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.     

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.     

Intracellular protein degradation in cultured bovine lens epithelial cells

Summary Although several proteases have been identified in homogenates of cultured epithelial cells of the eye lens and in lens tissues, there is little information regarding intracellular protein degradation in intact lens cells in vitro. Cultured lens cells may be useful in the study of intracellular protein degradation in the lens, a tissue with a wide range of protein half-lives. This is of interest because alterations in protein turnover in the lens have been implicated in cataract formation. This study examines intracellular protein degradation in cultured bovine lens epithelial cells (BLEC). Cell cultures were incubated with radiolabeled leucine to label intracellular proteins. Protein degradation was measured by monitoring the release of trichloroacetic-acid-soluble radioactivity into the culture medium. The average half-life of long-lived proteins (half-life >50 h) was typically about 57 h in serum-supplemented medium. Average rates of degradation of long-lived proteins increased by up to 73% when fetal bovine serum was withdrawn from the culture medium. Serum had no effect on the degradation of short-lived proteins (half-life <10 h). Degradation of long-lived proteins in the presence and absence of serum was further studied in cultured BLEC from population doubling level (PDL) 2 to 43. Average half-life of proteins in serum-supplemented medium was 52 to 58 h and did not vary significantly as a function of PDL. Degradation rates in serum-free medium increased approximately twofold up to PDL 7, but returned by PDL 25 to original levels, which were maintained through PDL 43. This work was supported in part by grants from U. S. Department of Agriculture contract 53-3K06-5-10, Massachusetts Lions Eye Research Fund, Inc., and the Daniel and Florence Guggenheim Foundation. D. A. E. is a recipient of a National Eye Institute postdoctoral fellowship.     

Degradation of proteins microinjected into IMR-90 human diploid fibroblasts

Erythrocyte ghosts loaded with 125I-labeled proteins were fused with confluent monolayers of IMR-90 fibroblasts using polyethylene glycol. Erythrocyte-mediated microinjection of 125I-proteins did not seriously perturb the metabolism of the recipient fibroblasts as assessed by measurements of rates of protein synthesis, rates of protein degradation, or rates of cellular growth after addition of fresh serum. A mixture of cytosolic proteins was degraded after microinjection according to expected characteristics established for catabolism of endogenous cytosolic proteins. Furthermore, withdrawal of serum, insulin, fibroblast growth factor, and dexamethasone from the culture medium increased the degradative rates of microinjected cytosolic proteins, and catabolism of long-lived proteins was preferentially enhanced with little or no effect on degradation of short-lived proteins. Six specific polypeptides were degraded after microinjection with markedly different half-lives ranging from 20 to 320 h. Degradative rates of certain purified proteins (but not others) were also increased in the absence of serum, insulin, fibroblast growth factor, and dexamethasone. The results suggest that erythrocyte- mediated microinjection is a valid approach for analysis of intracellular protein degradation. However, one potential limitation is that some microinjected proteins are structurally altered by the procedures required for labeling proteins to high specific radioactivities. Of the four purified proteins examined in this regard, only ribonuclease A consistently showed unaltered enzymatic activity and unaltered susceptibility to proteolytic attack in vitro after iodination.     

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.     

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.     

Stimulation of protein synthesis in stage IV Xenopus oocytes by microinjected insulin

The effects of intracellular insulin on protein synthesis were examined in intact cells and isolated, undiluted cellular components. [35S]Methionine incorporation into protein was measured in Stage IV oocytes from Xenopus laevis maintained under paraffin oil. Radiolabel and insulin were introduced into the cytoplasm by microinjection. After a short delay (approximately 15 min), injected insulin stimulated the rate of methionine incorporation. Stimulation was dose-dependent, increasing with injected doses in the 7-50-fmol range. Neither proinsulin nor insulin-like growth factor 1 were as effective as insulin in stimulating protein synthesis; microinjected epidermal growth factor and the A and B chains of insulin were without effect. When oocyte surface membranes were removed under oil, the resulting cytoplasm-nucleus samples exhibited methionine incorporation rates that were comparable to those found in intact cells. Microinjection of insulin increased rates of methionine incorporation in cytoplasm-nucleus samples; the effects of external (prior to transfer to oil) and internal (microinjection in oil) insulin exposure were additive. Cytoplasm samples (nuclei and surface membranes removed under oil) also synthesized protein and responded to microinjected insulin. However, insulin responses were reduced relative to cells and to cytoplasm-nucleus samples. 125I-Insulin was degraded rapidly after microinjection into oocytes. Degradation occurred in both the nucleus and cytoplasm. Degradation was delayed by injecting bacitracin into the cells and delaying degradation increased the effectiveness of a low dose of injected insulin. Together, the data show that insulin can act at external, nuclear, and cytoplasmic sites to stimulate protein synthesis in Xenopus oocytes. The signaling pathway activated by internal insulin does not involve plasma membrane-generated second messengers and appears to be separate from that activated by external hormone. Finally, although microinjected insulin is degraded rapidly, it is the intact hormone rather than a degradation product that stimulates protein synthesis.     

The diversity of protein turnover and abundance under nitrogen-limited steady-state conditions in Saccharomyces cerevisiae

To establish more advanced models of molecular dynamics within cells, protein characteristics such as turnover rate and absolute instead of relative abundance have to be analyzed. We applied a proteomics strategy to analyze protein degradation and abundance in Saccharomyces cerevisiae. We used steady-state chemostat cultures to ascertain well-defined growth conditions and nitrogen limited media, which allowed us to rapidly switch from (14)N to (15)N-isotope containing media and to monitor the decay of the (14)N mono-isotope signals in time. We acquired both protein abundance information and degradation rates of 641 proteins. Half-lives of individual proteins were very diverse under nitrogen-limited steady-state conditions, ranging from less than 30 min to over 20 h. Proteins that act as single physical complexes do not always show alike half-lives. For example the chaperonin-containing TCP-1 complex showed similar intermediate half-lives ranging from 7 to 20 h. In contrast, the ribosome exhibited a wide diversity of half-lives ranging from 2.5 to over 20 h, although their cellular abundances were rather similar. The stabilities of proteins involved in the central sugar metabolism were found to be intermediary, except for the glycolytic enzymes Hxk1p and Fba1p and the TCA-cycle proteins Lsc2p and Kgd1p, which showed half-lives of over 20 h. These data stress the need for inclusion of quantitative data of protein turn-over rates in yeast systems biology.     

The effect on butyryl-CoA dehydrogenase of reagents specific for nucleophilic sulphur.

The technique of erythrocyte-mediated microinjection has been successfully adapted for use with cultured muscle cells. Erythrocytes were fused with primary chick myotube cultures with poly(ethylene glycol), and fluorescent antibodies to haemoglobin demonstrated that this protein was injected into the sarcoplasm of myotubes. The microinjection treatment did not significantly alter protein metabolism in the muscle cells as monitored by rates of synthesis and degradation of muscle proteins. 125I-labelled ribonuclease A and bovine serum albumin were degraded with the expected exponential decay kinetics after microinjection into muscle cells, and the half-life of ribonuclease A (40 h) was approximately twice that of bovine serum albumin (17 h). The degradation of ribonuclease A in the muscle cells was enhanced 1.6-fold in the absence of horse serum and chick-embryo extract, whereas the degradation of bovine serum albumin was not altered during deprivation. These results are characteristic of the breakdown of microinjected ribonuclease A and bovine serum albumin in other cell types. Therefore, our experiments indicate the erythrocyte-mediated microinjection is a valid technique to study protein degradation in primary chick muscle cultures.     

A quantitative spatial proteomics analysis of proteome turnover in human cells

Measuring the properties of endogenous cell proteins, such as expression level, subcellular localization, and turnover rates, on a whole proteome level remains a major challenge in the postgenome era. Quantitative methods for measuring mRNA expression do not reliably predict corresponding protein levels and provide little or no information on other protein properties. Here we describe a combined pulse-labeling, spatial proteomics and data analysis strategy to characterize the expression, localization, synthesis, degradation, and turnover rates of endogenously expressed, untagged human proteins in different subcellular compartments. Using quantitative mass spectrometry and stable isotope labeling with amino acids in cell culture, a total of 80,098 peptides from 8,041 HeLa proteins were quantified, and their spatial distribution between the cytoplasm, nucleus and nucleolus determined and visualized using specialized software tools developed in PepTracker. Using information from ion intensities and rates of change in isotope ratios, protein abundance levels and protein synthesis, degradation and turnover rates were calculated for the whole cell and for the respective cytoplasmic, nuclear, and nucleolar compartments. Expression levels of endogenous HeLa proteins varied by up to seven orders of magnitude. The average turnover rate for HeLa proteins was ~20 h. Turnover rate did not correlate with either molecular weight or net charge, but did correlate with abundance, with highly abundant proteins showing longer than average half-lives. Fast turnover proteins had overall a higher frequency of PEST motifs than slow turnover proteins but no general correlation was observed between amino or carboxyl terminal amino acid identities and turnover rates. A subset of proteins was identified that exist in pools with different turnover rates depending on their subcellular localization. This strongly correlated with subunits of large, multiprotein complexes, suggesting a general mechanism whereby their assembly is controlled in a different subcellular location to their main site of function.     

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.     

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.     

Protein degradation in skin fibroblasts from patients with Duchenne muscular dystrophy.

The rates of degradation of [3H]leucine-labelled proteins have been measured in cultures of skin fibroblasts obtained from normal controls (five subjects) and patients with Duchenne muscular dystrophy (six subjects). Cultures were incubated with [3H]leucine (10 microCi/ml) for 60 min to label "short-lived" proteins, and with [3H]leucine (5 microCi/ml) for 60 h to label "long-lived" proteins. Optimal wash procedures were devised for removal of [3H]leucine from the extracellular space and from cell pools before beginning degradation measurements. Re-utilization of [3H]leucine released from degraded labelled proteins was prevented by supplementing the medium with 4mM-leucine. Rates of degradation did not depend on the growth state of the cells or on cell age over the range used (passages eight-20). Degradation of long-lived proteins was approximately linear over a 24h period, at a rate of 1.0% per h. 30% of short-lived protein was degraded within 6h. No differences were observed between protein degradation in normal fibroblasts and in those from patients with Duchenne muscular dystrophy.     

Protein synthesis and degradation in non-cultured and in-vitro cultured rabbit blastocysts

In 'pulse-chase' experiments synthesis and half-lives of leucine-labelled proteins were determined in rabbit blastocysts. Embryos were either non-cultured controls or were cultured for 24 h or 48 h in Ham's F-10 medium supplemented with homologous serum or uterine flushings. In control blastocysts protein synthesis increased by a factor of 10 between Day 4 and Day 5. Half-lives of newly synthesized proteins were 32 h in Day-4 and 99 h in Day-5 control blastocysts. In-vitro culture of Day-4 blastocysts led to dramatically shortened half-lives, amounting to 6-10 h. Blastocysts developing in uterine flushing-supplemented media differed significantly from those cultured in serum-supplemented media. Protein synthesis was enhanced and protein degradation was normal for culture times up to 24 h. These results demonstrate (1) that half-lives of proteins in rabbit blastocysts increase with advancing embryonic age, and (2) that a characteristic feature of the altered metabolism of cultured blastocysts is a dramatically accelerated protein degradation, which (3) can be prevented for some time by supplementation of the culture medium with uterine secretions.     

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.     

Microinjection of endogenous and exogenous proteins into primary cultures of rat hepatocytes and the degradation of the injected proteins

1. A method to microinject proteins into cells through packaging proteins to erythrocyte ghosts (erythrocyte-mediated microinjection) was modified partially in order to apply the method to primary cultures of rat hepatocytes. 2. Degradation of the microinjected proteins was examined employing the improved method. The mean half-life of the injected endogenous liver protein was 20 hr. The data suggested that the injected proteins are degraded through both lysosomal and non-lysosomal proteolytic pathways probably depending on their structure. 3. The present method to microinject exogenous proteins into primary cultures of rat hepatocytes can be employed usefully for the investigations of protein metabolism in liver.     

Purification and characterization of hepatic glutathione S-transferases of rhesus monkeys. A family of enzymes similar to the human hepatic glutathione S-transferases.

The uptake and degradation of radiolabelled rabbit muscle fructose-bisphosphate aldolase (EC 4.1.2.13) was studied in HeLa cells microinjected by the erythrocyte ghost fusion system. Labelled aldolase was progressively modified by treatment with GSSG or N-ethylmaleimide (NEM) before microinjection to determine whether these agents, which inactivate and destabilize the enzyme in vitro, affect the half-life of the enzyme in vivo. Increasing exposure of aldolase to GSSG or NEM before microinjection increased the extent of aldolase transfer into the HeLa cells and decreased the proportion of the protein that could be extracted from the cells after water lysis. Some degradation of the GSSG- and NEM-inactivated aldolases was observed in the ghosts before microinjection; thus a family of radiolabelled proteins was microinjected in these experiments. In spite of the above differences, the 40 kDa subunit of each aldolase form was degraded with a half-life of 30 h in the HeLa cells. In contrast, the progressively modified forms of aldolase were increasingly susceptible to proteolytic action in vitro by chymotrypsin or by cathepsin B and in ghosts. These studies indicate that the rate of aldolase degradation in cells is not determined by attack by cellular proteinases that recognize vulnerable protein substrates; the results are most easily explained by a random autophagic process involving the lysosomal system.     

Regulation of protein synthesis and degradation in L8 myotubes. Effects of serum, insulin and insulin-like growth factors.

We have examined the regulation of protein turnover in rat skeletal myotubes from the L8 cell line. We measured protein synthesis by the rates of incorporation of radiolabelled tyrosine into protein in the presence of a flooding dose of non-radioactive tyrosine. We monitored degradation of proteins labelled with radioactive tyrosine by the release of acid-soluble radioactivity into medium containing excess nonradioactive tyrosine. Extracellular tyrosine pools and intracellular tyrosyl-tRNA equilibrate rapidly during measurements of protein synthesis, and very little reutilization of the radiolabelled tyrosine occurs during degradation measurements. Measured rates of protein synthesis and degradation are constant for several hours, and changes in myotube protein content can be accurately predicted by the measured rates of protein synthesis and degradation. Most of the myotube proteins labelled with radioactive tyrosine for 2 days are degraded, with half-lives (t1/2) of approx. 50 h. A small proportion (less than 2.5%) of the radiolabelled proteins are degraded more rapidly (t1/2 less than 10 h), and, at most, a small proportion (less than 15%) are degraded more slowly (t1/2 greater than 50 h). A variety of agents commonly added to primary muscle cell cultures or to myoblast cell lines (18% Medium 199, 1% chick-embryo extract, antibiotics and antifungal agents) had no effect on rates of protein synthesis or degradation. Horse serum, fetal bovine serum and insulin stimulate protein synthesis and inhibit the degradation of long-lived proteins without affecting the degradation of short-lived proteins. Insulin-like growth factors (IGF)-1 and -2 also stimulate protein synthesis and inhibit protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation are of similar magnitude (a maximum of approx. 2-fold) and display similar sensitivities to a particular anabolic agent. Insulin stimulates protein synthesis and inhibits protein degradation only at supraphysiological doses, whereas IGF-1 and -2 are effective at physiological concentrations. These and other findings suggest that IGFs may be important regulators of skeletal muscle growth during the fetal and early neonatal periods.     

Evidence for the autophagy of microinjected proteins in HeLA cells

Rhodamine-conjugated proteins were microinjected into living HeLa cells. Fluorescence microscopy was then employed to study their segregation from the cytoplasm into lysosomes. Results obtained in this way were verified when the corresponding unconjugated proteins were localized by autoradiographic, histological, and antibody-staining methods after their microinjection. Most injected proteins were segregated into cytoplasmic granular structures during their removal from cells. As evidence that these were autophagic vacuoles, they were found to contain no detectable acid phosphatase activity upon formation, after which they moved to the juxtanuclear position of lysosomes and appeared to fuse with them. The segregation of microinjected proteins exhibited a high degree of selectivity. The half- times of placement of individual exogenous proteins into cytoplasmic granules varied from 3 h to nearly 3 days, and one protein, hemoglobin, was never observed to enter them. Furthermore, endogenous HeLa proteins in a size fraction near 200,000 daltons were segregated much more rapidly than those in a fraction near 40,000 daltons. In these studies, rapid protein segregation appeared to take place by a mechanism of exclusion of the injected protein from numerous cytoplasmic domains.     

Microinjection studies of protein transit across the nuclear envelope of human cells

Proteins of various molecular weights were conjugated with rhodamine and microinjected into the cytoplasm or nucleus of HeLa cells. The injected proteins were then localized within the cells at various times thereafter with fluorescence microscopy. Proteins below approx. 60 kD rapidly crossed the HeLa nuclear envelope. Some larger proteins also were able to pass into or out of the nucleus, while others were unable to do so, indicating the selective permeability of the HeLa nuclear envelope to large proteins. The nuclear protein HMG17 accumulated within the nucleus shortly after cytoplasmic microinjection.