Protein Turnover and Lysosome Function

Protein Turnover and Lysosome Function comprises the proceedings of a symposium under the same title held at the State University of New York at Buffalo on August 21-26, 1977. The book discusses mechanisms of protein turnover, as well as the identification and characterization of intracellular proteases. The text also describes the internalization of macromolecules into the intracellular digestive system; the types of specificity entailed; and the fate of the membrane material involved in the vacuolization process. Biochemists, pathologists, cell biologists, molecular biologists, and physiologists will find the book invaluable.

• Language: English
• Publisher: Academic Press
• Release date: Jun 28, 2014
• ISBN: 9781483220192

Book preview

York

INTRODUCTION

Harold L. Segal,     Division of Cell and Molecular Biology, State University of New York, Amherst, New York

Darrell Doyle,     Department of Molecular Biology, Roswell Park Memorial Institute, Buffalo, New York

A number of aspects of the mechanism and regulation of protein turnover, many of which are dealt with in the succeeding papers in this volume, may be illustrated by reference to the translocations and transformations illustrated in Fig. 1.

FIGURE 1 A schematic outline of the pathways of protein degradation.

In this schematic diagram are traced the possible pathways of degradation of an intact protein to its constituent amino acids. Two alternatives exist at the beginning. One entails uptake of the protein into the digestive compartment prior to the first degradative step (steps 2 and 3), and the other the inverse (steps 2’ and 3’).

Whichever precedes, uptake or proteolysis, a prior conversion of the protein to a susceptible form may be involved (step 1). The dashed arrow is meant to imply that this reaction may be reversible or irreversible. The latter type may entail denaturation, or a marking for destruction by a covalently added or removed tag, such as a phosphate, glycosyl, acetyl, or methyl group. Interesting recent examples have come from the work of Ashwell and Morell (1), in which it was shown that removal of sialic acid from certain circulating glycoproteins greatly increased their rate of uptake and ultimate digestion by the liver. Subsequent removal of some of the underlying galactose residues eliminated the rapid uptake. One may surmise that desialyation may be a physiological process and the rate-limiting step in the turnover of these serum glycoproteins.

Certain aspects of the degradation of these desialyated proteins by isolated hepatocytes are discussed by Berg (this volume). Other aspects of protein turnover in isolated hepatocytes are discussed by Wagle et al. (this volume). The use of isolated hepatocytes as a system for the study of protein turnover more amenable to experimental manipulation than the in situ rat liver is discussed by Seglen (this volume).

Reversible formation of a susceptible form of a protein may also imply that one of the equilibrium conformational states in which proteins are known to exist is the one susceptible to uptake or proteolytic attack, or may represent association or disassociation of a cofactor or other ligand. Examples of such cases with possible physiological relevance are the stabilization of phosphofructokinase by a peptide factor isolated from liver and the stabilization of arginase by Mn²+ or amino acids, discussed by Segal et al. (this volume). However, with other enzymes including alanine aminotransferase and tyrosine aminotransferase, coenzyme dissociation is not a significant determinant of the in vivo rate, according to the findings of Kenney and Lee (this volume). Rather, in the view of these workers, the cofactor dissociation rate and the intracellular stability may reflect structural features of the proteins that determine both properties.

Uptake of intact proteins (step 2) may represent a site of selectivity in turnover rates, whether uptake of extracellular or intracellular proteins is involved. Such selectivity implies a recognition capability, either broad or specific. Specific receptors on the external surface of the plasma membrane have been identified in the laboratories of Stahl (this volume) and Sly (this volume) for certain lysosomal enzymes that find their way into the circulation, and by Brown and Goldstein (this volume) for serum lipoproteins. Similarly, Michl and Silverstein (this volume) discuss the specific inhibition of FC and C3 plasma membrane receptor-mediated phagocytosis by 2-deoxy-D-glucose.

A broader specificity based on lipophilic affinities has been suggested by work of Dean (2, this volume), Bohley (this volume), and Segal et al. (this volume). In addition, Segal et al. have recently shown a general regulation of pinocytic uptake, using the rat yolk sac as a model system. The development and further properties of the rat yolk sac as a system for studying protein degradation are discussed by Lloyd and Williams (this volume).

The fates of both the interiorized protein and the unit of membrane involved in the uptake and recognition of extracellular proteins are discussed by several workers in this volume, including Brown et al. Doyle, and Kosmakos. Similarly, Atkinson (this volume) discusses the overall pathway of synthesis and degradation of membrane glycoproteins and other proteins using vesicular stomatitis virus-infected HeLa cells as a model system.

A major problem in understanding the uptake step is the disposition of the membrane unit of the endocytosed or autophagic vacuole after delivery of the contents to the lysosome. The rates of commitment of membrane material for the formation of such vacuoles greatly exceed the rates of membrane renewal, suggesting that recycling must occur. The work of Doyle (this volume), Reutter (this volume), and Tulkens (this volume) on membrane and vacuole formation is relevant to this aspect of the process.

Additional evidence implicating the lysosomes as the intracellular site for the degradation of exogenous proteins comes from the studies of Poole and Warburton (this volume), who used chloroquine to inhibit lysosomal function of macrophages and fibroblasts. In a similar approach utilizing chloroquine and other lysosomotropic drugs, Strauss and Flickinger (this volume) present evidence implicating a role for lysosomal degradation in corpus luteum function.

Strong evidence implicating the lysosome as a site for the degradation of at least some of the endogenous cell protein, particularly during nutritional step-down conditions, comes from the studies of Mortimore (this volume) using the perfused liver system. Data suggesting different mechanisms for basal degradation, perhaps not involving lysosomes, versus the lysosomal-type degradation occurring in nutritional step-down conditions are given by Poole and Warburton (this volume) and by Dice and Walker (this volume). The importance of protein degradation in the regulation of liver growth is discussed by Scornik (this volume), who proposes that the effect of growth on degradation is not peculiar to this physiological state but is a general one.

Selectivity in susceptibility of intact proteins to lysosomal proteases (step 3) has also been shown by the work of Bohley et al. (3, this volume), Dean (4, this volume), and Segal et al. (this volume). Furthermore, the proteolytic susceptibility among a population of proteins was in accord with their in vivo turnover rates. The nature and characterization of the lysosomal enzymes capable of degrading a vareity of cellular macromolecules is discussed by Barrett (this volume), Touster (this volume), Turk (this volume), and Kalnitsky (this volume), while Swank (this volume) describes experiments concerned with understanding the biogenesis of lysosomal enzymes themselves, using a genetic system as a tool to complement biochemical analysis. Schneider and Cornell (this volume) present evidence for the existence of an ATP-driven proton pump in lysosomes, which could function to provide the acid pH that is optimum for most lysosomal hydrolases.

However, a conceptual difficulty in attributing selectivity to the intralysosomal proteolytic step is that it implies a reversibility of previous steps, notably the uptake step. Some observations on the disposition of macromolecular markers subsequent to their uptake by the liver are consistent with a release from the lysosomal compartment, but the data are not yet sufficient to exclude other interpretations (Segal et al., this volume).

Limited extralysosomal proteolysis has been shown to occur by Katunuma’s laboratory for certain apopyroxidal proteins (5), and by Horecker with fructose bisphosphatase (this volume). In addition, Goll (this volume), Waxman and Krebs (this volume), Stracher (this volume), Bird (this volume), and Bhan and Hatcher (this volume) have studied nonlysosomal proteases of muscle which may be involved in the turnover of the myofibril in this tissue. The turnover rates of the myofibril proteins myosin and actin vary as a function of the physiological state of the cells, as reported by Nihei (this volume). Actually, total protein turnover in muscle varies markedly in a variety of dietary and hormonal growth conditions, as shown by the studies of Millward (this volume). Furthermore, the changes in muscle protein breakdown in anabolic or catabolic states are often opposite to those expected, suggesting that synthesis of muscle proteins also must undergo marked changes during adaptation. In this context, Lebherz et al. (this volume) studied the differential regulation of glycolytic enzymes in red and white muscle, which is primarily at the level of synthesis.

It also appears from work done in the laboratories of Goldberg (this volume) and of Hershko (this volume) that a nonlysosomal protease is involved in the selective degradation of abnormal proteins of the reticulocyte. Little is known about the normal degradation of the major erythrocyte proteins, but the turnover characteristics of the different globin chains are discussed by Garrick et al. (this volume).

Using another system, less complex than the animal cell, Holzer (this volume) discusses the control of proteolysis in yeast. In this system five different proteinases have been localized to yeast vacuoles. The activity of these proteinases may be controlled by one or more of three classes of inhibitor in the cytosol, demonstrating that even in a simple eukaryote the regulation of proteolysis is complex, possibly involving cascade-type mechanisms.

Although the first proteolytic step, wherever it occurs, would be expected to be irreversible and to commit the protein irrevocably to total hydrolysis, it need not necessarily lead to loss of function. It has been shown, for example, by Horecker (this volume) that an active species remains after proteolytic modification of fructose bisphosphatase, and the same is true for ribonuclease (6), ATP citrate lyase (7), and mung bean nuclease (8). Evidence obtained in the laboratory of Segal (this volume) suggests it may also be true for invertase subsequent to its ingestion by liver cells.

The development of new techniques and concepts such as highly resolving two-dimensional methods of separating proteins used by Larrabee (this volume), radioactive labeling of proteins to high specific activity by the method of Pine and Schimke (this volume), a better understanding of compartmentation and precursor pools as discussed by Khairallah (this volume), and the use of lipid vesicles to introduce specific macromolecules into animal cells as discussed by Papahadjopoulos et al. (this volume) promises continued advances toward our eventual understanding of the mechanism(s) of protein turnover.

The papers collected in this volume represent a major proportion of the current knowledge and direction of research in the fields that its subject addresses and should serve usefully to provide the general background from which further advances in this rapidly developing area of investigation will emerge.

REFERENCES

1. Ashwell, G., Morell, A. G. Adv. Enzymol. 1971; 41(99)

2. Dean, R. T. Biochem. Biophys. Res. Commun. 1975; 67(604)

3. Bohley, P., Miehe, C., Miehe, M., Ansorge, S., Kirschke, H., Langner, J., Wiederanders, B. Reta Biol. Med. Germ. 1971; 28(323)

4. Dean, R. T. Eur. J. Biochem. 1975; 58(9)

5. Katunuma, N. Rev. Physiol. Biochem. Pharmaco. 1975; 78(23)

6. Richards, F. M., Vithayathil, P. J. J. Biol. Chem. 1959; 234(1459)

7. Singh, M., Richards, E. G., Mukherjee, A., Srere, P. A. J. Biol. Chem. 1976; 251(5242)

8. Kowalski, D., Koreker, W. D., Laskowski, M. Biochemistry. 1976; 15(4457)

MECHANISMS AND REGULATION OF PROTEIN TURNOVER

Outline

Chapter 2: FACTORS INVOLVED IN THE REGULATION OF PROTEIN TURNOVER

Chapter 3: LYSOSOMAL MECHANISMS OF PROTEIN DEGRADATION

Chapter 4: SOME ASPECTS OF THE INTRACELLULAR BREAKDOWN OF EXOGENOUS AND ENDOGENOUS PROTEINS

Chapter 5: EVIDENCE FOR A PROTON PUMP IN RAT LIVER LYSOSOMES

Chapter 6: LYSOSOMAL PROCESSING OF INTRACELLULAR PROTEIN IN RAT LIVER AND ITS GENERAL REGULATION BY AMINO ACIDS AND INSULIN

Chapter 7: IN VIVO DETERMINATION OF RATES OF PROTEIN DEGRADATION IN LIVERS OF MEAL-FED RATS: IMPLICATIONS OF AMINO ACID COMPARTMENTATION

Chapter 8: THE GENERAL CHARACTERISTICS OF INTRACELLULAR PROTEIN DEGRADATION IN DIABETES AND STARVATION

Chapter 9: THE SIGNIFICANCE OF PROTEIN DEGRADATION IN THE REGULATION OF LIVER GROWTH

Chapter 10: TURNOVER OF THE MAJOR PROTEINS OF RAT ERYTHROCYTES

Chapter 11: MODE OF DEGRADATION OF ABNORMAL GLOBIN CHAINS IN RABBIT RETICULOCYTES

Chapter 12: SELECTIVE DEGRADATION OF ABNORMAL PROTEINS IN ANIMAL AND BACTERIAL CELLS

Chapter 13: MECHANISMS IN INTRACELLULAR TURNOVER OF STABLE AND LABILE ENZYMES

Chapter 14: STUDIES ON PROTEIN DEGRADATION IN ISOLATED HEPATOCYTES AND KUPFFER CELLS

Chapter 15: THE CHEMISTRY AND TURNOVER OF LYSOSOMAL ENZYMES

Chapter 16: GENETICS OF LYSOSOMAL FUNCTIONS

Chapter 17: MEASUREMENT OF PROTEIN SYNTHESIS AND TURNOVER IN ANIMAL CELLS WITH TRITIATED WATER

Chapter 18: TURNOVER OF SOLUBLE PROTEIN IN GROWING CULTURES OF ESCHERICHIA COLI

FACTORS INVOLVED IN THE REGULATION OF PROTEIN TURNOVER¹

Harold L. Segal, John A. Brown, George A. Dunaway, Jr.², James R. Winkler, Herman M. Madnick and David M. Rothstein,     Division of Cell and Molecular Biology, State University of New York, Amherst, New York

Three possible sites for the rate-limiting, regulatable step in protein degradation within cells are discussed, viz., transformation of the protein into a susceptible form, its uptake into the lysosomal digestive system, and proteolysis. Evidence relevant to all three processes is presented. The susceptibility to lysosomal proteolysis of both phosphofructokinase and arginase of liver was reduced in the presence of specific ligands; a peptide factor from liver in the former case and Mn ²+ in the latter. In both instances a relationship also exists in vivo between turnover of these enzymes and levels of the ligands. Uptake of macromolecular markers in the rat yolk sac system was inhibited by glucagon at levels around 10−8 M. In addition a correlation was shown in a protein pool between turnover in vivo and lipophilicity, as measured by affinity for hydrophobic columns, suggesting that relative affinity for membranes may be a factor in determining uptake and hence turnover rates. A correlation also existed in the protein pool between susceptibility to lysosomal proteolysis and in vivo turnover. The fate of yeast invertase subsequent to its uptake by rat liver was followed and certain progressive changes in its localization and physical properties were observed that may be relevant to the process of degradation of this protein.

In this paper we discuss some areas of research in our laboratory dealing with the pathways, mechanisms, and regulation of protein turnover. Several aspects of the problem are dealt with, including the possible existence of conformational states of proteins differing in their susceptibility to turnover, the relationship between in vivo rates and susceptibility to lysosomal proteases, the disposition of a marker protein subsequent to uptake into the lysosomal compartment, and the regulation of uptake of macromolecular markers by the rat yolk sac system.

The conceptual framework that guides our experimental approach to this problem is discussed in the Introduction to this volume and has been presented in detail in other publications (1,2).

RESULTS

Ligand Effects on Susceptibility to Lysosomal Proteolysis

Figure 1 demonstrates the presence in liver of a substance what stabilizes phosphofructokinase (PFK) against thermal inactivation (3). We have isolated the factor and determined a molecular weight for it of about 3500 (4). It is ninhydrin positive and is inactivated by pronase. We have some information regarding its chemistry but have not yet worked out its structure. The factor is also an activator of PFK and, relevant to the present context, it protects PFL against lysosomal digestion (Table I).

TABLE I

Protection by Stabilizing Factor against Lysosomal Inactivation of PFK-L2a

aIncubations were at 37° in the presence of (per ml) 0.35 unit of PFK-L2, 1 mM ATP, 10 mM dithiothreitol, 50 mM NaF, and 50 mM sodium phosphate, pH 6.0, with or without 24.1 mg of lysosmal protein (2.6 acid phosphatase units) and with or without 75 units of stabilizing factor. Lysosomes were prepared by a modification of the method of Ragab et al. (5). The times for 50% inactivation (tu½) were determined and first-order degradation rate constants (kd) were calculated from the relationship kd=0.69/t½. Taken from Dunaway and Segal (4).

FIGURE 1 , - no liver extract added; o, 1.0 ml of liver extract (20.3 mg protein) added. Bottom: liver extract contained 30.6 mg protein/ml. Taken from Dunaway and Segal 3.

The factor is specific in that it has no effect on the other regulated enzymes in glycolysis, glucokinase, and pyruvic kinase, or the minor liver isozyme of PFK, although it does inhibit the fructose bisphosphatase reaction (6), and it has protective effects on ATP citrate lyase, as reported by Osterlund and Bridger (7).

The factor is reduced in diabetes (Fig. 2), as is PFK itself, and rises after insulin administration prior to the rise in PFK. The situation is similar in the fasted liver (Fig. 3).

FIGURE 2 , an identical experiment with cycloheximide, except that each dose was 2.0 mg/kg body weight. In previous experiments it was found that the stabilizing factor activity peaked at 24 hours, while the PFK activity continued to rise (8). Taken from Dunaway and Segal (4).

FIGURE 3 , the 12-hour values for protective factor and PFK, respectively, in rats treated in addition with actinomycin (0.5 mg/kg body weight) 15 minutes before each glucose intubation. Taken from Dunaway and Segal(4).

Dunaway and Weber had previously shown that the elevations and declines in PFK in these states were a consequence primarily of altered degradation rates rather than of effects on synthesis (8). Thus we suggest that the factor mediates these changes in vivo by shifting the equilibrium between PFK conformers to the more proteolytically resistant form.

We have performed similar experiments with rat liver arginase (9). Figure 4 shows the effect of Mn²+ on the digestion of arginase by lysosomal extracts. As may be seen, Mn²+ binding converts the enzyme to a more resistant form. This correlates with a prior observation of Schimke, who found that Mn²+ inhibited arginase degradation in HeLa cells in culture (10).

FIGURE 4 Protection by Mn²+ of lysosomal inactivation of arginase. Incubations were at 37° in 167 mM Na citrate, pH 5.0, 40 mM β-mercaptoethylamine, 2 mg Triton X-100 per ml, and 1.8 mg lysosomal protein (1.3 acid phosphatase units) per ml. First-order rate constants of inactivation (k) are corrected for a slow rate of thermal inactivation at each Mn²+ concentration. Taken from Haider and Segal(9).

A related experiment deals with the factors involved in the diminished rate of liver arginase degradation in fasting (11). Table II shows a rather specific inhibition of arginase digestion by amino acids, which accumulate in the fasting liver. There is indication for a specificity of the amino acids in this regard as well, and further studies of these points would be useful.

TABLE II

Inhibition by Amino Acids of Lysosomal Inactivation of Arginasea

aIncubations were at pH 5.0 and 37° with partially purified arginase or crystalline alanine aminotransferase plus 10 mg lysosomal protein (8.7 acid phosphatase units). Values of the first-order degradation rate constants (kd) were calculated from the time course of activity loss and corrected for a slow rate of thermal inactivation in the case of arginase. Taken from Haider and Segal (9).

Relationship between Turnover Rates and Susceptibility to Lysosomal Proteolysis

Figure 5 and Figure 6 show the results of an experiment to test for a relationship between susceptibility to lysosomal proteolysis in vitro and turnover in vivo (12). The protein pool of a rat was double-labeled by injecting tritiated leucine, followed three days later by an injection of ¹⁴C leucine. Four hours later the rat was sacrificed and the soluble protein fraction of the liver prepared. During the three-day period between injections, the tritium was largely lost from the rapidly turning over proteins and remained predominatly in the slowly turning over proteins. The ¹⁴C on the other hand was primarily in the rapidly turning over proteins. This protein pool was then exposed to lysosomal extracts and the rate of tritium and ¹⁴C release measured. As can be seen, ¹⁴C-labeled proteins were hydrolyzed at a substantially higher rate than tritium-labeled proteins.

FIGURE 5 Differential rates of digestion by lysosomal proteases of rapidly (¹⁴C) and slowly turning over proteins of rat liver cytosol. A male rat was fed lab chow containing (³H) - leucine for four days followed by a four-day period without the label. Three hours before sacrifice the animal was injected with (¹⁴C)-leucine, and the high-speed supernatant fraction of the liver homogenate was prepared. The latter was incubated with disrupted lysosomes from another rat liver at pH 5.0, and the release of trichloroacetic acid-soluble ³H (O) and¹⁴C (O) was measured. Taken from Segal et al(12).

FIGURE 6 Change with time in ratio of percentage of C released to percentage of ³H released. The values are calculated from the data in Fig. 5. Taken from Segal et al. (12).

The conclusion from this experiment, which has been verified by others (13,14), is that there is a correlation within the pool of soluble rat liver proteins between susceptibility to lysosomal proteases and in vitro degradation rates. While the results do not prove that the bulk of protein degradation in vivo occurs in the lysosomes or that this is the rate-limiting step for such proteins, they are consistent with this hypothesis.

As discussed in the Introduction to this volume, for the lysosomal proteolysis step to be rate-limiting in overall protein degradation, the previous step of uptake must be a reversible one. Some results of possible relevance to this point are presented in Fig. 7. In this experiment yeast invertase was injected into the rat intravenously and its uptake into the liver and distribution between the particle fraction and soluble fraction of liver homogenates followed. As may be seen, the fraction in the soluble portion reached a level of about 45% of the total in about 20 hours, where it remained as the total liver invertase declined. We have separated, from livers containing invertase, the parenchymal from the nonparenchymal cells by the methods of Seglen (15) and Drevon et al. and found that most or all of the invertase was in the nonparenchymal cells (Table III). ¹²⁵I-Polyvinylpyrrolidone, on the other hand, was distributed approximately equally between parenchymal and nonparenchymal cells.

TABLE III

Distribution of Invertase and Polyvinylpyrrolidone between Parenchymal and Nonparenchymal cellsa

aYeast invertase (5,670 units) or ¹²⁵I-polyvinylpyrrolidone (PVP) (3.47 × 10⁶ dpm) was administered via the tail vein to rats weighing between 200 and 270 g. At various time intervals after injection the livers were perfused with collagenase for cell separation as described by Seglen (15) with slight modifications. Following the perfusion, the livers were weighed and a weighed portion removed and homogenized in eight volumes of 0.3 M sucrose adjusted to pH 7.0. These homogenates were assayed for invertase (17) or for ¹²⁵I in a Beckman Gamma Counter, Model 300, with an efficiency of 33%, corrected for decay to the same initial time. Glucose-6-phosphatase activity of the homogenate was also determined (18). The remaining perfused liver was dispersed in cold incubation medium (4°C) containing 2% charcoal-purified bovine serum albumin (16) to form the initial cell suspension, and the parenchymal cells separated by centrifugation (16). The purified parenchymal cells thus obtained in each case contained less than 3% nonparenchymal cells, as determined by differential counting in a Levy counting chamber, and the cell viability was between 74 and 91%, as determined by exclusion of 0.3% trypan blue. The purified parenchymal cells were pelleted by centrifugation at 1750 rpm for five minutes in an HB-4-rotor at O°C, resuspended in 0.3 M sucrose, pH 7.0, and homogenized until all the cells were disrupted (as determined microscopically). The amounts of the markers and of glucose-6-phosphatase were determined in the purified parenchymal cell homogenates as described above for the whole liver homogenates. The yield of parenchymal cells was determined from the recovery of G-6-pase in the purified parenchymal cell preparation, since G-6-pase is absent in nonparenchymal cells (19). The yields varied between 16 and 23%. The total amount of marker taken up by the parenchymal cells of liver was calculated from the amount of marker in the purified parenchymal cell preparation and the yield of parenchymal cells.

FIGURE 7 Time course of uptake and distribution in the liver of injected invertase. Each time point represents an average of two to four rats. (A) Total invertase present in the liver as percentage of injected dose. (B) Invertase in the high-speed supernatant fraction (HSS) of liver as percentage of the total present in the liver.

Figure 8 represents a second experiment plotted somewhat differently, showing the rapid clearance of invertase from the blood, the peak in the particulate fraction when the blood pool has declined to near zero, and the continued rise in the soluble fraction to a peak substantially later. It should be mentioned that in each case the level of the lysosomal marker enzyme, acid phosphatase, was monitored and a consistent 10–12% appeared in the soluble fraction.

FIGURE 8 Time course of distribution of injected invertase in blood and liver fractions, (A) Invertase in serum as percentage of injected dose. (B) Sedimentable invertase of liver as percentage of injected dose. (C) Invertase in high-speed supernatant fraction of liver as percentage of injected dose.

These results are amenable to at least two interpretations. (1) We may be observing a lysosomal maturation or aging process in which the particles containing the invertase become labile and are disrupted during homogenization. In this regard it can be noted that the results were not affected by different methods of homogenization, nor is it clear why the liberated invertase should reach only 50% of the total. (2) A second possible interpretation is that invertase is released from lysosomes into the cytosol and that a steady state of release and reuptake develops. At the moment we must leave the question undecided.

In regard to the invertase experiments, we have made some additional observations of interest. Invertase reextracted after various periods of residence in the liver exhibited a progressive increase in elution volume on gel filtration (Table IV). In addition, there were progressive changes in the stability of the invertase. Thus certain structural changes appear to be occurring that it would be useful to define.

TABLE IV

Relative Elution Volume of Invertase Reextracted from the Livera

aThe high-speed supernatant fractions of liver homogenates in 0.3 M sucrose were prepared and chromatogrammed on Sepharose 4B-CL (57.5 × 1.6 cm) in 60 mM K phosphate, pH 6.0

bVe is the elution volume of the invertase peak. Vo is the elution volume of the first blue dextran peak and equalled 38.4 ml.

cValue is for the stock invertase solution.

Uptake as a Site of Discrimination and Regulation in Turnover

Another potential site of discrimination in the turnover process is at the uptake step (see Introduction to this volume), for either intracellular or extracellular proteins. Plasma membrane receptors for a few specific circulating proteins have been demonstrated (20, in this volume, chapters by Stahl and Sly), which provide a sharp descrimination between such proteins taken up by adsorptive pinocytosis and those taken up much more slowly by fluid pinocytosis (21). A reasonable further question is whether there is a range of intermediate uptake rates based on nonspecific adsorption of a lipophilic type, as proposed by Jacques (22). We have found that gradations in lipophilic affinity do exist and that they are correlated with in vivo turnover rates (Fig. 9) (23), again using the double-labeled pool of rat liver soluble proteins as the test material.

FIGURE 9 , nonadsorbed protein (or, in the case of the first open circle, the protein applied to the first column). Arrows lead from the original protein or the nonadsorbed protein to the fractions obtained from it at the next step. Taken from Segal et al(23).

For further studies of uptake selectivity and regulation we have recently employed the rat yolk sac system (17) developed by Lloyd and his co-workers (24,25). In Fig. 10 is shown the uptake of invertase into the yolk sac as a function of time. Each point represents a separate yolk sac. The system is highly reproducible and linear with time for six hours or longer. This figure also shows that no redistribution of invertase into the soluble portion of homogenates occurs, unlike the case in the liver (Fig. 7). As shown in Fig. 11, the uptake process is nonsaturable and nonspecific (see below), and therefore apparently one of fluid pinocytosis.

FIGURE 10 , the total invertase activity; •, the soluble invertase activity per mg of homogenate protein. Taken from Brown and Segal(17).

FIGURE 11 Uptake rate of invertase as a function of its concentration in the medium. Yolk sacs were incubated for four hours at the invertase concentrations shown. Taken from Brown and Segal(17).

An interesting finding in this process is that it was markedly inhibited by glucagon (Fig. 12). Half-maximum effect was at about 3 × 10−8 M (Fig. 13), and the inhibition was readily reversible upon removal of the glucagon (Fig. 14). The same effect was observed on the uptake of another marker as well, namely, ¹²⁵I-polyvinylpyrrolidone (Fig. 15). The clearance rates of invertase and PVP were comparable and very similar to those reported by Lloyd for a series of nonspecific macromolecular substances (26). The possibility was considered that glucagon might be promoting the release of invertase rather than inhibiting its uptake. The linearity of uptake in the presence (as well as in the absence) of glucagon argues against that possibility; however, it was more conclusively eliminated by a direct experiment. Yolk sacs were allowed to take up invertase for two hours, then transferred to an invertase-free medium, either with or without glucagon, and the invertase loss measured. There was a slight loss of invertase during the second incubation period, which was not affected by glucagon.

FIGURE 12 Inhibition of invertase uptake by glucagon. Yolk sacs were incubated with 68 units/ml of invertase without additions (0) or with 10−6 M glucagon (•). Taken from Brown and Segal(17).

FIGURE 13 Concentration dependence of glucagon inhibition. Yolk sacs were incubated for four hours with 68 units/ml of invertase at the concentrations of glucagon shown. The points around the inflection are averages from three different animals. (Taken from Brown and Segal(17).

FIGURE 14 ) or in the presence of 10−7 M glucagon (•). After two hours (arrow) the remaining yolk sacs in both groups were rinsed in three changes of glucagon-free medium, and then further incubated without glucagon. Taken from Brown and Segal(17).

FIGURE 15 ) or with 10−6 M glucagon (•). Taken from Brown and Segal(17).

It seemed probable to us that the glucagon effect was mediated by cyclic AMP, and that is supported by two observations. First, the levels of cyclic AMP were measured in the tissue and found to be higher in the glucagon-treated yolk sacs (3.61 and 2.48 pmol/mg protein for duplicate controls, and 6.17 and 6.69 pmol/mg protein for duplicate samples exposed to 10−6 M glucagon for two hours). Second, dibutyryl cyclic AMP was able to reproduce the inhibitory effect (Fig. 16). As may also be seen in Fig. 16, there was an inhibitory effect of epinephrine, but only at concentrations several orders of magnitude greater than with glucagon, and the epinephrine inhibition was augmented by theophylline.

FIGURE 16 ). Taken from Brown and Segal(17).

Insulin, chloroquine, carbachol, ascorbic acid, and dibutyryl cyclic GMP had no effect on uptake in this system.

Studies of a number of other systems have appeared that relate certain cell functions to intracellular levels of cyclic nucleotides and indicate a relationship of the latter to normal microtubular status, including the release of lysosomal enzymes from leucocytes as reported from Weissmann’s laboratory (27,28) and concanavalin A-induced cap formation in leucocytes as reported from Oliver’s laboratory (29,30). Therefore, we tested the effect of microtubule disrupting agents on pinocytosis in the yolk sac. As may be seen in Fig. 17, both colchicine and vinblastine at the 10−6 M level were potent inhibitors of uptake in this system, supporting the idea of an involvement of microtubules in this process as well.

FIGURE 17 ). Taken from Brown and Segal (17).

DISCUSSION

The most obvious possibilities for rate-limiting, regulatable steps in protein turnover are those involving the transformation of the protein to a susceptible form, its uptake into the lysosomal digestive system, and the proteolytic step(s).

Evidence supporting all of these possibilities has been developed, some of which is discussed in this chapter. Such findings are not necessarily in conflict. Some time ago we proposed a model (31) based on a kinetic treatment of the steps in the pathway of protein degradation (see Introduction to this volume). Taking into account the possibility of an equilibrium between susceptible and nonsusceptible states, the following equation emerges:³

where ksp is the fraction of the component removed per unit time, keq the equilibrium constant of the corresponding reactions, and the other k’s rate constants of the corresponding reactions (see Introduction to this volume).

Clearly this equation allows for effects on ksp via Keq, k2, or k3, which correspond to the possible sites or regulation referred to above. It also offers an explanation for the observation that some conditions that promote overall protein turnover affect predominantly the slowly turning over proteins (32, 33; in this volume chapters by Poole & Warburton, Dice & Walker, since turnover of this class is dependent upon k2, and k3, while that of rapidly turning over proteins is not (31). Thus the possibility of discriminatory effects on these two classes exists in situations that either increase k3 (intralysosomal proteolysis) or decrease k2 (escape from the lysosome).

However much this model can explain, a major difficulty in applying it with confidence is that it depends heavily on an as yet entirely hypothetical process, viz., that of escape of intact proteins from the lysosomal system. No convincing evidence for such a process has been adduced. On the other hand, as pointed out by Tulkens (this volume), some mechanism for recycling of the membrane material of vesicles must exist. If the membrane material of vesicles can detach itself from secondary lysosomes and return to the cellular compartment from which it arose, it seems reasonable to suppose that some of the lysosomal contents may be recycled in the same way.

REFERENCES

1. Segal, H. L.Dingle, J.T., Dean, R.T., eds. Lysosomes in Biology and Pathology; 4. North-Holland Publ, Amsterdam, 1975:295.

2. Segal, H. L.Horecker, B.L., Stadtman, E.R., eds. Current Topics in Cellular Regulation; 11. Academic Press, New York, 1976:183

3. Dunaway, G. A., Jr., Segal, H. L. Biobibem. Biophys. Res. Commun. 1974; 56(689)

4. Dunaway, G. A., Jr., Segal, H. L. J. Biol. bibem. 1976; 251(2323)

5. Ragab, H., Beck, C., Dillard, C., Tappel, A. L. Biobibim. Biophys. Acta. 1967; 148(501)

6. Sankaran, L., Proffitt, R. T., Pogell, B. M., Dunaway, G. A., Jr., Segal, H. L. Biobibem. Biophys. Res. Commun. 1975; 67(220)

7. Osterlund, B., Bridger, W. A. Biobibem. Biophys. Res. Commun. 1977; 76(1)

8. Dunaway, G. A., Jr., Weber, G. Arbib. Biobibem. Biophys. 1974; 162(629)

9. Haider, M., Segal, H. L. Biobibem. Biophys. 1972; 148(228)

10. Sbibimke, R. T. Nat. Cancer Inst. Monogr. 1964; 13(197)

11. Sbibimke, R. T. J. Biol. bibem. 1964; 239(3808)

12. Segal, H. L., Winkler, J. R., Miyagi, M. P. J. Biol. bibem. 1971; 249(6364)

13. Bohley, P., Miehe, C., Miehe, M., Kirsbibke, S., Kirsbibke, H., Langner, J., Wiederanders. Acta Biol. Med. Germ. 1972; 58(9)

14. Dean, R. T. Eur. J. Biobibem. 1975; 58(9)

15. Seglen, P. O. Meth. Cell Biol. 1976; 13(29)

16. Drevon, C. A., Berg, T., Horum, K. R. Biobibim. Biophys. Acta. 1977; 487(122)

17. Brown, J. A., Segal, H. L. J. Biol. bibem. 1977; 252(715)

18. Baginski, E. S., Foa, P. P., Zak, B.Bergmeyer, H.U., eds. Methods of Enzymatic Analysis, 2nd; 2. Acadamic Press, New York, 1974:776.

19. Crips, D. M., Pogson, C. I. Biobibem. J. 1972; 126(1009)

20. Ashwell, G., Morell, A. G. Advan. Enzymol. 1974; 41(99)

21. Silverstein, S. C., Steinman, R. M., Cohn, Z. A. Ann. Rev. Biobibem. 1977; 46(669)

22. Jacques, P. J.Dingle, J.T., Fell, H.B., eds. Lysosomes in Biology and Pathology; 2. North-Holland Publ, Amsterdam, 1969:395.

23. Segal, H. L., Rothstein, D. M., Winkler, J. R. Biobibem. Biophys. Res. Commun. 1976; 73(79)

24. Williams, K. E., Kidston, E. M., Beck, F., Lloyd, J. B. J. Cell Biol. 1975; 64(113)

25. Williams, K. E., Kidston, E. M., Beck, F., Lloyd, J. B. J. Cell Biol. 1975; 64(123)

26. Lloyd, J. B.Robbins, D.W., Brew, K., eds. Proteolysis and Physiological Regulation. Academic Press, New York, 1976:371.

27. Zurier, R. B., Weissmann, G., Hoffstein, S., Kammerman, S., Tai, H. H. J. Clin. Invest. 1974; 53(297)

28. Hoffstein, S., Goldstein, J. M., Weissmann, G. J. Cell Biol. 1977; 73(242)

29. Oliver, J. M., Zurier, R. B., Berlin, R. D. Nature. 1975; 253(471)

30. Oliver, J. M., Zurier, R. B. J. Clin. Invest. 1976; 57(1239)

31. Segal, H. L., Matsuzawa, T., Haider, M., Abraham, G. J. Biobibem. Biophys. Res. Commun. 1969; 36(764)

32. Warburton, M. J., Poole, B. Proc. Nat. Acad. Sci. 1977; 74(2427)

33. Bradley, M. O. J. Biol. bibem. 1977; 252(5310)

---

¹Supported by grants from the National Institutes of Health (AM–18187 and AM–08873).

²Present address: Department of Medical Sciences, Southern Illinois University School of Medicine, Springfield, Illinois 62708

³If the conversion to the susceptible form is irreversible, ksp is simply kl.

LYSOSOMAL MECHANISMS OF PROTEIN DEGRADATION

Roger T. Dean,     Division of Cell Pathology, Clinical Research Centre, Harrow, Middlesex, England

Evidence from studies on mouse peritoneal macrophages using the inhibitor pepstatin confirms lysosomal involvement in basal protein degradation, and extends its relevance to degradation of long half-life and analog-containing proteins. Studies on the ability of MRC-5 (a limited-life-span fibroblast line) cells to selectively degrade analog-containing proteins are described. These indicate that this capacity is retained even in very old cells; indeed such cells show an increased proportion of rapidly degradable proteins. Analog-containing proteins bind preferentially to lysosomal membranes, and like liver cytosol proteins of short half-life, are selectively endocytosed and degraded by certain cells in culture. Thus membrane binding allowing selective entry to the lysosomal system may be important in controlling rate of degradation of both intracellular and extracelluar protein. A method potentially allowing for determination of the rate of autophagy in cells is described. This should enable further assessment of the quantitative involvement of lysosomes in protein degradation. A possible mechanism for the recovery of membrane entering the lysosomal interior during autophagy is