Vanadate inhibits the ATP-dependent degradation of proteins in reticulocytes without affecting ubiquitin conjugation

Reticulocytes contain a nonlysosomal, ATP-dependent system for degrading abnormal proteins and normal proteins during cell maturation. Vanadate, which inhibits several ATPases including the ATP-dependent proteases in Escherichia coli and liver mitochondria, also markedly reduced the ATP-dependent degradation of proteins in reticulocyte extracts. At low concentrations (K1 = 50 microM), vanadate inhibited the ATP-dependent hydrolysis of [3H]methylcasein and denatured 125I-labeled bovine serum albumin, but it did not reduce the low amount of proteolysis seen in the absence of ATP. This inhibition by vanadate was rapid in onset, reversed by dialysis, and was not mimicked by molybdate. Vanadate inhibits proteolysis at an ATP-stimulated step which is independent of the ATP requirement for ubiquitin conjugation to protein substrates. When the amino groups on casein and bovine serum albumin were covalently modified so as to prevent their conjugation to ubiquitin, the derivatized proteins were still degraded by an ATP-stimulated process that was inhibited by vanadate. In addition, vanadate did not reduce the ATP-dependent conjugation of 125I-ubiquitin to endogenous reticulocyte proteins, although it markedly inhibited their degradation. In intact reticulocytes vanadate also inhibited the degradation of endogenous proteins and of abnormal proteins containing amino acid analogs. This effect was rapid and reversible; however, vanadate also reduced protein synthesis and eventually lowered ATP levels in the intact cells. Vanadate (10 mM) has also been reported to decrease intralysosomal proteolysis in hepatocytes. However, in liver extracts this effect on lysosomal proteases required high concentrations of vanadate (K1 = 500 microM) and was also observed with molybdate, unlike the inhibition of ATP-dependent proteolysis in reticulocytes.     

Rabbit red blood cell hexokinase. Decay mechanism during reticulocyte maturation

In rabbit reticulocytes, the hexokinase (EC 2.7.1.1)-specific activity is 4-5 times that of corresponding mature red cells. Immunoprecipitation of hexokinase by a polyclonal antibody made in vitro shows that this maturation-dependent hexokinase decay is not due to accumulation of inactive enzyme molecules but to degradation of hexokinase. A cell-free system derived from rabbit reticulocytes, but not mature erythrocytes, was found to catalyze the decay of hexokinae activity and the degradation of 125I-labeled enzyme. This degradation is ATP-dependent and requires both ubiquitin and a proteolytic fraction retained by DEAE-cellulose. Maximum ATP-dependent degradation was obtained at pH 7.5 in the presence of MgATP. MgGTP could replace MgATP with a relative stimulation of 0.90. 125I-Hexokinase incubated with reticulocyte extract in the presence of ATP forms high molecular weight aggregates that reach a steady-state concentration in 1 h, whereas the degradation of the enzyme is linear up to 8 h, suggesting that the formation of protein aggregates precedes enzyme catabolism. These aggregates are stable upon boiling in 2% sodium dodecyl sulfate, 3% mercaptoethanol and probably represent an intermediate step in the enzyme degradation with hexokinase and other proteins covalently conjugate to ubiquitin. That hexokinase could be conjugated to ubiquitin was shown by the formation of 125I-ubiquitin-hexokinase complexes in the presence of ATP and the enzymes of the ubiquitin-protein ligase system. Thus, the decay of hexokinase during reticulocyte maturation is ATP- and ubiquitin-dependent and suggests a new physiological role for the energy-dependent degradation system of reticulocytes.     

Subcellular distribution of abnormal proteins in rabbit reticulocytes. Effects of cellular maturation, phenylhydrazine and inhibitors of ATP synthesis

Inhibitors of ATP synthesis (cyanide, dinitrophenol, fluoride) inhibited proteolysis of pulse-labelled abnormal proteins in rabbit reticulocytes and caused an accumulation of the aberrant polypeptides in the cell debris fraction in a manner analogous to phenylhydrazine-induced Heinz bodies. When the reticulocytes were separated into age-groups by sedimentation through discontinuous gradients of bovine serum albumin, the ability of the cells to degrade puromycin peptides decreased with increasing cellular maturity and, in the more mature cells, up to 40% of the labelled abnormal polypeptide remained associated with the cell debris fraction at the end of the chase period. It is suggested that the association of the abnormal polypeptide with the cell debris fraction is a consequence of a maturation-induced loss, or an inhibitor-induced inactivation of the cellular proteolytic activity.     

A soluble ATP-dependent system for protein degradation from murine erythroleukemia cells. Evidence for a protease which requires ATP hydrolysis but not ubiquitin

A soluble ATP-dependent system for protein degradation has been demonstrated in reticulocyte lysates, but not in extracts of nucleated cells. We report that extracts of undifferentiated murine erythroleukemia (MEL) cells contain a labile ATP-stimulated proteolytic system. The addition of ATP to MEL cell extracts at alkaline pH enhances degradation of endogenous cell proteins and various radiolabeled exogenous polypeptides from 2-15-fold. Nonhydrolyzable ATP analogs had no effect. In reticulocytes, one role of ATP in proteolysis is for ubiquitin conjugation to protein substrates. MEL cells also contain ubiquitin and extracts can conjugate 125I-ubiquitin to cell proteins; however, this process in MEL cells seems unrelated to protein breakdown. After removal of ubiquitin from these extracts by DEAE- or gel chromatography, the stimulation of proteolysis by ATP was maintained and readdition of purified ubiquitin had no further effect. In addition, these extracts degraded in an ATP-dependent fashion casein whose amino groups were blocked and could not be conjugated to ubiquitin. After gel filtration or DEAE-chromatography of the MEL cell extracts (unlike those from reticulocytes), we isolated a high molecular weight (600,000) ATP-dependent proteolytic activity, which exhibits many of the properties of energy-dependent proteolysis seen in crude cell extracts. For example, both the protease and crude extracts are inhibited by hemin and N-ethylmaleimide and both hydrolyze casein, globin, and lysozyme rapidly and denatured albumin relatively slowly. The protease, like the crude extracts, is also stimulated by UTP, CTP, and GTP, although not as effectively as ATP. Also, nonhydrolyzable ATP analogs and pyrophosphate do not stimulate the protease. Thus, some mammalian cells contain a cytosolic proteolytic pathway that appears independent of ubiquitin and involves and ATP-dependent protease, probably similar to that found in Escherichia coli or mitochondria.     

Red blood cells contain a pathway for the degradation of oxidant-damaged hemoglobin that does not require ATP or ubiquitin

It is generally accepted that ATP is required for intracellular protein breakdown. Reticulocytes contain a soluble ATP-dependent pathway for the degradation of highly abnormal proteins and for the elimination of certain proteins during cell maturation. Reticulocytes and erythrocytes also selectively degrade proteins damaged by oxidation. When these cells were exposed to oxidants, such as phenylhydrazine or nitrite, they showed a large increase in protein breakdown. This oxidant-induced proteolysis was not inhibited in cells depleted of ATP. However, ATP depletion did prevent the degradation of pre-existent cell proteins. In reticulocyte extracts, phenylhydrazine-treated hemoglobin is also degraded rapidly by an ATP-independent process, unlike endogenous proteins and many exogenous polypeptides. This lack of an ATP requirement means that the degradation of oxidant-damaged proteins does not require ligation to ubiquitin (even though phenylhydrazine treatment does make hemoglobin a very good substrate for ubiquitin conjugation). In many respects, the pathway for breakdown of oxidant-treated hemoglobin differs from the ATP-dependent process. The latter has a much higher activation energy than the degradation of oxidized proteins. The ATP-dependent process is inhibited by hemin, 3,4-dichloroisocoumarin, diisopropylfluorophosphate and N-ethylmaleimide. The ATP-independent pathway is less sensitive to N-ethylmaleimide, hemin, and 3,4-dichloroisocoumarin and is not affected by diisopropylfluorophosphate. In addition, only the ATP-dependent proteolytic process is inactivated by dilution or incubation at 37 degrees C in the absence of nucleotides. Reticulocytes thus contain multiple soluble systems for degrading proteins and can rapidly hydrolyze certain types of abnormal proteins by either an ATP-independent or ATP-dependent process. Erythrocytes lack the ATP-dependent process present in reticulocytes; however, erythrocytes retain the capacity to degrade oxidant-damaged hemoglobin. These two processes probably are active in the elimination of different types of abnormal proteins.     

Proteolysis in the obligate methylotroph Methylophilus methylotrophus

This report represents the first demonstration of degradation of intracellular protein in the obligate methylotroph, Methylophilus methylotrophus. Proteolysis in batch culture was followed by a pulse-chase protocol which included chloramphenicol during the chase period to prevent re-incorporation of the radio-label, l-[4,5-³H] isoleucine. Starvation for a nitrogen source mildly stimulated proteolysis whereas starvation for the carbon source (0.5% v/v methanol) inhibited proteolysis by over 50%. Respiratory inhibitors (e.g. 2,4-DNP) caused a rapid decline in both intracellular ATP concentration and protein catabolism. Proteins synthesized after the addition of methanol (5% v/v) and ethanol (5% v/v) to the growth medium were subject to rapid degradation. Breakdown of abnormal proteins generated by treatment with dihydrostreptomycin and puromycin was also inhibited by inhibitors of respiration and deprivation of carbon source. The stability of an heterologous gene product, interferon -2, was also investigated; loss of immunoreactivity was reduced in the absence of methanol but not prevented.     

A ubiquitin C-terminal isopeptidase that acts on polyubiquitin chains. Role in protein degradation.

In the ubiquitin (Ub) pathway, proteins are ligated with polyUb chains and then are degraded by a 26 S protease complex. We describe an enzyme, called isopeptidase T, that acts on polyUb chains. It is a monomeric Ub-binding protein abundant in erythrocytes and reticulocytes. The activity of the isopeptidase is inhibited by iodoacetamide and Ub aldehyde. Treatment of the enzyme with Ub aldehyde increased its affinity for free Ub, indicating the existence of two different Ub-binding sites and cooperativity between the two sites. Isopeptidase T acts on polyUb-protein conjugates, but not on conjugates in which the formation of polyUb chains was prevented by the use of reductively methylated Ub or on abnormal polyUb chains formed with a mutant Ub that contains a Lys----Arg substitution at residue 48. The enzyme converts high molecular mass polyUb-protein conjugates to lower molecular mass forms with the release of free Ub, but not of free protein substrate. The lower molecular mass Ub-protein conjugate products are resistant to further action of the enzyme. Isopeptidase T stimulates protein degradation in a system reconstituted from purified enzyme components. The enzyme also stimulates the degradation of proteins ligated to polyUb chains by the 26 S protease complex. Preincubation of polyUb-protein conjugates with the isopeptidase did not much increase their susceptibility to proteolysis by the 26 S complex. On the other hand, preincubation of conjugates with the 26 S protease complex and ATP increased the release of free Ub upon further incubation with the isopeptidase. It thus seems that a role of this isopeptidase in protein breakdown is to remove polyUb chain remnants following the degradation of the protein substrate moiety by the 26 S complex.     

Macroxyproteinase (M.O.P.): a 670 kDa proteinase complex that degrades oxidatively denatured proteins in red blood cells

Erythrocytes and reticulocytes are shown to undergo rapid rates of protein degradation following exposure to oxidative stress. Experiments with ATP depletion revealed that, unlike the proteolysis of many other abnormal proteins, the degradation of oxidatively modified proteins is an ATP-independent process. Ion exchange chromatography (DEAE Sepharose CL-6B), ammonium sulfate precipitation, gel filtration chromatography (Sephacryl S-300 or Sepharose CL-6B), and a second ion exchange step were used to resolve the activity responsible for degrading oxidatively modified proteins from (dialyzed) cell-free extracts of erythrocytes and reticulocytes. Gel filtration studies revealed that some 70-80% of the activity in erythrocytes, and some 60-70% of the activity in reticulocytes, is expressed by a 670 kDa proteinase complex that is not stimulated by ATP (in fact, ATP is slightly inhibitory). This proteinase complex is inhibited by sulfhydryl reagents, serine reagents, and transition metal chelators, and has a pH optimum of 7.8. We propose the trivial name "macroxyproteinase" or "M.O.P." (abbreviated from Macro-Oxy-Proteinase) for the complex because of its large size, substrate preference (oxidatively modified proteins), and inhibitor profile (which indicates multiple catalytic sites). Electrophoresis studies of the 670 kDa M.O.P. complex revealed the presence of 8 distinct polypeptide subunits with the following apparent molecular sizes: 21.5, 25.3, 26.2, 28.1, 30.0, 31.9, 33.3, and 35.7 kDa. The large molecular size of the M.O.P. complex, its ATP- and ubiquitin-independence, its inhibitor profile, its distinctive subunit banding pattern in denaturing electrophoresis gels, its pH optimum, and its proteolytic profile with fluorogenic peptide substrates all indicate that M.O.P. is identical to 600-700 kDa neutral/alkaline proteinase complexes that have been isolated from a wide variety of eucaryotic cells and tissues, but for which no function has previously been clear. We propose that macroxyproteinase is responsible for catalyzing most of the selective degradation of oxidatively denatured proteins in red blood cells. We further suggest that M.O.P. may perform the same function in other eucaryotic cells and tissues.     

Ubiquitin-dependent proteolysis of native and alkylated bovine serum albumin: effects of protein structure and ATP concentration on selectivity

The susceptibility of bovine serum albumin to degradation by the ubiquitin-dependent system of proteolysis depends on the severity of the iodination conditions [Wilkinson, K.D., & Audhya, T.K. (1981) J. Biol. Chem. 256, 9235-9241]. To evaluate if other modifications of the protein changed its susceptibility to degradation, chemically modified derivatives of bovine serum albumin have been synthesized, characterized, and tested as substrates for the ubiquitin-dependent system. Serum albumin was reduced or reduced and alkylated with iodoacetic acid or iodoacetamide. Only the alkylated derivatives exhibit saturation kinetics. Both alkylated proteins competitively inhibit the degradation of the other. These substrates are useful for assay of the intact proteolysis system in crude extracts and in assays for other substrates using competitive alternate substrate inhibition. The physical properties of these proteins suggest that charge, denaturation, or aggregation is not correlated with the degradation rate of these proteins by this system. However, the selectivity of the ubiquitin-dependent proteolysis depends strongly on the ATP concentration. At saturating substrate concentrations, both alkylated substrates are degraded equally. At low ATP concentrations, there is a 2.4-fold difference in the degradation rates of the alkylated proteins. The results presented here indicate that the ubiquitin-dependent protein degradation system is selective and responsive to ATP concentrations and that not all abnormal proteins are equally preferred substrates. Thus, the system may be more selective than previously thought.     

ATP serves two distinct roles in protein degradation in reticulocytes, one requiring and one independent of ubiquitin

Protein degradation in rabbit reticulocytes is a nonlysosomal process requiring ATP. Recently, appreciable evidence has been presented that ATP is required for the covalent binding of the polypeptide ubiquitin to epsilon-amino groups on protein substrates. To test whether linkage of ubiquitin to substrates is required for ATP-dependent proteolysis, the amino groups of 3H-methyl-casein and denatured 125I-bovine serum albumin (BSA) were completely (93-99%) blocked by methylation, acetylation, carbamylation, or succinylation. In each case, the proteins lacking amino groups were still degraded by an ATP-stimulated process, although these various treatments altered absolute rates of proteolysis and reduced the magnitude of the ATP stimulation (two- to fourfold) below that seen measured with the unmodified substrates. When ubiquitin was removed by ion exchange chromatography, ATP still stimulated breakdown of casein and carbamylated casein twofold. The addition of ubiquitin in the presence of ATP caused a further twofold increase in the hydrolysis of unmodified casein but did not affect the degradation of casein lacking amino groups. Thus ubiquitin conjugation to substrates appears important in the breakdown of certain substrates (especially of BSA), but this reaction is not essential for ATP- stimulated proteolysis. The ATP-activated step that is independent of ubiquitin probably is also involved in the degradation of unblocked proteins, since both processes require Mg++ and ATP hydrolysis and are inhibited by hemin but not by protoporphyrin IX. These results suggest that ATP has distinct roles at different steps in the degradative pathway.     

ATP-dependent dissociation of non-disulfide-linked aggregates of coagulation factor VIII is a rate-limiting step for secretion

Deficiency in coagulation factor VIII leads to the bleeding disorder hemophilia A. Previous studies demonstrated that factor VIII secretion is limited due to an ATP-requiring step early in the secretory pathway. In this report, we identified that this ATP-dependent rate-limiting step involves the dissociation of non-disulfide-linked aggregates within the endoplasmic reticulum (ER). In contrast to the numerous examples of interchain disulfide-linked aggregates, factor VIII is the first protein characterized to form non-disulfide-linked high molecular weight aggregates within the ER. Approximately a third of newly synthesized factor VIII was detected in high molecular weight aggregates. These aggregates disappeared over time as functional factor VIII appeared in the medium. The aggregated complexes did not require proteasomal degradation for clearance. Aggregate formation was enhanced by ATP depletion, and upon restoration of metabolic energy, these aggregates were dissociated and secreted. With the coexpression of von Willebrand factor (vWF), a small portion of vWF coaggregated with factor VIII. However, vWF dissociated from the aggregates more rapidly than factor VIII, supporting that these aggregates are dynamic. An increase in the factor VIII expression level elicited a corresponding increase in the fraction of factor VIII that was aggregated. In addition, a 110 amino acid sequence containing a hydrophobic beta-sheet within factor VIII was identified that may predispose factor VIII to aggregation. These data show that formation and ATP-dependent dissolution of nondisulfide-linked factor VIII aggregates is a dynamic, rate-limiting step during the folding process in the early secretory pathway. In summary, we have identified an unprecedented requirement for protein transport out of the ER that involves an ATP-dependent dissociation of non-disulfide-linked aggregates within the ER.     

ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent

beta-Thalassemia is an inherited human disorder which is characterized by a deficient production of hemoglobin beta chains and an attendant accumulation of structurally normal alpha chains in the erythropoietic cells. The objective of this work is to understand the mechanism of intracellular proteolysis of these excess alpha chains. Dialyzed stroma-free hemolysates (32 mg/ml hemoglobin) of blood reticulocytes from four individuals with beta-thalassemia intermedia were incubated with human hemoglobin 3H-alpha chains (0.13 mg/ml) at 37 degrees C in a reaction mixture supporting protein degradation. In the presence of ATP and an ATP-generating system, the fraction of alpha chain 3H radioactivity made acid-soluble after 4 h ranged from 4 to 12% among the different hemolysates; in the absence of ATP or when hemolysates of normal human erythrocytes were used, only 1 to 2% of the 3H-alpha chains were degraded. It is likely that the ATP-dependent proteolysis of 3H-alpha chains in the beta-thalassemic hemolysates corresponds to the ATP-dependent turnover of newly synthesized soluble alpha chains in intact beta-thalassemic reticulocytes observed previously (Shaeffer, J. (1983) J. Biol. Chem. 258, 13172-13177) because of the following similarities between the two systems: (a) free 3H-alpha chains, but not 3H-labeled tetrameric hemoglobins, were readily degraded; (b) the rate of 3H-alpha chain proteolysis in the cell-free system was at least one-half of that observed for the turnover of newly synthesized alpha chains (t1/2 approximately 6 h) in intact cells; and (c) the ATP-dependent proteolytic activity of both systems was inhibited substantially by certain chemical agents (orthovanadate, N-ethylmaleimide, o-phenanthroline, and phenylmethylsulfonyl fluoride) but only slightly, if at all, by others (epsilon-aminocaproic acid and leupeptin). When excess human erythrocyte ubiquitin was added to the beta-thalassemic cell-free systems, a stimulation in ATP-dependent proteolysis of 3H-alpha chains ranging from 30 to 58% was observed. Conversely, addition of from 1.25 to 2.50 mg/ml affinity-purified rabbit antiubiquitin inhibited almost all (greater than 90%) of the ATP-dependent 3H-alpha chain proteolysis; in control experiments, antiubiquitin neutralized with excess ubiquitin inhibited only 13 to 30% of the total (including ubiquitin-stimulated) ATP-dependent proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

A novel ATP-requiring protease from skeletal muscle that hydrolyzes non-ubiquitinated proteins

Previously, we isolated an ATP-dependent proteolytic pathway in muscle, liver, and reticulocytes that requires ubiquitin and the enzymes which conjugate ubiquitin to proteins. We report here that skeletal muscle contains another soluble alkaline energy-dependent (but ubiquitin-independent) proteolytic activity. The cleavage of non-ubiquitinated protein substrates by the partially purified protease requires ATP hydrolysis since ATP in the absence of Mg2+, nonhydrolyzable ATP analogs, and pyrophosphate all fail to stimulate proteolysis. Proteolytic activity is also stimulated by UTP, CTP, and GTP, although not as effectively as by ATP (Km(ATP) = 0.027 mM). The enzyme is inactivated by the serine protease inhibitors diisopropyl fluorophosphate and 3,4-dichloroisocoumarin, but not by specific inhibitors of aspartic, thiol, or metalloproteases. It is maximally active at pH 8 and has a molecular weight of approximately 600,000. This new activity differs from the 720-kDa multicatalytic proteinase, but resembles the soluble ATP-dependent proteolytic system that we previously isolated from murine erythroleukemia cells.     

An investigation of mistranslation in vivo induced by streptomycin by an examination of the susceptibility of abnormal proteins to degradation.

Proteolysis rates in vivo were measured in Escherichia coli cultures during treatment with dihydrostreptomycin and under various other conditions. Dihydrostreptomycin treatment caused an increase in the proteolysis rate, compared to untreated controls. The proteolytic system in vivo responsible for the elevated proteolysis in the early stages of dihydrostreptomycin treatment, or that during canavanine and puromycin treatment, were not inhibited by addition of phenylmethanesulphonyl fluoride. This agent did inhibit proteolysis rates in cultures whose growth was inhibited by starvation, or had been completely stopped by dihydrostreptomycin. It seems, therefore, that the extremely high proteolysis rates in cultures at this stage of dihydrostreptomycin treatment were due to the action of two protease systems: the one concerned with the breakdown of abnormal proteins, and the other concerned with normal protein turnover and active during a non-specific decline of growth. The proteolytic rate at complete growth inhibition brought about by dihydrostreptomycin was intermediate between those induced by canavanine and puromycin at the same stage of treatment. This indicated a similar hierarchy in the extent and nature of abnormality in the proteins synthesized under these conditions. The relationship between the abnormality of proteins induced by dihydrostreptomycin and the importance of this in the antibiotic mechanism is discussed.     

ATP-stimulated degradation of endogenous proteins in cell-free extracts of BHK 21/C13 fibroblasts. A key role for the proteinase, macropain, in the ubiquitin-dependent degradation of short-lived proteins.

Baby hamster kidney (BHK) 21/C13 cell proteins, labeled with [35S]methionine, [14C]leucine or [3H]leucine in intact cells, were degraded in soluble, cell-free extracts by an ATP-stimulated process. The stimulatory effect of ATP appeared to require ATP hydrolysis and was mediated to a large extent by ubiquitin. Although the cell extracts contained endogenous ubiquitin, supplementation with exogenous ubiquitin increased ATP-dependent proteolysis by up to 2-fold. Furthermore, antibodies against the E1 ubiquitin conjugating enzyme specifically inhibited both conjugation of [125I]ubiquitin to endogenous proteins and ATP/ubiquitin-dependent proteolysis. Addition of purified E1 to antibody-treated extracts restored conjugation and proteolysis. Proteins containing the amino acid analogues canavanine and azatryptophan were also degraded in vitro by an ATP/ubiquitin-dependent process but at a rate up to 2-fold faster than normal proteins. These results indicate that soluble, cell-free extracts of BHK cells can selectively degrade proteins whose rates of degradation are increased in intact cells. Treatment of cell-free extracts with antibodies against the high molecular weight proteinase, macropain, also greatly inhibited the ATP/ubiquitin-dependent degradation of endogenous proteins. Proteolysis was specifically restored when purified macropain L was added to the antibody-treated extracts. Treatment of cell extracts with both anti-macropain and anti-E1 antibodies reduced ATP/ubiquitin-dependent proteolysis to the same extent as treatment with either antibody alone. Furthermore, proteolysis could be restored to the double antibody treated extracts only after addition of both purified E1 and macropain. These results provide strong evidence for an important role for macropain in the ATP/ubiquitin-dependent degradation of endogenous proteins in BHK cell extracts.     

Infulence of hormones and medium composition on the degradation of phosphoenolpyruvate carboxykinase (GTP) and total protein in Reuber H35 cells.

Reuber H35 cells were pulse-labeled with radioactive leucine and the influence of hormones, serum, and amino acids on protein degradation was investigated during a subsequent chase period. Radioactive, immunoprecipitable phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32) had a half-life of 5 to 6 hours which was not influenced by either N6, O2-dibutyryl adenosine 3':5'-monophosphate, dexamethasone, or insulin. The rate of phosphoenolpyruvate carboxykinase degradation was the same under steady state conditions as during the approach to a new steady state following hormonal induction or deinduction of the enzyme. Therefore, hormonal regulation of enzyme activity in vivo is the result of changes in the rate of enzyme synthesis. The rate of proteolysis for total cell proteins was increased under nutritional step-down conditions produced by the removal of serum or amino acids, or both, from the medium. This effect was completely prevented by insulin. Cycloheximide and puromycin, but not actinomycin D or cordycepin, inhibited protein degradation under step-down conditions but did not further decrease the basal rate of proteolysis measured in the presence of either insulin or serum plus amino acids. There was a good correlation between changes in proteolysis produced by serum and amino acids and changes in the degradation rate of phosphoenolpyruvate carboxykinase. Also, inhibition of proteolysis with cycloheximide and puromycin was accompanied by a decrease in the degradation rate for enzyme antigen. It is suggested that nutritional step-down leads either to the synthesis or activation of a proteolytic system.     

Interaction between membrane properties and protein synthesis: In vitro synthesis of membrane proteins during erythropoiesis

Membrane protein synthesis was investigated by incubating rabbit reticulocytes, in vitro, with radioactive amino acids. The kinetics of membrane protein synthesis showed linear incorporation for approx. 15 min, after which there was only a slight increase in incorporation. On the other hand, intracellular protein synthesis was linear for an incubation period of 60 min. Membranes isolated from such rabbit reticulocytes were analysed on sodium dodecyl sulfate (SDS)-polyacrylamide gels. Two major radioactive bands were found in the 50–60 000 D region, whilst another labelled band had a molecular weight of 43 000 D. This latter band had an electrophoretic mobility identical with rabbit muscle actin (and chick brain actin), when run on one-dimensional SDS polyacrylamide gels. Absolute identity between rabbit brain actin and a newly synthesized reticulocyte membrane protein was shown by comigration on a two-dimensional (first dimension isoelectric focusing and second dimension SDS gel) electrophoresis system. Another band that was radioactively labelled was found to have a molecular weight of approx. 32 000 D. Separation of reticulocytes into different age groups showed that young reticulocytes synthesized a membrane protein species that was not radioactively labelled in the old reticulocyte population.     

Effects of puromycin and hydroxynorvaline on net production and intracellular degradation of collagen in human fetal lung fibroblasts

Amino acid substitutions in collagen that impair folding of the triple helix result in significant increases in intracellular degradation of newly synthesized collagen. We have studied the effects of agents that cause other kinds of defects in collagen: hydroxynorvaline, a threonine analog that interferes with association of pro-alpha chains; and puromycin, an antibiotic that causes premature release of nascent polypeptides. cis-Hydroxyproline and cycloheximide, whose effects on collagen synthesis and degradation have already been studied and reported, were employed as reference compounds. Human fetal lung fibroblasts were used in these experiments. All the agents inhibited total protein production, and all except cycloheximide inhibited percentage collagen production. Intracellular collagen degradation was increased in cultures exposed to puromycin, hydroxynorvaline, and cis-hydroxyproline, but not in cultures exposed to cycloheximide. These results suggest that pro-alpha chains that were either unassociated (due to hydroxynorvaline) or shortened (due to puromycin) were recognized as abnormal and degraded to the same extent as chains that contained cis-hydroxyproline. However, the increases in degradation could not account completely for the decreases in collagen production (except when cis-hydroxyproline was used at low concentrations). These findings indicate that, in addition to rendering newly synthesized procollagen molecules or partial polypeptide chains more susceptible to intracellular degradation, puromycin, hydroxynorvaline, and cis-hydroxyproline significantly inhibited collagen synthesis.     

The ClpPX protease is required for radioresistance and regulates cell division after gamma-irradiation in Deinococcus radiodurans

Protein degradation in bacteria is involved in diverse cellular responses to environmental stimuli and in removing potentially toxic damaged proteins or protein aggregates. ATP-dependent proteases play a key role in these processes. Here, we have individually inactivated all the ATP-dependent proteases belonging to the Clp or Lon families in Deinococcus radiodurans. The mutants were tested for survival after gamma-irradiation and for sensitivity to the tRNA analogue puromycin in order to assess the impact of each disruption on radioresistance, as well as on proteolysis of misfolded proteins. We found that inactivation of the ClpPX protease significantly decreased cell survival at elevated gamma-irradiation doses, while inactivation of Lon1 and Lon2 proteases reduced resistance to puromycin, suggesting that they play a role in eliminating damaged proteins. Mutants devoid of ClpPX protease displayed altered kinetics of DNA double-strand break repair and resumed cell division after an exceedingly long lag phase following completion of DNA repair. During this stasis period, most of the DeltaclpPX irradiated cells showed decondensed nucleoids and abnormal septa and some cells were devoid of DNA. We propose that the ClpPX protease is involved in the control of proper chromosome segregation and cell division in cells recovering from DNA damage.