Lysosomal degradation of microinjected proteins.
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Cytosol↗
Biomedical subjects
J M Backer
Publications and source records attributed to J M Backer.
Regulation of catabolism of microinjected ribonuclease A. Identification of residues 7-11 as the essential pentapeptide.
We have identified a pentapeptide region of microinjected ribonuclease A that is required for enhanced degradation of this protein during serum withdrawal. We introduced reductively methylated [3H]ribonuclease A, [3H]ribonuclease S-protein (residues 21-124), and [3H]ribonuclease S-peptide (residues 1-20) into the cytosol of human fibroblasts by red cell-mediated microinjection and osmotic lysis of pinosomes. The degradative rates of ribonuclease A and ribonuclease S-peptide are increased 2-fold upon withdrawal of serum, while catabolism of ribonuclease S-protein is not regulated in this manner. Certain fragments of ribonuclease S-peptide are also degraded in a serum-dependent fashion (residues 1-14 and 4-13), while other fragments are not (residues 1-10 and 2-8). [3H]Ribonuclease S-peptide is cleaved into two smaller radioactive peptides during loading into red cell ghosts. We tentatively identified the larger fragment as residues 7-11 based on its molecular weight determined by Sephadex chromatography in the presence of 8 M urea combined with sequential Edman degradation to identify the position of radioactive lysines. The smaller peptide fragment appears to be the amino-terminal dipeptide, Lys-Glu, and/or residues 7-8, Lys-Phe. After microinjection into fibroblasts, the pentapeptide is degraded at an enhanced rate in the absence of serum, while degradation of the dipeptide is not affected. We confirmed that residues 7-11 constitute the larger hydrolysis product of S-peptide by synthesizing this pentapeptide and radiolabeling it by reductive methylation. It migrated at the expected position after Sephadex chromatography in 8 M urea and was further hydrolyzed only slightly during loading into red cells. Finally, degradation of this pentapeptide after injection into fibroblasts was enhanced 2-fold upon serum withdrawal. These results, combined with our other recent studies (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993), suggest that the pentapeptide, Lys-Phe-Glu-Arg-Gln, targets microinjected ribonuclease A to lysosomes for enhanced degradation during serum deprivation.
Cell Line↗
Protein phosphorylation in isolated mitochondria and the effects of protein kinase C.
When isolated intact rat liver mitochondria are incubated with [gamma-32P]ATP the major phosphorylated proteins are those of 47 and 36 kDa. Phosphorylation of the 47 kDa protein, but not of the 36 kDa protein, is inhibited by carboxyatractyloside, an inhibitor of mitochondrial ATP uptake, while phosphorylation of the 36 kDa protein is inhibited by various uncouplers and an inhibitor of mitochondrial respiration. Addition of purified protein kinase C to the isolated mitochondria leads to the phosphorylation of 69, 37 and 17 kDa proteins. As with other substrates for protein kinase C, phosphorylation of these proteins is dependent on Ca2+ and markedly stimulated by various tumor promoters.
Adenosine Triphosphate↗
Proteins encoded by ras oncogenes stimulate or inhibit phosphorylation of specific mitochondrial membrane proteins.
We have examined the effects of a series of purified p21 proteins encoded by ras oncogenes and synthesized in E. coli via recombinant DNA methods, on the phosphorylation of proteins associated with isolated rat liver mitochondria. We find that these proteins markedly enhance the phosphorylation of a 36KD protein and inhibit phosphorylation of a 17KD protein. The phosphorylated residues on the 36KD protein are hydrolyzed by mild acid, suggesting that they involve phosphoamide bonds. These results suggest that p21 proteins may play a role in vivo by altering the phosphorylation of certain proteins.
Animals↗
Covalent linkage of ribonuclease S-peptide to microinjected proteins causes their intracellular degradation to be enhanced during serum withdrawal.
The amino-terminal 20 amino acids are required for microinjected ribonuclease A (RNase A) to be taken up by lysosomes and degraded at an enhanced rate during serum withdrawal. We used water-soluble carbodiimides to covalently attach the RNase S-peptide (residues 1-20) to [3H]RNase S-protein (residues 21-124) at unspecified locations. We then measured catabolism of the [3H]S-protein-S-peptide conjugate after its microinjection into human diploid fibroblasts. The attached S-peptide caused the degradation of S-protein to be enhanced 2-fold in the absence of serum. Control experiments showed that degradation of [3H]RNase S-protein remained unresponsive to serum after conjugation with the inactive fragment, RNase S-peptide (residues 1-10). Covalent attachment of RNase S-peptide had a similar effect on the catabolism of two other proteins. Degradation rates of microinjected 125I-labeled lysozyme and 125I-labeled insulin A chain are normally unresponsive to serum withdrawal. However, breakdown rates of microinjected 125I-labeled lysozyme-S-peptide and 125I-labeled insulin A chain-S-peptide conjugates were increased 2-fold during serum deprivation. We suggest that RNase S-peptide acts as a "single sequence" that directs cytosolic proteins to lysosomes through a pathway that is activated by deprivation conditions.
Cell Line↗
p21 ras proteins and guanine nucleotides modulate the phosphorylation of 36- and 17-kilodalton mitochondria-associated proteins.
We have found that, when isolated rat liver mitochondria are incubated with [gamma-32P]ATP, there is phosphorylation of 36- and 17-kDa proteins. These proteins together with their protein kinase(s) are released as a complex by incubation of the isolated rat liver mitochondria at 20 degrees C for 30 min with 10 mM glucose 6-phosphate, 0.5 mM inositol phosphate, or 0.01 mM inositol triphosphate. Phosphorylation of the 36- and 17-kDa proteins in this soluble protein fraction is modulated by p21 proteins encoded by ras oncogenes and synthesized in Escherichia coli via recombinant DNA methods. A normal p21 ras protein stimulates phosphorylation of the 36-kDa protein and inhibits phosphorylation of the 17-kDa protein, whereas two transforming p21 ras proteins inhibit phosphorylation of both the 36- and 17-kDa proteins. Although GDP and 5'-guanylyl imidodiphosphate also influence the phosphorylation of these proteins, we present evidence that the effects of p21 ras protein are not simply due to their bound GDP. This novel system may be useful for further studies on the biochemical functions of the p21 ras proteins.
Animals↗
Regulation of catabolism of ribonuclease A microinjected into human fibroblasts.
We are using ribonuclease A (RNase A) as a model protein to study how the degradative rates of proteins are regulated within cells. RNase A and several derivatives can be microinjected into confluent cultures of human fibroblasts using red cell-mediated microinjection. The half-life of RNase A is 80-100 hrs in cells maintained in the presence of serum, and the degradative rate is enhanced approximately two-fold upon serum withdrawal. The ability of fibroblasts to regulate breakdown of this protein depends on a small peptide region within the amino terminal twenty amino acids. This amino terminal peptide from RNase A can be covalently attached to unrelated proteins and will cause their catabolism to become serum responsive. The mechanism of degradation of RNase A involves lysosomal pathways both in the presence and absence of serum, and the enhanced catabolism during serum deprivation results from a two-fold increase in the rate of uptake of the protein by lysosomes. These findings suggest that autophagy, or some other process occuring in serum-deprived cells, can be highly selective.
Fibroblasts↗
Regulation of catabolism of microinjected ribonuclease A requires the amino-terminal 20 amino acids.
RNase A introduced into the cytoplasm of IMR-90 human diploid fibroblasts by erythrocyte-mediated microinjection is degraded with a half-life of approximately equal to 75 hr in the presence of fetal bovine serum. In response to serum deprivation the degradative rate of microinjected RNase A is enhanced 2-fold. RNase S protein (amino acids 21-124) is degraded with a half-life similar to that of RNase A in the presence of serum, but its catabolism is not increased during serum withdrawal. Reconstitution of RNase S protein with RNase S peptide (amino acids 1-20) restored full enzymatic activity to the S protein as well as the ability of fibroblasts to increase its catabolism during serum deprivation. Finally, RNase S peptide microinjected alone shows the full 2-fold increase in degradative rate during serum withdrawal. These results show that recognition of RNase A for enhanced breakdown during serum deprivation is based on some feature of its amino-terminal 20 amino acids. Furthermore, our results indicate that the enhanced protein catabolism during serum deprivation can be highly selective.
Amino Acid Sequence↗
When do carcinogen-treated 10T1/2 cells acquire the commitment to form transformed foci?
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7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Inhibition of respiration by a phorbol ester tumor promoter in murine cultured cells.
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Animals↗
Interaction of benzo(a)pyrene and its dihydrodiol-epoxide derivative with nuclear and mitochondrial DNA in C3H10T 1/2 cell cultures.
To analyze the distribution of radioactive carcinogens and [3H]thymidine between nuclear DNA (nDNA) and mitochondrial DNA (mtDNA), we have developed a simple and rapid method for the separation of nDNA and mtDNA using gel electrophoresis of cell lysates. Using this method, we found that, when C3H10T 1/2 cells are exposed to either 0.5 microM [3H]-(+/-)-7 beta,8 alpha-dihydroxy-9 alpha,10 alpha-epoxy-7,8,9,10-tetrahydrobenzo(a)pyrene ([3H]BPDE) or 1 microM [3H]benzo(a)pyrene, the mtDNA contains a major fraction of the total adducts formed with cellular DNAs. Deoxynucleoside adducts formed between benzo(a)pyrene and mtDNA in intact C3H10T 1/2 cells or between BPDE and isolated rat liver mtDNA were analyzed by high-performance liquid chromatography, and were found to be much more heterogeneous than those present in nDNA of C3H10T 1/2 cells. The extensive modification of mtDNA in BPDE in C3H10T 1/2 cells is associated with preferential inhibition of the incorporation of [3H]thymidine into mtDNA, when compared to incorporation of [3H]thymidine into nDNA. To analyze the factors responsible for the extensive modification of mtDNA by BPDE, we investigated the role of a lipid phase utilizing liposome:DNA complexes as a model system. We found that the liposomes protect BPDE from spontaneous hydrolysis and enhance the extent of DNA modification at low DNA concentrations. These findings extent previous evidence suggesting that the mitochondria may be important cellular targets in the process of chemical carcinogenesis.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
Mechanism of cholesterol exchange between phospholipid vesicles.
The kinetics of cholesterol exchange between two populations of small unilamellar vesicles has been investigated. There is no change in the initial rate of this exchange process over a 100-fold change in the acceptor vesicle concentration at a constant donor concentration. These results are not consistent with a collision-dependent exchange mechanism. In support of transfer via the aqueous phase, the inclusion of a negatively charged lipid into the vesicles did not affect the exchange rate. Evidence for a water-soluble pool of cholesterol that had partitioned ut of the vesicle was obtained. Finally, cholesterol exchange was observed when donor and acceptor membranes were separated by a barrier through which neither could pass. These data together support our contention that the exchange of cholesterol between these vesicles involves a water-soluble intermediate.
Cholesterol↗
The rapid transbilayer movement of thiocholesterol in small unilamellar phospholipid vesicles.
Cholesterol is a major component of biological membranes, yet there is very little information concerning its distribution across the membrane. Recent experiments in our laboratory, using cholesterol oxidase, have demonstrated that cholesterol can undergo a rapid transbilayer movement in lecithin-cholesterol vesicles in a half-time of 1 min or less at 37 degrees C. In order to support this conclusion, we have sought other approaches to the measurement of this process. We now report our finding that the transbilayer movement of thiocholesterol in phospholipid vesicles occurs in a half-time of 1 min or less at 20 degrees C.
Cholesterol↗
Transmembrane movement of cholesterol in small unilamellar vesicles detected by cholesterol oxidase.
Greater than 90% of the cholesterol in small unilamellar vesicles composed of egg lecithin and cholesterol (molar ratio 1:0.7) was oxidized by a cholesterol oxidase from Brevibacterium sp., with a single time constant and a half-time of 1 min at 37 degrees C. The enzyme preparation used was at least 95% pure and possessed no detectable phospholipase C activity. Since cholesterol is present in both halves of the bilayer, it was concluded that transmembrane movement of cholesterol in these vesicles occurs with a half-time of 1 min or less at 37 degrees C.
3-Hydroxysteroid Dehydrogenases↗
Mitochondrial DNA is a major cellular target for a dihydrodiol-epoxide derivative of benzo[a]pyrene.
When mammalian cell cultures are exposed for 2 hours to (+/-)-7 beta, 8 alpha-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, a mutagenic and carcinogenic derivative of benzo[a]pyrene, the extent of covalent modificationof mitochondrial DNA is 40 to 90 times greater than that of nuclear DNA. Evidence is presented that this reflects the lipophilic character of the derivative and the very high ratio of lipid to DNA in mitochondria. These results suggest that mitochondrial DNA may be an important cellular target of chemical carcinogens.
7,8-Dihydro-7,8-dihydroxybenzo(a)pyrene 9,10-oxide↗
The rapid transmembrane movement of cholesterol in small unilamellar vesicles.
The exchange of cholesterol between two populations of small unilamellar vesicles has been investigated using a new system. Uniformly sized egg lecithin-cholesterol vesicles containing [3H]cholesterol and the glycolipid N-palmitoyl-DL-dihydrolactocerebroside were used as donors, whereas similar vesicles containing unlabelled cholesterol and no glycolipid were used as cholesterol acceptors. The two populations of vesicles were separated with the castor bean lectin Ricinus communis. It was found that greater than 90% of the cholesterol in the donor vesicle could be exchanged with a single time constant, the half-time for the completion of this exchange process being 1.5 h at 37 degrees C. Therefore, the rate of transmembrane movement or flip-flop of cholesterol in these vesicles must be at least as fast as the intermembrane exchange process. Similar results were obtained using hemoglobin-free human erythrocyte ghosts as the acceptor membrane. If the molecular-sieve chromatography step used to fractionate the vesicles was omitted, a non-exchangeable pool of cholesterol was detected which was shown not to be due to the presence of multilamellar vesicles.
Biological Transport↗
Kinetic evidence for multiple binding sites of nucleoside triphosphates in Escherichia coli RNA polymerase.
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Binding Sites↗
Detection of the kinetics of biochemical reactions with oxygen using exchange broadening in the ESR spectra of nitroxide radicals.
To detect changes in the oxygen concentration during biochemical reactions, the exchange broadening in the ESR spectra of nitroxide radicals caused by the dissolved oxygen, has been used. The measurements have been carried out using changes in the width either of the proton hyperfine structure components or of the nitrogen hyperfine structure line with an unresolved proton structure. Detection of mitochondrial respiration in a volume of about 10(-3) cm 3 and respiration for 100 +/- 5 liver cells in a volume of about 10(-4) cm3 has been carried out.
Electron Spin Resonance Spectroscopy↗