anchor of the full-length forms. Insect cells infected with each of these baculoviruses produced both secreted and cell-associated AAPs. Full-length constructs produced secreted derivatives which were COOH-terminally cleaved within the beta-protein domain at Gln15 or Lys16, essentially identical to previous reports utilizing mammalian cell systems. Rare secreted forms (less than 5%) appeared to extend to Lys28. Secretion constructs produced these same forms, but in different ratios. Most (approximately 60%) terminated at Gln15 or Lys16, while the remainder apparently extended to Lys28. AAPs containing the Kunitz-type serine protease inhibitory domain (AAP-751 and -770) were shown to be active inhibitors. No differences were observed in the inhibitors activities of these two forms. The similarities in AAP processing by insect and mammalian systems, together with the large amounts of recombinant protein produced by baculovirus expression, make this an attractive system for studies of AAP processing and biochemical properties.     

Overproduction and purification of SulA fusion protein in Escherichia coli and its degradation by Lon protease in vitro

To overproduce extremely unstable SulA protein, which is the cell-division inhibitor of Escherichia coli, we fused the sulA gene to the maltose-binding protein (MBP) fusion vectors with or without the signal sequence (plasmids pMAL-p-SulA and pMAL-c-SulA respectively). The amount of the full-length fusion protein expressed from the plasmid pMAL-p-SulA (pre-MBP-SulA) in E. coli was much larger than that expressed from the plasmid pMAL-c-SulA (MBP-SulA). A major amount of the pre-MBP-SulA fusion protein was expressed in a soluble form and affinity-purified by amylose resin. Since site-specific cleavage of the fusion protein with factor Xa resulted in the precipitation of SulA protein, the pre-MBP-SulA fusion protein was used to study the degradation of SulA protein by E. coli Lon protease in vitro. It was found that only the SulA portion of the fusion protein was degraded by Lon protease in an ATP-dependent manner. This result provides direct evidence that Lon protease plays an important role in the rapid degradation of SulA protein in cells.