Degradation of structurally characterized proteins injected into HeLa cells. Basic measurements

Thirty-five proteins of known x-ray structure were labeled by chloramine-T radioiodination or by reaction with 125I-Bolton-Hunter reagent and introduced into HeLa cells using red cell-mediated microinjection. Degradation rates of the injected proteins were then determined over the next 50 h by measuring the release of soluble isotope to the culture medium. Control experiments demonstrated that the measured rates were not compromised by proteolysis within RBCs, the presence of unfused RBCs, or degradation of protein released from RBCs to the medium. Degradation of some injected proteins was faster during the first 12 h after fusion than at later times, apparently a response of HeLa cells to trypsinization. However, all proteins exhibited first-order degradation rates between 24 and 48 h post injection. Except for seven proteins, stabilities measured during this interval were unaffected by the labeling procedure. Reductive methylation was used to choose among the seven discordant values, and half-lives for the 35 proteins ranged from 16 h for lysozyme to 214 h for yeast alcohol dehydrogenase. Since half-lives for six of the injected proteins closely match values obtained by in vivo measurements, we consider our estimates of the metabolic stabilities of the injected proteins to be generally accurate. Therefore, the half-lives obtained by microinjection should prove useful in the search for relationships between protein structure and intracellular stability.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in CHO cells

CHO cells can incorporate thialysine and selenalysine in their proteins in substitution of lysine. Data are reported in the present paper showing that proteins containing either thialysine or selenalysine are unstable and quite rapidly degraded. The degradation rate is strictly related to the extent of protein lysine substitution. At similar extent of substitution, selenalysine-containing proteins are more unstable that thialysine-containing ones.     

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.     

Degradation of structurally characterized proteins injected into HeLa cells. Tests of hypotheses

We have compared sequence and structural features of 35 proteins to their metabolic stabilities in HeLa cells. No relationship was observed between the half-life of an injected protein and its subunit molecular weight, isoelectric point, hydrophobicity, thermostability, surface charge density, or N-terminal residue. Other properties, including susceptibility to oxidation, specific combinations of amino acids, secondary structure composition, and solvent exposed residues, also failed to correlate with protein stability. Although a weak inverse correlation was obtained when stability was compared to asparagine and glutamine content, we conclude that the degradation of an injected protein is unlikely to be related to any single structural parameter. Rather, we hypothesize that it results from an interplay between subcellular location and still poorly defined surface features of the injected proteins.     

Degradation of abnormal proteins in growing yeast

Abstract Degradation of abnormal proteins in Saccharomyces cerevisiae , synthesized in an ethanol medium in the presence of 0.1 mM canavanine, proceeded more rapidly than that of the normal ones, the degradation constant of the 'long-lived' fraction being most affected. Higher concentrations of the analogue had an adverse effect on protein degradation during subsequent growth. Degradation of normal and abnormal proteins was suppressed by glucose but was increased substantially in cell-free extracts. This suggests that their internalization into the digestive organelle may be an important step in protein catabolism in yeast.     

Methylation of ribosomal proteins in HeLa cells.

Methylated amino acids from both 40 and 60S subunit proteins of HeLa cytoplasmic ribosome were analyzed. It was observed that methylation of ribosomal proteins occurs in both subunits with N^G,N^G-dimethylarginine as the major methylated amino acid. The presence of N^G,N^G-dimethylarginine has been identified by high-voltage paper electrophoresis, by paper chromatography, and by amino acid analysis. In addition, both ribosomal subunits contain methylated lysines with ?-N-trimethyllysine being the predominant one, followed by ?-N-dimethyllysine. Little, if any ?-N-monomethyllysine was detected in either subunit. The cytoplasmic 60S ribosomal subunit contains much more ?-N-trimethyllysine compared to the 40S ribosomal subunit. The possible biological significance of methylation was discussed.     

Degradation of abnormal proteins in peptidase-deficient mutants of Salmonella typhimurium.

The degradation of abnormal proteins produced as a result of incorporation of the arginine analog L-canavanine or generated by exposure to puromycin was studied in wild-type and multiply peptidase-deficient strains of Salmonella typhimurium. Both types of abnormal protein were rapidly degraded during growth of Pep+ strains of this organism. Peptidase--deficient mutants (lacking peptidases N, A, B, and D) could also degrade these abnormal proteins, although the rate of production of trichloroacetic acid-soluble degradation products was slower in the mutant strain than in a strain carrying a normal complement of peptidases. Analysis of these trichloroacetic acid-soluble degradation products of ion-exchange chromatography showed that free amino acid was the major breakdown product produced by the wild-type strain. The acid-soluble degradation product produced by the mutant strain, however, was a complex mixture that contained a variety of small peptides as well as free amino acids. These results indicate that the same group of peptidases shown previously to function in the degradation of exogenously supplied peptides and in protein turnover during carbon starvation also lie on the pathway by which abnormal proteins are degraded.     

Degradation of thialysine- or selenalysine-containing abnormal proteins in E. coli

Thialysine and selenalysine can be utilized for protein synthesis by lysine-requiring E. coli cells even in the absence of lysine. Protein synthesis has been determined as labeled leucine incorporation into acid-insoluble material, as increase of cell proteins and as protein-lysine substitution by the analog. Either analog can be incorporated into proteins, in the absence of lysine, for a limited time interval after which cells stop to duplicate. Proteins synthesized during this period contain most of their lysine residues substituted by the analog. Moreover, it has been shown that the analog-containing proteins are unstable and rapidly degraded. Their instability would account for the inability of lysine-requiring E. coli cells to utilize the analog as growth factor.     

Degradation of structurally characterized proteins injected into HeLa cells. Comparison with their stability in rabbit reticulocyte lysate

We recently reported (Rogers, S. W., and Rechsteiner, M. (1988) J. Biol. Chem. 263, 19833-19842) the metabolic stabilities of 35 structurally characterized proteins following their injection into HeLa cells. In this study 22 proteins from that set were radioiodinated, and their susceptibilities to proteolysis were measured in reticulocyte lysate. Degradation rates varied from less than 1% per h to almost 25% per h. ATP stimulated the degradation of 21 of the proteins with nucleotide enhancement typically in the range 2- to 3-fold. When structural features of the 22 proteins were compared with their degradation rates in lysate, no correlation was found with respect to charge, size, thermal stability, or N-terminal acylation. Furthermore, relative rates of proteolysis in lysate correlated only marginally with the metabolic stabilities of the 22 proteins as measured 24-48 h after injection into HeLa cells. Degradation rates for the 22 proteins in lysate did, however, show a strong correlation with their rates of turnover immediately after injection into the human cells. Since the enhanced proteolysis observed for many proteins soon after injection is thought to reflect disruption of HeLa cytoskeletal assemblies, this correlation provides further evidence that diffusibility or location can affect intracellular protein stability.     

Ribosomal proteins of HeLa cells

Ribosomal proteins from HeLa cells were analyzed by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt-Wittmann) and dodecylsulfate polyacrylamide gel electrophoresis (Laemmli). 35 proteins are associated with the small ribosomal subunit and 47 proteins with the large ribosomal subunit. The HeLa ribosomal proteins S6, S32, L40b,c, L41 and L42 are phosphorylated in vivo and in vitro. Minor differences between HeLa and rat liver ribosomal proteins were revealed by their direct coelectrophoresis.     

Non-ribosomal nucleolar proteins in HeLa cells

The study of nucleolar macromolecular components has previously emphasized ribosomal precursor and mature RNA, or ribosomal structural proteins. In this work, we have stressed the purification of nucleoli, and have studied their protein composition. The results indicate that there exists in highly purified nucleoli of HeLa cells, a class of high molecular weight polypeptides which have been identified by means of size, kinetics of turnover, and metabolic behavior to be non-ribosomal nucleolar-specific proteins. The possibility that these proteins play a part in the assembly of mature ribosomes is discussed.     

Identification of nuclear cap specific proteins in HeLa cells.

Two polypeptides of apparent molecular mass of 20 and 115 kilodaltons in nuclear fractions from HeLa cells were shown to recognize and be crosslinked to the cap structure of eukaryotic mRNAs in a cap-dependent fashion. Crosslinking of the 20 and 115 kDa polypeptides was sensitive to inhibition by low concentrations of the cap analogue m7GDP and resistant to inhibition by high KCl concentrations. In addition, crosslinking of these polypeptides to the cap structure occurred in nuclear extracts prepared from poliovirus-infected cells, under conditions where cytoplasmic cap binding proteins were incapable of interacting with the mRNA cap structure. The possible function of nuclear cap binding proteins is discussed.     

Degradation of structurally characterized proteins injected into HeLa cells. Effects of intracellular location and the involvement of lysosomes

Thirty-five proteins of known x-ray structure were radioiodinated and injected into HeLa cells. The cells were then cultured in the presence or absence of the lysosomotropic agents, ammonium chloride and chloroquine. These compounds did not inhibit the degradation of an injected protein unless its half-life was greater than 45 h. Among the more stable proteins the extent of inhibition was proportional to their half-lives. These results indicate that all injected proteins are transferred to lysosomes at comparable rates such that the fraction of a specific protein degraded in lysosomes depends upon its rate of degradation in the cytosol. That is, basal autophagy is nonselective in HeLa cells. The intracellular location of each injected protein was measured by homogenization of injected cells in sucrose and differential sedimentation or by extraction in buffers containing Triton X-100. Solubilities of the injected proteins ranged from 6 to 89%, and stabilities of 10 proteins, originally extracellular in function, were inversely proportional to their solubility. These results illustrate the potential importance of subcellular location on protein stability in the cytosol.     

Affinity purification of histidine-tagged proteins transiently produced in HeLa cells.

In order to produce eukaryotic proteins in a functional state, it is often necessary to use eukaryotic instead of prokaryotic expression systems. We have designed vectors which can be employed to express either N- or C-terminally histidine-tagged proteins in transiently transfected eukaryotic cells. The histidine tag allows the rapid enrichment of these proteins by metal chelate affinity chromatography in a native and functional state. Yields of up to 5 micrograms protein/5 x 10(7) cells were achieved.     

Initial characterization of heat-induced excess nuclear proteins in HeLa cells.

Exposure of mammalian cells to hyperthermia is known to cause protein aggregation in the nucleus. The presence of such aggregates has been detected as the relative increase in the protein mass that is associated with nuclei isolated from heated cells. We have characterized these excess nuclear proteins from the nuclei of heated HeLa cells by two-dimensional gel electrophoresis. The abundance of cytoskeletal elements which co-purify with the nuclei did not increase with exposure to hyperthermia, indicating that these proteins are not part of the excess nuclear proteins. In contrast, several specific polypeptides become newly bound or increase in abundance in nuclei isolated from heated cells. Members of the hsp 70 family were identified as a major component of the excess nuclear proteins. Among the other excess nuclear proteins we identified ten that had apparent molecular weights of 130, 95, 75, 58, 53, 48, 46, 37, 28, and 26 kilodaltons. Since hsp 70 is mainly cytoplasmic in non-heated cells, its association with nuclei in heated cells indicates that one mechanism accounting for the heat-induced excess nuclear proteins is the movement of cytoplasmic proteins to the nucleus. We also obtained evidence that increased binding of nuclear proteins is another mechanism for this effect. No overall increase or decrease in the phosphorylation of nuclear proteins was found to be associated with such altered binding or movement from the cytoplasm to the nucleus.     

ADP-ribosylation of nucleolar proteins in HeLa tumor cells

ADP-ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with ³²P-NAD; then the nucleolar proteins were analyzed by 1-dimensional and 2-dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE-cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP-ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP-ribosylation.     

Degradation of (ADP-ribose)n in permeabilized HeLa cells

Determination of (ADP-ribose)n degradation rates in permeabilized HeLa cells, measured as loss of acid-insoluble radioactivity from permeabilized cells previously incubated with [3H]NAD+, showed bi-phasic kinetics. The majority of label was lost within 20 min at pH 6.0 and 37 degrees C and has a half-life of about 12-15 min. The minor ADP-ribose component was either removed very slowly, or appeared to be stable over an 80 min incubation. The degradation rate of the labile component was directly proportional to the initial amount of ADP-ribose present, and was independent of the experimental conditions used to create various elevated levels. The degradation rates of monomeric and oligo/polymeric ADP-ribose were the same, surprising since different enzymes catalyse the respective reactions. The more stable ADP-ribose component could be more inaccessible to degrading enzymes and/or might represent a different linkage to protein, the cleavage of which is slow.     

Degradation of proteins microinjected into cultured mammalian cells.

Iodinated proteins were degraded after injection into HeLa cells at first-order rates with half-lives varying from three hours for the trout monhistone chromosomal protein, HMG-T, -to 60 hours for whale myoglobin. Fluoresceinated-bovine serum albumin (fl-BSA) was degraded almost twice as fast as unmodified BSA. The rate of degradation of 125I-BSA was very similar in eight cell lines of mouse, human, monkey and rat origin. Microinjected proteins were analyzed on SDS-acrylamide gels after injection, and for BSA and immunoglobin G, all remaining intracellular 125I migrated at the molecular weight of the injected proteins. By contrasting, more than 80% of the extracellular 125I chromatographed as iodotyrosine. With the exception of fl-BSA, which exhibited perinuclear accumulation in approximately one-half of the injected cells, autoradiography showed that throughout the period of study the injected proteins remained dispersed in the cytoplasm.