Evidence for a particulate location of ubiquitin conjugates and ubiquitin-conjugating enzymes in rabbit brain.

Conjugate ubiquitin was previously found in the nucleus, cytoplasm, and membranes of eukaryotic cells while the enzymes of the ubiquitin-conjugating system appear to be cytoplasmic. We have prepared the mitochondrial fraction from rabbit brain by discontinuous density gradient ultracentrifugation and by Western blotting, using a specific antibody against conjugate ubiquitin, showing that it contains ubiquitin conjugates in a very wide molecular weight range. Electron microscopy and measurement of specific enzyme markers show that this fraction not only contains mitochondria but also some endoplasmic reticulum vesicles. Immunostaining with anti-ubiquitin IgG followed by immunodecoration with colloidal gold particles provides evidence for the presence of conjugate ubiquitin both in mitochondria and in the endoplasmic reticulum. Furthermore, this "mitochondrial fraction" shows a pronounced ATP-dependent ability to conjugate 125I-ubiquitin into a number of endogenous proteins as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. Addition of E1, E2, and E3, the enzymes of the ubiquitin conjugating system purified from rabbit reticulocytes, does not further increase this ubiquitination nor incorporate 125I-ubiquitin into additional protein bands. The same mitochondrial fraction is not able to carry out any ATP-dependent degradation of 125I-albumin; however, it contains an isopeptidase activity able to release the covalently incorporated 125I-ubiquitin and is also able to conjugate 125I-ubiquitin to exogenous proteins as oxidized RNase. By affinity chromatography on ubiquitin-agarose of fraction II of a crude Triton X-100 extract of the mitochondrial fraction, several proteins corresponding in Mr to the E1 and E2s enzymes were obtained. These proteins were also able to form specific ubiquitin-thiol ester bounds on sodium dodecyl sulfate-polyacrylamide gels and to support 125I-ubiquitin conjugation to oxidized RNase. Detergent fractionation of the mitochondrial fraction provided evidence for a possible localization of the ubiquitin conjugating activity in the mitochondrial external membrane and endoplasmic reticulum. The presence of an active ubiquitin protein conjugating system in mitochondria and endoplasmic reticulum may be related to the turnover of organelle proteins as well as to specific cell functions such as import of proteins into mitochondria and ubiquitination of externally oriented membrane-bound proteins.     

High performance liquid chromatography resolution of ubiquitin pathway enzymes from wheat germ

The highly conserved protein ubiquitin is involved in several cellular processes in eukaryotes as a result of its covalent ligation to a variety of target proteins. Here, we describe the purification of several enzymatic activities involved in ubiquitin-protein conjugate formation and disassembly from wheat germ (Triticum vulgare) by a combination of ubiquitin affinity chromatography and anion-exchange high performance liquid chromatography. Using this procedure, ubiquitin activating enzyme (E1), several distinct ubiquitin carrier proteins (E2s) with molecular masses of 16, 20, 23, 23.5, and 25 kilodaltons, and a ubiquitin-protein hydrolase (isopeptidase) were isolated. Purified E1 formed a thiol ester linkage with ¹²⁵I-ubiquitin in an ATP-dependent manner and transferred bound ubiquitin to the various purified E2s. The ubiquitin protein hydrolase fraction was sensitive to hemin, and in an ATP-independent reaction, was capable of removing the ubiquitin moiety from both ubiquitin ¹²⁵I-lysozyme conjugates (ε-amino or isopeptide linkage) and the ubiquitin 52-amino acid extension protein fusion (α-amino or peptide linkage). Using this procedure, wheat germ represents an inexpensive source from which enzymes involved in the ubiquitin pathway may be isolated.     

Aging and cellular maturation cause changes in ubiquitin-eye lens protein conjugates

The eye lens is a useful tissue for studying phenomena related to aging since it can be separated into differentially aged or matured zones. This work establishes correlations between ubiquitin-lens protein conjugating capabilities and age, as well as the stage of maturation of bovine lens tissue. When exogenous 125I-ubiquitin was combined with supernatants of epithelial (least mature), cortex, and core (most mature) tissue, ATP-dependent conjugation of 125I-ubiquitin to lens proteins was most effective with the epithelial tissue preparation. Conjugate formation was greatest when lenses were obtained from young animals. Supernatants from cultured bovine lens epithelial (BLE) cells conjugated more 125I-ubiquitin to lens proteins than any tissue preparation. In all cases the predominant conjugates formed in these cell-free assays were of high molecular mass, although conjugates with masses in the 25-70 kDa range were also observed. Lens tissue and cultured BLE cell preparations were also probed with antibodies to ubiquitin to detect in vivo ubiquitin-lens protein conjugates. There was more free ubiquitin and ubiquitin conjugates in tissue from young as compared with older lenses. The greatest levels of conjugates were observed in cultured BLE cells. Specificity in the ubiquitination system is indicated since some of the conjugates formed in vivo appear identical to those formed in the cell-free assays and in reticulocytes using exogenous 125I-ubiquitin. Upon development and maturation of lens tissue (i.e., core as opposed to epithelium), there is accumulation of lower molecular mass conjugates.     

Changes in ubiquitin and ubiquitin-protein conjugates during seed formation and germination

Changes in both free ubiquitin and ubiquitin-protein conjugateswere followed in cotyledons of lupin (Lupinus albus L.) duringthe course of seed formation, from the flower to the dry seed,and during germination and seedling growth, from the dry seedto the senescing cotyledons. The observed levels of ubiquitinconjugates, detected by immunoblotting using antiubiquitin antibodiesand by autoradiography using ¹²⁵I-labelled ubiquitin, suggestan intense involvement of the ubiquitin-mediated proteolyticpathway during the highly regulated phases of seed formationand germination. High amounts of free ubiquitin are presentat all stages in all tissues examined. With the exception ofthe dry seed, the high molecular mass ubiquitin-protein conjugatesare also present at all stages. Higher amounts of these conjugateswere found during the initial stages of pod development andseed germination and during the most active phases of storageprotein deposition and degradation. Germination and seedlinggrowth in total darkness not only delays the degradation ofthe storage proteins, but also extends the period characterizedby the presence of a high amount of these conjugates. No suchconjugates were detected in the dry seeds, probably reflectingthe extremely low metabolic activity observed in these organs.A number of smaller molecular mass polypeptides were also detectedat different stages of seed development, germination and seedlinggrowth. Of particular interest is the abrupt accumulation ofan abundant 20 kDa polypeptide in the cotyledons during the4th day after imbibition, which is maintained in high amountsin these organs, rapidly declining after about 12–14 d.The pattern of accumulation of the 20 kDa polypeptide is controlledneither by light nor by the embryo axes, and large variationsin its concentration are observed during heat shock. Key words: Ubiquitin, ubiquitin-protein conjugates, seed storage proteins, protein synthesis, protein degradation     

The Use of Chemical and Infra-red Spectral Analyses to Verify the Identification of Two Species of Duckweed, Lemna minor L. and Lemna gibba L. Grown Under Defined Conditions

Flat forms of the fronds of Lemna gibba L., used widely in laboratoryexperiments, can be easily confused with Lemna minor Chemicalanalyses of the two species, grown under defined laboratoryconditions, showed clear distinctions between the two speciesin relation to total protein content and the content of individualmonosaccharides. Infra-red spectral analysis confirmed the chemicaldata for protein content and also revealed differences in thetypes of proteins present in the two species. Chemical and infra-redspectral analyses, particularly the amide I: carbohydrate absorbanceratio, provide a rapid and reliable means of verifying betweenthe two species, which are often difficult to identify withcertainty from morphological differences. Key words: Gibbosity, infra-red spectroscopy, Lemna gibba, Lemna minor, monosaccharides, proteins     

Influence of nutrients on the development of gibbosity in fronds of the duckweed Lemna gibba L.

The duckweeds Lemna gibba L. and Lemna minor L. only grew wellin undisturbed culture under axenic conditions in low lightintensity when provided with a suitable energy source such asglucose. In media containing N0₃-N gibbosity (a convex ventralsurface) was induced in the presence of the chelating agentethylene-diamine-di-o-hydroxyphenylacetic acid (EDDHA). In nutrientsolutions containing NO₃-N as the only N source, but withoutEDDHA, L. gibba occasionally exhibited gibbosity in culturesolutions of 40 cm³ volumes. More fronds were induced to exhibitgibbosity when the volume of the culture medium was increasedfrom 40 cm³ to 200 cm³. Gibbosity was never induced in L. minor,neither was it induced in L. gibba in media containing NH₄-N,even in the presence of NO₃-N. There was no direct correlationbetween the occurrence of gibbosity and frond growth rate, butgibbosity occurred only when there was good frond growth. In the absence of a sugar, frond growth was enhanced by bubblingair through the culture solution in the light. Increasing theCO₂ concentration in the air up to 1% enhanced growth and inducedgibbosity. Carbon dioxide did not induce gibbosity in mediacontaining NH₄-N. Key words: Ammonium-N, carbon dioxide, gibbosity, Lemna, nitrate-N     

Demonstration of ATP-Dependent, Ubiquitin-Conjugating Activities in Higher Plants

Ubiquitin is a highly conserved, 76-amino acid polypeptide with several important regulatory functions in both plants and animals that all arise from its covalent ligation to other cellular proteins. Here, we demonstrate that higher plants have the capacity to conjugate ubiquitin to other plant proteins in vitro. Using ¹²⁵I-labeled human ubiquitin as a substrate, conjugating activities were observed in crude etiolated tissue extracts from all species tested, including oats, rye, barley, corn, zucchini squash, pea, soybean, and sunflower. The reaction has a soluble distribution, is specific for ATP, and requires the protease inhibitor, leupeptin, to protect ubiquitin from inactivation during the assay. Conjugation is inhibited by N-ethylmaleimide and high concentrations of 2-mercaptoethanol suggesting that the mechanism of ubiquitin ligation in plants involves a similar thiolester intermediate to that found in the mammalian pathway. The conjugating activity in etiolated oat extracts is extremely labile with a half-life of about 20 minutes at 30°C. Detectable but low ATP-stimulated, conjugating activities were also observed in extracts from dry seeds and green leaves of oats. In addition to this conjugating activity, crude plant extracts have the capacity to degrade ubiquitin-protein conjugates formed in vitro. These results demonstrate that higher plants contain several of the enzymic activities necessary for ubiquitin's functions and provide a method for assaying ubiquitin conjugation in vitro.     

The Isolation and Examination of Protein Samples of Known Age for the Study of Selectivity of Protein Degradation in Lemna minor

The proposal that the selectivity of protein degradation isdetermined by particular physical properties of proteins hasbeen examined in Lemna minor. A method for isolating proteinof known age is described. Density-labelled proteins of knownage, isolated by the procedure, showed a weak correlation betweenSH content and degradation, and little or no correlation betweenmolecular weight and degradation. An alternative method of examining possible correlations betweenthe rate of degradation of proteins and their physical propertiesis also described. This method which is based on labelling proteinswith ³H₂O gave results suggesting that there was little or nocorrelation between rates of degradation and the molecular weightor charge of proteins. These weak or non-existent correlations are compared with previousreports and lead to the suggestion that the half-life of proteinsis determined by the sum effect of many physical propertiesrather than by a single ‘signal’ property. Key words: Lemna minor, Protein isolation, Protein degradation     

The eye lens has an active ubiquitin-protein conjugation system

Using exogenous 125I-ubiquitin, ubiquitin-lens protein conjugation was observed with supernatants of cultured rabbit lens epithelial cells and lens cortex tissue. Conjugation was ATP-dependent with the greatest variety and amount of conjugates larger than 150 kDa. In vivo production of ubiquitin-protein conjugates in cultured rabbit and beef lens epithelial cells and rabbit lens tissues of different developmental age was established using immunological detection. There were limited similarities between conjugates found in youngest as opposed to oldest tissue. Cultured rabbit cells contained 27 pmol/mg free ubiquitin and 18 pmol/mg conjugated ubiquitin. Levels of free ubiquitin in lens tissue epithelium, cortex, and core were 36, 5, and 5 pmol/mg, respectively. There were only 2 pmol/mg conjugated ubiquitin in each of these tissues. Hydrolysis of 125I-ubiquitin was catalyzed by supernatants of cultured lens cells, beef and human lens tissues, and reticulocytes. Degradation was greatest in epithelial tissues, and least in core. This corroborates studies which show that proteolytic capabilities are attenuated in older tissue. Decreased initiation of proteolysis by ubiquitination as well as diminished proteolysis in older lens tissue may be related to the accumulation of damaged proteins in aging lens tissue.     

The Potassium Relations of Lemna minor L.: II. THE MECHANISM OF POTASSIUM UPTAKE

The experiments described in this paper concern the nature ofthe K⁺-influx mechanism in Lemna minor L. It is establishedthat K⁺ influx is ‘site restricted’ and that theaffinity of the system involved is in close agreement with thatof the well-documented System I mechanisms that are found inmany plants (Epstein, 1972). Experiments with ATP and CCCP suggestthat K⁺ influx is an active process, that the influx machineryresides at the plasmalemma and that this machinery containsan ATPase activity. Membrane vesicles isolated from the plantsabsorb K⁺(Rb⁺) with an affinity comparable to that of the systeminvolved in physiological K⁺ influx.     

Proteolytic Processes Underlying Molt-Induced Claw Muscle Atrophy in Decapod Crustaceans

The claw muscles of large-clawed decapod crustaceans undergoa programmed atrophy in preparation for molting, or ecdysis.This is mediated by five cytosolic proteinases organized intotwo proteolytic pathways: calcium-dependent and ubiquitin/proteasome-dependent.The calcium-dependent system consists of four calcium-dependentcysteine proteinases (CDPs I, IIa, IIb, and III; native masses310, 125, 195, and 59 kDa, respectively) that completely degrademyofibrillar proteins and are activated in atrophic muscles.Immunological analysis shows that the active-site sequence inCDP IIa (60-kDa subunit mass) is similar to that in mammalianCDPs (calpains), and that CDP IIb is homologous to a calpain-likegene isolated from Drosophila cDNA libraries. Increased intracellullarCa²⁺ stimulates proteolysis in situ, indicating CDPs play animportant role in muscle protein catabolism. The ubiquitin/proteasome-dependentsystem involves the ATP-dependent conjugation of multi-ubiquitinchains to protein by ubiquitin-conjugating enzymes. This actsas a signal for substrate degradation by the 26S proteasome,a multi-subunit complex consisting of a 20S proteasome catalytic"core" and two PA700 (19S) regulatory complexes. PolyubiquitinmRNA, ubiquitin-protein conjugates, and 20S proteasome are elevatedabout 5-, 8-, and 2-fold, respectively, during atrophy. A heat-inducedform of the 20S proteasome hydrolyzes myosin, troponin, andtropomyosin to large fragments in vitro. Biochemical studiesidentified the branched-chain amino acid-preferring (BrAAP)activity, one of six distinct catalytic components in the complex,as the activity that carries out these initial cleavages. Theseresults indicate that the ubiquitin/proteasome pathway is involved,but its precise role remains to be resolved.     

MODULATION OF THE HEAT SHOCK UBIQUITIN POOL IN SKELETONEMA COSTATUM (BACILLARIOPHYCEAE)1

Immunoblotting experiments performed with an anti-ubiquitin antibody revealed that Skeletonema costatum (Grev.) Cleve cells contained free ubiquitin as well as ubiquitin conjugated to various endogenous proteins. A temperature shift from 18° to 30°C greatly increased the total amount of ubiquitin and particularly the ubiquitin fraction in high molecular mass conjugates. A solid-phase immunoassay indicated values of 0.031 ± 0.004 pmol·10^?6 cells for free ubiquitin and 0.046 ± 0.004 pmol·10^?6 cells for conjugated ubiquitin for cells grown at 18°C, and 0.056 ± 0.008pmol·10^?6cells and 0.21 ± 0.03 pmol·10^?6cells, respectively, after a temperature increase from 18° to 30°C. Cell-free extracts of S. costatum were equally able to form thiol ester linkages with ¹²⁵I-ubiquitin in an adenosine triphosphate–dependent manner at 18° C and at 30°C. Cell-free extracts were also able to conjugate ¹²⁵I-ubiquitin to endogenous proteins, but the ubiquitin conjugation rate at 30°C was lower than at 18°C. Incubation of S. costatum for 3 h at 30°C and then for 3 h at 18°C resulted in the formation of high amounts of ubiquitin conjugates, suggesting that partially inactive or denaturated proteins accumulate during heat stress. These denaturated proteins are then conjugated to ubiquitin very efficiently when the physiological temperature is restored. Thus, S. costatum cells contain ubiquitin and an active ubiquitin conjugation system responding to stress conditions (temperature stress). The intracellular concentration of ubiquitin conjugates is most likely limited by the availability of protein substrates to be conjugated rather than by ubiquitin-conjugating activity.     

Conjugation of ubiquitin to proteins from green plant tissues

Conjugation of the polypeptide ubiquitin to endogenous proteins was studied in oat (Avena sativa L.) plants, and particularly in green tissues. Conjugating activity in leaf extracts was different from that in root extracts, and in both was less than in etiolated tissue. The conjugates were identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and their formation was both time- and ATP-dependent and had a pH optimum of about 8.2. The assay had a high affinity for ATP with a probable K_m of less than 50 micromolar. The ubiquitin conjugating system was also shown to be present in isolated chloroplasts, and ubiquitin could be conjugated to endogenous proteins of lyzed chloroplasts in which the ATP concentrations were reduced by preincubation or desalting. SDS-PAGE analysis led to the suggestion that the large and small subunits of ribulose-1,5-bisphosphate carboxylase (RuBPCase) may be able to be ubiquitinated, and we have shown that ubiquitin can stimulate the in vitro breakdown of ¹²⁵I-labeled RuBPCase. These results invite the speculation that ubiquitin may be involved in the regulation of protein turnover in green plants.     

Flower-inducing Effect of Benzoic and Salicylic Acids in Various Strains of Lemna paucicostata and L. minor

The flower-inducing activities of benzoic and salicylic acidsadded to the medium differ with the species (Lemna paucicostataand L. minor), and even with the strains used. The type andpH of the medium used, full or 1/10 strength M medium at pH3.8, 4.4 or 5.1, or 1/2 or 1/20 strength NH₄⁺-free Hutner'smedium at pH 5.0, 6.0 or 7.0, also modify their activity. L.paucicostata, strain 151 is the most sensitive of the strainsused to both benzoic and salicylic acids followed by strain381. Such dramatic flowering responses were not obtained withthe other strains, but even strain 321, reportedly insensitiveto benzoic acid, could be induced to flower by adding benzoicacid to a modification of the medium. Benzoic acid is more effectivethan salicylic acid for all strains of L. paucicostata, butthe contrary is true for two L. minor strains tested. A higherpercentage of flowering is obtained in L. paucicostata in 1/2strength NH₄⁺-free Huter'sn medium than in M medium, exceptfor strain 151. When diluted, both media enhance flowering inall L. paucicostata strains. Generally, a lower concentrationof benzoic acid or salicylic acid is enough to induce floweringwhen the pH of the medium is lower. (Received March 30, 1981; Accepted May 16, 1981)     

Reconstituting Ubiquitination Reactions with Affinity-Purified Components andP-Ubiquitin

The discovery of protein ubiquitination in a broad range of organisms and experimental settings has raised the need for a straightforward way to characterize the mechanism of substrate targeting, using purified components. The mechanism of ubiquitin conjugation to proteins has been extensively studied and is mediated by a family of evolutionarily conserved proteins. Wehave used previously described expression systemsto purify the relevant targeting components of the ubiquitin system. These methods yielded substantial amounts of highly purified and catalytically active enzymes that permitted their use in reconstituting protein ubiquitination. We monitored ubiquitination reactions with³²P-ubiquitin rather than¹²⁵I-ubiquitin. This advance makes the procedure accessible to a broader range of experimentalists, since it eliminates the additional concerns involved in handling¹²⁵I-isotope. Furthermore, the strategies described here can be used to investigate the effects of specific mutations introduced into ubiquitin or the targeting components (E1, Ubc/E2, and E3) of this pathway.     

Heat Shock Induced Change in Protein Ubiquitination in Chlamydomonas

Ubiquitin was purified from pea (Pisum sativum L.) and its antibodywas produced. Western blot analysis showed that the antibodycross-reacted with ubiquitins from a green alga Chlamydomonasreinhardtii, a brown alga Laminaria angustata and a red algaPorphyridium cruentum but not with ubiquitin from a blue-greenalga Synechococcus sp. In Chlamydomonas, the antibody also reactedwith some ubiquitinated proteins including 28- and 31-kDa polypeptides.The isoelectric points of Chlamydomonas ubiquitin and the 28-and 31-kDa ubiquitinated proteins were 8.0, 8.9 and 10.3, respectively.The ubiquitinated proteins, including the 28- and 31-kDa polypeptideswere detected after in vitro ATP-dependent ubiquitination ofChlamydomonas cell extract with ^l25I-labeled bovine ubiquitin.Heat treatment of Chlamydomonas cells (>40°C) causeddrastic increase of ubiquitinated proteins with high mol wt(>60kDa), and coordinated redistribution or decrease of otherubiquitinated proteins and free ubiquitin. Quantitative analysisrevealed that the 28- and 31-kDa ubiquitinated proteins showeddifferent responses against heat stress, i.e. the former beingmore sensitive than the latter. (Received July 10, 1988; Accepted October 4, 1988)     

Dual pattern of DL-leucine absorption by duckweed root tips

The rates of cellular absorption of DL-leucine from solutionsof different concentrations indicate the presence of dual uptakemechanisms in root tips of Lemna minor, one in the low (0.01to 0.2 mM) and the other in the high (5 to 25 mM) concentrationrange. The absorption pattern for the lower concentrations conformsto Michaelis-Menten kinetics. ¹Supported in part by a Grant-in-Aid of Research from the Societyof Sigma Xi. (Received June 19, 1973; )     

Metabolism of Glycollate by Lemna minor L. Grown on Nitrate or Ammonium as Nitrogen Source

Marques, I. A., Oberholzer, M. J. and Erismann, K. H. 1985.Metabolism of glycollate by Lemna minor L. grown on nitrateor ammonium as nitrogen source.—J. exp. Bot. 36: 1685–1697. Duckweed, Lemna minor L., grown on inorganic nutrient solutionscontaining either NH₄⁺ or NO₃^– as nitrogen source wasallowed to assimilate [1-¹⁴C]- or [2-¹⁴C]glycollate during a20 min period in darkness or in light. The incorporation ofradioactivity into water-soluble metabolites, the insolublefraction, and into the CO₂ released was measured. In additionthe extractable activity of phosphoenolpyruvate carboxylasewas determined. During the metabolism of [2-¹⁴C]glycollate in darkness, as wellas in the light, NH₄⁺ grown plants evolved more ¹⁴CO₂ than NO₃^–grown plants. Formate was labelled only from [2-¹⁴C]glycollateand in NH₄⁺ grown plants it was significantly less labelledin light than in darkness. In NO₃^– grown plants formateshowed similar radioactivity after dark and light labelling.The radioactivity in glycine was little influenced by the nitrogensource. Amounts of radioactivity in serine implied that thefurther metabolism of serine was reduced in darkness comparedwith its metabolism in the light under both nitrogen regimes.In illuminated NH₄⁺ plants, serine was labelled through a pathwaystarting from phosphoglycerate. After [1-¹⁴C]glycollate feedingNH₄⁺ grown plants contained markedly more radioactive aspartateand malate than NO₃^– plants indicating a stimulated phosphoenolpyruvatecarboxylation in plants grown on NH₄⁺. Key words: Photorespiration, glycollate, nitrogen, Lemna     

Isolation and Identification of L-Pipecolic Acid and Nicotinamide as Flower-Inducing Substances in Lemna

Flower-inducing factors in extracts of flowering Lemna gibbaG3 were investigated using Lemna paucicostata 151 as the bioassayplant. Fractions with flower-inducing activity were obtainedafter several purification steps. Two of the active substanceswere identified as L-pipecolic acid and nicotinamide by MS andNMR analyses. Both L-pipecolic acid and nicotinamide exhibited flower-inducingactivity in L. paucicostata 151 grown on one-tenth-strengthM medium containing benzyladenine, the former being ten timesas active as the latter. L-Pipecolic acid was active even at0.01 ppm (7.8 ? 10^–8 M). The effect of L-pipecolic acidon flowering strongly depended upon the presence of exogenouscytokinin. The coexistence of cytokinin seemed to be essentialfor L-pipecolic acid to exhibit flower-inducing activity. Incontrast, the effect of nicotinamide on flowering was basicallythe same as that of benzoic acid or nicotinic acid. (Received February 9, 1987; Accepted May 21, 1987)