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M Rechsteiner

Publications and source records attributed to M Rechsteiner.

At least 91 records · Page 5Linked to original sources
PubMed1987

Do myc, fos and E1A function as protein phosphatase inhibitors?

The oncogenic proteins myc, fos and E1A bear striking resemblance to protein phosphatase inhibitors 1 and 2. Both sets of proteins possess several regions rich in proline (P), glutamic acid (E), serine (S) and threonine (T). In addition to PEST sequences four of the five proteins contain clusters of arginine-arginine pairs. On the basis of these similarities, I suggest that myc, fos and E1A are protein phosphatase inhibitors.

Adenovirus Early Proteins↗
PubMed1987

Microinjection of ubiquitin: intracellular distribution and metabolism in HeLa cells maintained under normal physiological conditions.

Radioiodinated ubiquitin was introduced into HeLa cells by erythrocyte-mediated microinjection. Subsequent electrophoretic analyses revealed that the injected ubiquitin molecules were rapidly conjugated to HeLa proteins. At equilibrium, 10% of the injected ubiquitin was conjugated to histones and 40% was distributed among conjugates of higher molecular weight. Although the remaining ubiquitin molecules appeared to be unconjugated, the free pool of ubiquitin decreased by one-third and additional conjugates were present when electrophoresis was performed at low temperature under nonreducing conditions. Molecular weights of these labile conjugates suggest that they are ubiquitin adducts in thiolester linkage to activating enzymes. Despite the fairly rapid degradation of injected ubiquitin (t1/2 approximately 10-20 h), the size distribution of ubiquitin conjugates within interphase HeLa cells remained constant for at least 24 h after injection. The intracellular locations of ubiquitin and ubiquitin conjugates were determined by autoradiography, by differential sedimentation of subcellular fractions in sucrose, and by extraction of injected cells with buffer containing Triton X-100. Free ubiquitin was found mostly in the cytosolic or Triton X-100-soluble fractions. As expected, histone conjugates were located predominately in the nuclear fraction and exclusively in the Triton X-100-insoluble fraction. Although high molecular weight conjugates were enriched in the Triton X-100-insoluble fraction, their size distribution was similar to that of soluble conjugates. When injected HeLa cells were exposed to cycloheximide to inhibit protein synthesis, the size distribution of ubiquitin conjugates was similar to that found in untreated cells. Moreover, high molecular weight conjugates decreased less than 20% after inhibition of protein synthesis. These results indicate that most ubiquitin conjugates are not newly synthesized proteins which have been marked for destruction.

Erythrocytes↗
PubMed1987

Microinjection of ubiquitin: changes in protein degradation in HeLa cells subjected to heat-shock.

Ubiquitin was radiolabeled by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using erythrocyte-mediated microinjection. The injected cells were then incubated at 45 degrees C for 5 min (reversible heat-shock) or for 30 min (lethal heat-shock). After either treatment, there were dramatic changes in the levels of ubiquitin conjugates. Under normal culture conditions, approximately 10% of the injected ubiquitin is linked to histones, 40% is found in conjugates with molecular weights greater than 25,000, and the rest is unconjugated. After heat-shock, the free ubiquitin pool and the level of histone-ubiquitin conjugates decreased rapidly, and high molecular weight conjugates predominated. Formation of large conjugates did not require protein synthesis; when analyzed by two-dimensional electrophoresis, the major conjugates did not co-migrate with heat-shock proteins before or after thermal stress. Concomitant with the loss of free ubiquitin, the degradation of endogenous proteins, injected hemoglobin, BSA, and ubiquitin was reduced in heat-shocked HeLa cells. After reversible heat-shock, the decrease in proteolysis was small, and both the rate of proteolysis and the size of the free ubiquitin pool returned to control levels upon incubation at 37 degrees C. In contrast, neither proteolysis nor free ubiquitin pools returned to control levels after lethal heat-shock. However, lethally heat-shocked cells degraded denatured hemoglobin more rapidly than native hemoglobin and ubiquitin-globin conjugates formed within them. Therefore, stabilization of proteins after heat-shock cannot be due to the loss of ubiquitin conjugation or inability to degrade proteins that form conjugates with ubiquitin.

Autoradiography↗
PubMed1987

Ubiquitin-mediated pathways for intracellular proteolysis.

Ubiquitination is one of several ways in which cells modify their proteins. As for phosphorylation or acetylation, there are distinct enzymes for adding and removing Ub from the surfaces of protein substrates. The dynamic equilibration of Ub with cellular proteins is also typical of most posttranslational modifications. Ubiquitination differs, however, in that the added group is large compared to acetate or phosphate. Its size must provide great potential for recognition by other cellular proteins. Ub may be the cell's reversible cross-linking reagent, covalently bound to protein substrates at one end and noncovalently associated with various Ub binding proteins at the other. It is likely that one ubiquitin binding protein is a component of the 26S ATP-dependent protease. The presence of Ub on histones and on the lymphocyte homing receptor suggests that ubiquitination does not serve exclusively to mark proteins for degradation. There are probably various ubiquitin binding proteins since Ub appears to be a multifunctional protein that affects chromatin structure, intracellular proteolysis, cellular interactions, and the stress response. This abundant protein may serve as an intracellular barometer whose distribution among several pools regulates a variety of processes.

Animals↗
PubMed1986

Degradation of proteins microinjected into HeLa cells. The role of substrate flexibility.

Increasing the flexibility of a protein enhances its susceptibility to defined proteases in vitro. To ascertain whether flexibility also affects protein stability in vivo, radioiodinated proteins with similar structures, but dissimilar flexibilities, were introduced into HeLa cells using red cell-mediated microinjection. Intracellular proteolysis was then measured as the rate of release of 125I-tyrosine into the medium. Ribonuclease A was considerably more resistant to degradation by purified proteases or in reticulocyte lysate than its flexible derivatives ribonuclease S and S-protein. In contrast, all three proteins were equally stable within HeLa cells. Like the results obtained for RNases, the rates of degradation of trypsin inhibitors, trypsin analogs, and their complexes correlated with flexibility in reticulocyte lysate. However, the intracellular half-lives of anhydrotrypsin and various proteinaceous trypsin inhibitors were not affected upon formation of enzyme-inhibitor complexes. Furthermore, trypsinogen was degraded more slowly than the structurally similar anhydrotrypsin in HeLa cells, although trypsinogen has additional segmental flexibility in its activation domain. Electrophoretic analyses revealed that trypsin-inhibitor complexes remained intact following injection into HeLa cells, and that neither free inhibitors nor anhydrotrypsin formed Triton-stable complexes with soluble cytoplasmic proteins. The observation that the components of the trypsin-inhibitor complexes were degraded simultaneously indicates that neither constituent unfolded prior to the onset of proteolysis. These studies provide evidence that RNases, trypsin, and trypsin inhibitors are degraded by an intracellular proteolytic pathway(s) which recognizes surface features of the folded proteins.

Glycoproteins↗
PubMed1986

Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis.

The amino acid sequences of ten proteins with intracellular half-lives less than 2 hours contain one or more regions rich in proline (P), glutamic acid (E), serine (S), and threonine (T). These PEST regions are generally, but not always, flanked by clusters containing several positively charged amino acids. Similar inspection of 35 proteins with intracellular half-lives between 20 and 220 hours revealed that only three contain a PEST region. On the basis of this information, it was anticipated that caseins, which contain several PEST sequences, would be rapidly degraded within eukaryotic cells. This expectation was confirmed by red blood cell-mediated microinjection of 125I-labeled caseins into HeLa cells where they exhibited half-lives of less than 2 hours. The rapid degradation of injected alpha- and beta-casein as well as the inverse correlation of PEST regions with intracellular stability indicate that the presence of these regions can result in the rapid intracellular degradation of the proteins containing them.

Amino Acid Sequence↗
PubMed1986

The degradation of guanidinated lysozyme in reticulocyte lysate.

Egg white lysozyme, treated with O-methylisourea to convert lysine to homoarginine residues, was used as a substrate for the ATP-dependent proteolytic pathway in rabbit reticulocyte lysates. Although guanidinated lysozyme was degraded by an ATP-dependent, hemin-sensitive process, ubiquitin conjugates of this protein were present at less than 5% the level of conjugates between ubiquitin and nonguanidinated lysozyme. When lysates were chromatographed on DEAE-cellulose to produce Fractions I and II of (Hershko et al. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 3107), ubiquitin-depleted Fraction II was capable of degrading nonguanidinated lysozyme, but the degradation of guanidinated lysozyme was markedly reduced or abolished. Glycerol-stabilized Fraction II, on the other hand, supported the degradation of both proteins in an ATP-dependent process stimulated by ubiquitin. The degradation of the two proteins differed, however, in that guanidinated lysozyme was more sensitive to competitive substrates, and higher concentrations of ubiquitin were required for its maximal proteolysis. Despite ubiquitin stimulation of guanidinated lysozyme degradation, only trace amounts of higher molecular weight species of guanidinated lysozyme attributable to ubiquitin conjugation were observed in ubiquitin-supplemented, glycerol-stabilized Fraction II even when special precautions were employed to preserve labile covalent bonds. These results indicate that covalent attachment of ubiquitin to the epsilon-amino group of substrate lysines is not mandatory for ATP-dependent proteolysis in rabbit reticulocyte lysates. The observation that ubiquitin stimulates proteolysis of guanidinated lysozyme, without extensive conjugation to it, suggests that ubiquitin may have essential functions for proteolysis other than direct marking of the protein substrate.

Adenosine Triphosphate↗
PubMed1986

Ubiquitin-lysozyme conjugates. Purification and susceptibility to proteolysis.

To produce ubiquitinated substrates for studies on ATP-dependent proteolysis, 125I-lysozyme was incubated in hemin-inhibited rabbit reticulocyte lysates. A portion of the labeled molecules became linked to ubiquitin in large covalent complexes. When these were partially purified and returned to uninhibited lysates containing ATP, the conjugated lysozyme molecules were degraded 10 times faster than free lysozyme. Purification of covalently modified lysozyme from hemin-inhibited lysates containing 125I-ubiquitin and 131I-lysozyme confirmed that both molecules were present in the complexes. The doubly labeled conjugates also permitted us to determine the fate of each molecule in uninhibited lysates. Besides degradation of lysozyme, there was a progressive release of intact lysozyme molecules from the complexes. This disassembly, which was the only fate of the complexes in the absence of ATP, proceeded through a series of smaller intermediates, several having molecular weights expected for ubiquitin-lysozyme conjugates, and eventually free lysozyme was regenerated. The behavior of labeled ubiquitin was similar, though not identical, to that of lysozyme. Even in lysates containing ATP ubiquitin emerged from the complex undegraded. Furthermore, ubiquitin was present in a greater number of species than was lysozyme. The demonstration that ubiquitin-lysozyme conjugates are rapidly degraded provides support for the hypothesis of Hershko, Rose, Ciechanover, and their colleagues that a key function of ubiquitin is to modify the proteolytic substrate. Further support for the hypothesis is presented in the following paper where we show that the conjugated lysozyme molecules are substrates for an ATP-dependent protease that does not degrade free lysozyme.

ATP-Dependent Proteases↗
PubMed1986

Ubiquitin-lysozyme conjugates. Identification and characterization of an ATP-dependent protease from rabbit reticulocyte lysates.

Ubiquitin-lysozyme conjugates have been used as substrates to identify an ATP-dependent protease from rabbit reticulocyte lysates. The enzyme, which has been partially purified by DEAE chromatography and glycerol gradient centrifugation, has an apparent molecular weight greater than 600,000 based on sedimentation and gel filtration. Whereas it degrades conjugated lysozyme molecules in the presence of ATP, the protease does not degrade free lysozyme molecules even upon addition of ubiquitin, lysozyme-ubiquitin conjugates, and ATP. Degradation of lysozyme conjugates is independent of added ubiquitin and occurs in fractions incapable of ubiquitin conjugation. Proteolysis is maximal at pH 7.8, inhibited by hemin, N-ethylmaleimide, or aurintricarboxylic acid, and proceeds with an apparent Arrhenius activation energy in the range of 27 +/- 5 kcal/mol. These properties are similar to those observed for the degradation of lysozyme conjugates in lysates indicating that the partially purified protease catalyzes the "second" ATP-utilizing reaction identified previously (Hough, R., and Rechsteiner, M. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 90-94; Hershko, A., Leshinsky, E., Ganoth, D., and Heller, H. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 1619-1623; Tanaka, K., Waxman, L., and Goldberg, A. L. (1983) J. Cell Biol. 96, 1580-1585).

ATP-Dependent Proteases↗
PubMed1986

Dynamic behavior of histone H1 microinjected into HeLa cells.

Histone H1 was purified from bovine thymus and radiolabeled with tritium by reductive methylation or with 125I using chloramine-T. Red blood cell-mediated microinjection was then used to introduce the labeled H1 molecules into HeLa cells synchronized in S phase. The injected H1 molecules rapidly entered HeLa nuclei, and a number of tests indicate that their association with chromatin was equivalent to that endogenous histone H1. The injected molecules copurified with HeLa cell nucleosomes, exhibited a half-life of approximately 100 h, and were hyperphosphorylated at mitosis. When injected HeLa cells were fused with mouse 3T3 fibroblasts less than 10% of the labeled H1 molecules migrated to mouse nuclei during the next 48 h. Thus, the intracellular behavior of histone H1 differs markedly from that of high mobility group proteins 1 and 2 (HMG1 and HMG2), which rapidly equilibrate between human and mouse nuclei after heterokaryon formation (Rechsteiner, M., and L. Kuehl, 1979, Cell, 16:901-908; Wu, L., M. Rechsteiner, and L. Kuehl, 1981, J. Cell Biol, 91: 488-496). Despite their slow rate of migration between nuclei, the injected H1 molecules were evenly distributed on mouse and human genomes soon after mitosis of HeLa-3T3 heterokaryons. These results suggest that although most histone H1 molecules are stably associated with interphase chromatin, they undergo extensive redistribution after mitosis.

Animals↗
PubMed1985

Relationship between the structure of chromosomal protein HMG1 and its accumulation in the cell nucleus.

When microinjected into the cytoplasm of cultured mammalian cells, non-histone chromosomal protein HMG1 migrates into the nucleus and binds to the chromatin. To define the features of the HMG1 molecule which are essential for this activity, fragments of HMG1 and chemically modified HMG1 molecules were injected into HeLa cells and the capacity of each of these probes to accumulate in the nucleus was measured by an autoradiographic technique. Fragments representing the C-terminal and central portions of HMG1 did not concentrate in the nucleus; a fragment which consisted of the N-terminal two-thirds of the molecule and which lacked the 41 consecutive aspartate and glutamate residues located near the C-terminal end of the molecule accumulated to about the same extent as intact HMG1. When the amino groups of HMG1 were chemically modified, there was a progressive loss in the ability of the protein to accumulate in the nucleus; derivatization of one-fourth of the total amino groups reduced the concentration of microinjected protein in the nucleus relative to that in the cytoplasm to one-half of the original value. In contrast, modification of one-fourth of the total carboxyl groups did not significantly affect the capacity of HMG1 to accumulate in the nucleus, although further modification resulted in decreased nuclear accumulation. Iodination of tyrosine residues was without effect and modification of the cysteine residues had only a modest effect on the ability of HMG1 to concentrate in the nucleus.

Amino Acids↗
PubMed1985

RBC-mediated microinjection of chromatin components into cultured mammalian cells.

Radiolabeled DNA fragments or nuclear proteins were encapsulated within human erythrocytes, and the erythrocytes were then fused with cultured mammalian cells using Sendai virus. Autoradiography revealed that 125I-labeled DNA fragments remained dispersed in the cytoplasm and disappeared with a half-life of 24 hours. In contrast, the nuclear proteins, HMG1, HMG2, HMG17 and histone H1, rapidly localized within HeLa nuclei and exhibited half lives greater than 80 hours. Several biochemical criteria indicate that the association of the injected nuclear proteins with chromatin faithfully mimics the behavior of their endogenous counterparts.

Animals↗
PubMed1984

What determines the degradation rate of an injected protein?

The fusion of cultured mammalian cells to red blood cells loaded with specific proteins provides a powerful system for the study of intracellular proteolysis. During the past four years the degradation rates of more than 30 proteins have been examined after their injection into HeLa cells. Results from these studies support the legitimacy of the microinjection approach. They also provide insight into the mechanism of intracellular proteolysis.

Animals↗
PubMed1984

Effects of temperature on the degradation of proteins in rabbit reticulocyte lysates and after injection into HeLa cells.

Bovine serum albumin, pyruvate kinase, hemoglobin, and the Fc fragment of IgG were labeled and introduced into HeLa cells by erythrocyte-mediated microinjection. Degradation of the injected proteins was then measured in cells cultured at temperatures between 6 degrees C and 37 degrees C. Arrhenius plots revealed a constant Ea of 27 +/- 5 kcal/mol over this temperature interval. Similarly, the apparent Ea for the degradation of long-term endogenously labeled HeLa proteins was 22-26 kcal/mol. Both local protein unfolding and proteolysis by defined enzymes, such as trypsin or papain, proceed with EaS between 5 and 15 kcal/mol. The 2-fold higher values obtained in this study indicate that protein unfolding or simple proteolysis is not rate limiting in the degradation of injected or long-lived endogenous HeLa proteins. Moreover, the relatively uniform EaS suggest that a similar biochemical event is rate limiting in the degradation of a specific protein independent of its half-life. This event may involve a reaction in the ATP-dependent proteolytic pathway from rabbit reticulocyte lysates because we observed that EaS for ATP-dependent proteolysis in this system were also 27 +/- 5 kcal/mol.

Animals↗
PubMed1983

Degradation of microinjected proteins: effects of lysosomotropic agents and inhibitors of autophagy.

HeLa cells, injected with radioiodinated proteins by fusion with RBC ghosts, were exposed to inhibitors of lysosomal proteolysis and autophagy. The degradation of injected [125I]bovine serum albumin (BSA) was unaffected by chloroquine, NH4Cl, nocodazole, colcemid, puromycin, cycloheximide, or enucleation. Although degradation of [125I]lactate dehydrogenase (LDH) and [125I]pyruvate kinase (PK) was inhibited one-third by chloroquine or ammonia, their degradation was unaffected by the other compounds. In contrast, enhanced degradation of 125I-PK resulting from depriving injected HeLa cells of amino acids and serum was inhibited 70% by colcemid and abolished by chloroquine or ammonia. Similarly, degradation of [14C]sucrose-labeled BSA-polylysine conjugates that entered HeLa cells by endocytosis was inhibited as much as 80% by chloroquine and ammonia. Sensitivity of both enhanced proteolysis and degradation of exogenous proteins to ammonia or chloroquine indicates they are effective inhibitors of lysosomal proteolysis in HeLa cells. Failure of ammonia or chloroquine to inhibit degradation of injected 125I-BSA and the modest inhibition of degradation of injected 125I-LDH or 125I-PK indicates that virtually all BSA molecules and most PK or LDH molecules are degraded by a nonlysosomal proteolytic system. Components of this degradative system are present in vast excess or are long lived, since inhibition of protein synthesis for 20 hr had no effect on the degradation of injected proteins.

Ammonium Chloride↗
PubMed1983

Genome intermixing and sister chromatid exchange in newly-formed HeLa-3T3 Hybrid cells.

Mouse and human genomes occupy distinct regions within hybrid interphase nuclei following division of HeLa-3T3 heterokaryons. With subsequent cell division the proportion of interphase cells displaying separation of human and mouse genomes decreases. Examination of several hundred hybrid colonies revealed a linear relation between the log of the fraction of interphase cells with separated genomes and the log of clone size. This indicates that there is a constant probability that separated genomes will intermingle at each mitosis. Human and mouse chromosomes can also occupy distinct sectors in metaphase spreads derived from heterokaryons. Computer analysis of the distribution of chromosomes within 548 hybrid metaphases showed that mouse and human chromosomes are randomly intermixed within several divisions and before the onset of rapid chromosome loss. Sister chromatid exchange (SCE) rates were also measured in mass populations of newly-formed HeLa-3T3 hybrid cells. For most hybrid metaphases there was not significant change in SCE rates within the human chromosome set. In a small minority of hybrid metaphases, characterized by asynchronous condensation of chromosome sets, there was a 50-fold increase in SCE. However, chromosomes are progressively lost from all hybrid cells. Thus, the two processes examined in the present studies, the distribution of human chromosomes at metaphase and SCE, are not implicated in the preferential loss of human chromosomes from HeLa-3T3 cells.

Animals↗
PubMed1983

Intracellular distribution and degradation of immunoglobulin G and immunoglobulin G fragments injected into HeLa cells.

Intact rabbit immunoglobulin G molecules (IgGs) and their papain or pepsin fragments were radio-iodinated and injected into HeLa cells. Whole IgGs, Fab2, and Fc fragments were degraded with half-lives of 60-90 h, whereas half-lives of Fab fragments were 110 h. These results indicate that proteolytic cleavage in the hinge region of the IgG molecule is not the rate-limiting step in its intracellular degradation. The hingeless human myeloma protein, Mcg, was degraded at the same rate as bulk human IgG, providing further evidence that the proteolytically susceptible hinge region is not important for intracellular degradation of IgG molecules. SDS acrylamide gel analysis of injected rabbit IgG molecules revealed that heavy and light chains were degraded at the same rate. Injected rabbit IgGs and rabbit IgG fragments were also examined on isoelectric focusing gels. Fab, Fab2, and Fc fragments were degraded without any correlation with respect to isoelectric point. Positively charged rabbit IgGs disappeared more rapidly than their negative counterparts, contrary to the trend reported for normal intracellular proteins. The isoelectric points of two mouse monoclonal antibodies were essentially unchanged after injection into HeLa cells, suggesting that the altered isoelectric profile observed for intact rabbit IgG resulted from degradation and not protein modification. The intracellular distributions of IgG fragments and intact rabbit IgG molecules were determined by autoradiography of thin sections through injected cells. Intact IgG molecules were excluded from HeLa nuclei whereas both Fab and Fc fragments readily entered them. Thus, for some proteins, entry into the nuclear compartment is determined primarily by size.

Animals↗