Significance of the rapid degradation of newly synthesized proteins in mammalian cells: A working hypothesis

J. theor. Biol. (1982) 98,283-300.

Significance of the Rapid Degradation of Newly Synthesized Proteins in Mammalian Cells: A Working Hypothesis DENYS N. WHEATLEY Cell Pathology Laboratory, Department of Pathology, University Medical Buildings, Foresterhill, Aberdeen AB9 220, Scotland SANTIAGO

GRISOL~A

AND JOSE HERNANDEZ-YAGO

Instituto de Investigaciones Citoldgicas de la Caja de Ahorros de Valencia, Amadeo de Saboya 4, Valencia 10, Spain (Received 22 December 1981, und in revised form 6 April 1982) The decay curve for labelled proteins in living tissue (biomass)is the integral of the different slopesfor every speciesof protein present.While this heterogeneitymay accountfor someof the complexity of degradation kinetics, a factor which must play an important role-especially where discontinuitiesin exponential decay curves occur-is the stabilization of newly synthesized(nascent)proteins. These metastableproteins remain at risk and experience a high rate of proteolysis until they become integrated into the biomass. Following the initial period of stabilization, integrated proteins randomly but steadily emergeunder controlled conditions, reflecting their individual turnover rate in the biomass.Destabilized proteins will be susceptibleonce again to proteolysis, but the rate of their degradation will be slower than in the initial phasebecausethe frequency of their re-emergencefrom their sitesof integration hasbecomethe rate limiting factor. Irreversible conformationalchangesand denaturation may further influencetheir rate of removal. Recognition of the two distinct phasesof proteolysis offers a better explanation of the kineticsof degradationof endogenousproteins.Several important implicationsarisingout of thesefindingsare discussed. “More difficult to explain, is the fact that turnover is a continual and extensive processunder normal steady-state nutritional conditions in animal tissues,a seeminglywastefulprocess”(Schimke& Bradley, 1975).

1. Introduction Protein degradation proceeds much faster immediately after synthesis than at later times in cultured cells. This declining susceptibility does not follow 283

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simple kinetics in any system (Goldberg, 1972; Bradley, Hayflick & Schimke, 1976; Garlick, Fern & McNurlan, 1978; Amenta, Sargus & Brother, 1980), and there is usually clear evidence of a discontinuity in the curves (Poole & Wibo, 1973; Knowles et al., 1975; Knowles & Ballard, 1976; Bradley et al., 1976; Wheatley et al., 1979). Recent experiments were designed to look more closely at the kinetics and discontinuities so often seen in degradation rates (Wheatley, Giddings & Inglis, 1980). More accurate estimates of rates in the early phase of proteolysis in mammalian cells were also sought. The results have led to revision of the present hypothesis about the susceptibility of protein to degradation, which is the main purpose of this communication. Certain points in the text which require further elaboration are dealt with in footnotes to avoid interrupting the flow of the discussion. Most of the data concerns mammalian cells, but other systems are considered because a plausible hypothesis should apply to all living organisms. 2. Kinetics

of Early Degradation

Many experiments on protein degradation have been concerned with its biological aspects, especially the putative role of the lysosome in the hydrolysis of endogenous as well as exogenous proteins (Dean & Barrett, 1976; Segal, Winkler & Miyagi, 1974; Ward & Mortimore, 1978; Apenta & Brother, 1981). Others have analysed the arsenal of hydrolytic enzymes in the cell (see Barrett, 1977). But few have looked at the process as a simple chemical reaction proceeding within a very complicated milieu, or how it fits with the overall physiology of the cytoplasm at any given time. The conventional pulse-chase procedures used in the majority of studies have revealed only the tail-end of a much more extensive and steadily on-going process of intracellular proteolysis than hitherto suspected. The significance of continual, rapid and widespread hydrolysis of newly synthesized proteins (Wheatley et al., 1980) should be examined in greater detail. The simple model proposed previously was as follows: PROTEIN Metastable ______* (high susceptibility) I

Stable(low susceptibility) DEGRADATION AMINO

1

ACIDS

Destabilized (high susceptibility) 1

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Some recent experiments (Wheatley, 1982) involved intervals of a few seconds between time points after the administration of labelled amino acid. Because of technical difficulties, pulse-chase experiments of similarly short intervals are very difficult to perform without disturbing the cells through extra manipulation, creating excessively labelled pools compared with protein labelling, and increasing reutilization of the labelled amino acid. However, the recent studies of Wheatley et al. (1980) achieved a 1 min interval, from which it was possible to calculate that nascent protein hydrolysis approached 40% per hour in HeLa cells. Cells labelled for 5 min showed about 30% hydrolysis, whereas those labelled for 30 min gave only a 12% degradation in the first hour of chase. Two important facts emerged: (i) newly synthesized proteins are quickly destroyed, and (ii) the shorter the pulse, the more marked is the discontinuity between the rate of early proteolysis and that found subsequently. The familiar “dog-leg” curve seen in studies on mammalian cells (Knowles et al., 1975; Bradley et al., 1976; Amenta et al., 1980) has also been observed in bacteria (Goldberg, 1972; Kemshead & Hipkiss, 1974). Where long labelling periods preceded the chase, degradation curves became shallower without evidence of discontinuity. The kinetics for most organisms appear to follow a very similar pattern [Fig. l(a)], suggesting an underlying mechanism(s) common to all living things. As explained in Fig. 1 (see legend), a typical curve for the degradation of HeLa cell proteins following a 5-min pulse-labelling cannot be described by a single exponential function. Furthermore, the rate of change of the degradation rate with time does not follow a simple exponential [Fig. l(a)]. These results strongly indicate the existence of more than one mechanism. A similar situation occurs in uiuo. Garlick et al. (1978) estimated that about 77% of rat liver proteins which had been pulse labelled for 1 h were destroyed within the first day of existence, whereas only 15% were destroyed in the next 29 days. Another example is that of collagen synthesis and degradation by hepatocytes (Diegelmann, Cohen & Guzelian, 1980). These rather extreme changes of rate have been attributed to the heterogeneity of the proteins in the labelled population [see Fig. l(b)]. In the simplest treatment of the data obtained for total cell proteins in the usual type of degradation experiment, proteins have been divided into those tending to have short half-lives and those with long half-lives (Poole 8~ Wibo, 1973). Unfortunately this explanation is based on the assumption that those proteins in the heterogeneous mixture which are removed most promptly are the ones known to have the shortest half-lives, which has not in fact been demonstrated. If this were the correct explanation, then it could also have been inferred that there would be a qualitative difference

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‘ij

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1,, , , , ,

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Cd)

1 I

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FIG. 1. This composite figure explores the possible interpretations of the degradation curve normally produced in mammalian cells by a 5 min pulse-labelling procedure (a) on semilogarithmic plots. In (b), it is presumed that there are three large groups of proteins labelled, of long (l), medium (2) and short (3) half-lives with the thickness of individual lines indicating the relative amounts present. A weighted mean (dashed line) would nevertheless produce a single exponential curve over this time scale (see text). In (c), the data is examined as if the decline in labelled proteins follows classical enzyme kinetics, i.e. as an inverted hyperbola on a semi-log plot (l), which seems to approximate a simple exponential until it is compared with a true exponential (2). It is a smooth function which curves the opposite way to that seen in (a). In (d), the degradation curve of (a) has been split into two components, one with a steep exponential function (1st exponential), the other with a shallow exponential function (2nd exponential). Their summation provides a perfectly good description of the overall curve. Figure 2 will elaborate upon this as a means of analysis.

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in the spectrum of labelled proteins before and immediately after the early chase period. This is not the case (Wheatley et al., 1980), however, for the degradation of proteins destroyed in the early chase period appears to be random, which allows one to apply straightforward statistical treatment to the probability with which a protein is removed. In reality, all proteins lost in the early chase times have had relatively short lives, even though some species will later show short hulf-lives and others long half-lives.? If the heterogeneity of protein half-lives was the explanation of the complex kinetics of degradation, then the decay curves might follow those depicted in Fig. l(b). Garlick et al. (1978) obtained a good approximation to the observed degradation curve by arbitrarily dissecting it into fast, medium and slow turning-over proteins. Taking this to its logical conclusion, the closest approximation would be the integration of the slopes of every single species of protein, i.e. several hundred or thousand [Fig. l(b)]. Accepting that this must in reality be the case, and that each species of protein decayed with a characteristic half-life by strictly first order kinetics, the integrated curve for all species of protein as a weighted mean [Fig. l(b)] would also follow first order kinetics until a significant number of short-lived proteins were lost. There would be no obvious discontinuity, simply a slight curvature away from the straight line. Both the kinetic data [Fig. l(a)] and the fact that random hydrolysis occurs in the early chase period suggest that some other mechanism is responsible. Classical enzyme kinetics should apply when proteases break down labelled substrates. Attempts to plot the percentage of substrate remaining on semi-log plots assuming normal hyperbolic kinetics of hydrolysis do not generate the expected degradation curves, as shown in Fig. l(c). In MS cells, Knecht et al. (1980) showed that two simple exponential curves, one of a steep gradient (fast decay) and the other shallow (slow decay), combine to describe essentially the same curve as the experimental one for these proteins. The sharp discontinuity in these curves indicates that the proteins themselves must undergo a change of their susceptibility to proteolysis since the cell itself is in a steady state condition. Previous data (Wheatley et al., 1979, 1980) has been consistently plotted with two components, one a fast exponential out of which a second emerges at a shallower gradient in the later chase period. Recent results using this analysis [Figs l(d) and 2] show this in operation. Analysis of the “pools” of proteins which are short- or long-lived in relation to the discontinuities described above can be carried out according + It is essential to distinguish between short liued and short half-lioed in this discussion. The former is a literal description of the fate of a protein, whether it is a highly unstable or a stable structure. The latter is a statistical property describing the longevity of a population of one species of protein.

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IOC

I-

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)I

(0) I 1

I 2 Time

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(hr)

FIG. 2. (a) Semi-log ploi showing the loss of counts (absolute) from HeLa cells labelled with [3H]leucine as described in Wheatley et al. (1980), for 1 min (A), 5 min (H), 30 min (0) and 120 min (0). The 1200 min (20 hr) labelling gives a straight line (without symbols). Using the procedure of Knecht et al. (1980), the parallel lines from about 1 hr were extrapolated back to the ordinate. These slopes were subtracted from the whole curves. In this way it is possible to analyse the first 45 min for the three lower curves, the first 20 min for the fourth curve from the bottom, but there was no difference left for plotting in the very long-labelling case (20 hr). The subtracted values were normalized to 100% at zero time and replotted on another semi-log plot (b). (b) Demonstration that the subtracted values obtained as described above give straight lines, i.e. pure exponentials. It is also of interest that the same curve was applicable in all cases, which shows that the population of “metastable” protein molecules at high risk in each of the different pulse treatments were subject to the same random rate of proteolysis, their half-life being about 53 min. In contrast, the half-life of stabilized proteins was estimated by extrapolation as between 31 and 32 hr, i.e. about a 36 times slower rate of proteolysis.

to Knecht et al. (1980). Assuming steady state conditions for the pools, it is possible to calculate the proportion of protein belonging to each pool (i.e. of short- and long-lived proteins). It is necessary to measure the radioactivity associated with the cell protein lost during the chase and then to calculate the apparent average half-lives and the, rac;ioactivity present in the total protein in each peal at the end of the pulse time. The percentage of protein in the pools may be calculated as follows: dn*=

kNdt-kn*dt=k(N-n*)dt

(1)

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where the constant k expresses the turnover rate (k = V/N); V = dn/dt is the velocity of protein turnover in the pool; N is the total protein of the pool; kN dt is the label incorporated into the pool protein in time dt; n* is the labelled protein present in the pool at t of pulse; kn* dt is the label lost from the pool protein in time dt. Intregration of equation (1) (if n * = 0 at t = 0) yields n* = N(l -eakr) = N(l -2-‘/‘1/2) (2) since tl12 = (In 2)/k. NL and Ns refers to the protein in the long-lived pool (tlj2=) and in the short-lived pool (t1,2S), respectively, while n? and ns* are the labelled proteins of the long-lived and of the short-lived pools, respectively, at the end of the pulse time. The ratio NL/Ns can be calculated by applying equation (3): NL E

n;(l = N;

(1

-2-‘425) _ 2-‘4m)

(3)

'

The bases for these calculations are illustrated in Fig. 2 and examples are presented in Table 1. As is clearly shown in Table 1, the percentage of short-lived proteins is very small, although, as already indicated, their turnover is very fast. TABLE

1

Calculated ratio NL/NS, as well as percentage of short-lived and long-lived proteins for three cell types Pulse Cell type WI-3t3t HeLaS MS§

Labelled Protein Total Total Cycloheximideinsensitive

25 5 60

81 77.5 50

25 27 144

19 22.5 50

0.68 0.9 1.6

129 100 73

0.76 0.99 1.35

t From Bradley et al. (1976). f From Fig. 2 of this review. 8 From Knecht ef al. (1980). Data obtained from the analysis of cell protein-associated radioactivity decay for each cell type, as described by Knecht et al. (1980).

3. The Discontinuity of Degradation Curves

The crucial problem is to find the explanation for the discontinuities of degradation curves. In mammalian cells, for example, the discontinuity

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nascent proteins

/ FIG. 3. Schematic diagram of flux of proteins in relation to growth and maintenance of biomass. For simplicity, the situation relating to only one protein is considered after its synthesis, in this case a structural protein. Although it is physiologically useful in a conformation denoted by a square, the protein oscillates between this and another form represented by a diamond, the latter being of no physiological value. The biomass can integrate squares when slots are available (A), which results in the protein being stabilized, i.e. protected from proteolysis. Those not stabilized remain at risk to the diffuse proteolytic system, which degrades them with random probability. Through the dynamicity of the biomass, squares will also be lost with approximately random probability, mainly because of alteration in the sites in which they are lodged (B). Alternatively the proteins themselves may become altered through some reaction they carry out or by spontaneous change induced by extraneous influences (C). These will also result in the squares returning to a higher risk of proteolysis. The long dashed line down the centre does not indicate compartmentation, but is drawn so that both nascent proteins and estabilized proteins can be seen to occur in a common “pool” in which the molecules will be of equal susceptibility to proteolysis. The only factor which

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appears about 40-60 min after chasing of cells labelled for 5-10 min.t The shorter the pulse, the more exaggerated the discontinuity (Wheatley et al., 1980), since the initial slopes become steeper whereas the subsequent slopes are parallel for all pulse lengths (Fig. 2). This important experimental finding has been instrumental in formulating the model. Our hypothesis rests on the fact that the cell is a self-assembly machine which produces proteins for its development, maintenance and growth. It is highly improbable that the ribosomal machinery can match the exact requirements for proteins throughout the cell at all times, especially as environmental factors may be incessantly altering them (Grisolia, 1964). Equally there is no known mechanism which guarantees that every protein which has been synthesized will be ushered into an appropriate “slot”, always assuming there is one available. For example, there is no known pathway which ensures that each histone molecule released from the ribosome will become associated with the chromatin of the nucleus, particularly as it must be translocated over considerable molecular distances. Even if one were prepared to entertain this absurdity, i.e. of determinism operating at the molecular level, such a mechanism of integrating proteins would require enormously complex machinery to ensure the correct transportation and fitting of newly made components.S Furthermore, the end result would be a cell devoid of all flexibility and adaptability. The alternative is for newly synthesized proteins to be “sorted” largely, if not completely, on a random basis; the major factor determining the probability with which a protein survives is the frequency of occurrence of conditions and/or places within the biomass into which it can be functionally or structurally integrated (Fig. 3). t Accurate measurements of the inflection points in discontinuous decay curves are difficult to obtain because they alter with the length of labelling, the chase conditions and many other factors. Long pulses are of little use because these correspond to a combination of continued labelling and chasing throughout the whole period of labelling. The difficult problem of allowing for the chasing of already labelled proteins during long pulse periods can be surmounted by studying degradation in cells which have been injected with labelled proteins, as carried out by Wallace and co-workers (Dehn & Wallace, 1973; Wallace & Hollinger, 1979). This may provide a technical solution to the problem, but the procedure of injecting proteins may disturb the normal proteolytic response inside the cell. Nevertheless, proteins which have been introduced (vitellogenin, bovine serum albumin) undergo a two-phase degradation process with a marked discontinuity after the hydrolysis of about 40% of the injected material. The technique of cell fusion is now being used in similar studies (Hendil, 1980). $ This machinery would, in turn, require more complex machinery for its construction, and so on ad absurdum. modifies this is that some of the destabilized protein will have become denatured (Cl) or otherwise altered in conformation, so that they do not necessarily constitute an identical population to the nascent proteins.

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Iioteins which are not assimilated remain at risk and are rapidly destroyed by proteolysis.? Those which are assimilated are protected or stabilized in the sense that their risk of being hydrolysed is greatly reduced (cf. Diegelmann et al., 1980). The association of an enzyme with its substrate(s), the polymerization of a tubulin molecule into a microtubule or the association of histones with chromatin leads to proteins entering a different phase of existence from the metastable state of their nascent form3 In some cases, the population of a particular, newly synthesized species of polypeptide may have only a certain probability of attaining a physiologically “useful” conformation,§ and this could explain early proteolysis as being the elimination of its useless forms. This would also include polypeptide chains which undergo premature termination in the presence of puromycin (Kernshead & Hipkiss, 1974), error proteins, etc., although it has been calculated that mistranslated proteins would not normally t The argument as to whether there is a dispersed system of proteases responsible for basal hydrolysis of endogenous proteins as opposed to the lysosomal system need not concern us here. However, the randomness of proteolysis both in the early rapid phase and in the slower destabilisation phase is more difficult to explain on a purely lysosomal basis, a weakness acknowledged by some adherents of the lysosomal system (Kirschke et al., 1978). $ Many of the older reports in the literature suggest that the interaction of an enzyme with its substrate, a polymerizable protein with its co-polymers, etc., would tend to stabilize them against proteolysis. Some recent examples include Mori et al. (1980) and Raymond & Shore (1981), who found that assimilation of carbamyl phosphate synthetase into the mitochondrion protects it from degradation. Kruse, Frevert & Kind1 (1981) have shown that the assimilation of malate synthase mto glyoxysomes stabilizes it, whereas enzyme molecules not taken into these bodies are rapidly degraded. The literature contains much further evidence that rapid degradation of surplus molecules OCCUISin many different types of cell. In HeLa cells, ribosomal proteins titrate stoicheiometrically with nascent ribosomes in the nucleolus (Lastick & McConkey, 1976; Phillips & McConkey, 1976), but the existence of a pool which allowed this to occur had almost certainly been missed because it would be too labile to be picked up in the “short” pulse period used in these experiments, which amounted to 12 hr. Their otherwise elegant studies strongly support the notion that the integration of proteins into the biomass during the “sorting” phase leads to a population of stablized molecules, which should in their turn bear stoicheiometric relationships with other proteins and molecules comprising the living substance of cytoplasm. Alhanaty er al. (1981) found a cyclic AMP-dependent protein kinase in rats which was degraded more rapidly in its native subunit form than after heat denaturation, a procedure which generally renders proteins particularly susceptible to hydrolysis. The dimerized enzyme (normally found) allows it to retain its molecular integrity and catalytic activity. Obviously a stabilizing interaction under one set of conditions can become a labilizing interaction under another, and be important in the regulation of protein (enzyme) level in cells. In some instances, the stability of proteins (enzymes) is reduced in the presence of their substrates (Grisolia, 1964; Citri, 1973), which is contrary to the above-mentioned rule. But in general the principle of interaction as a means to stabilization seems to operate. 5 Pace, Fisher & Cupo (1982) have calculated that the globular forms of proteins are often only marginally more stable than their extended forms, with which they may osillate quite freely. Very small changes in primary sequence, therefore, can easily affect their susceptibility in either direction.

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account for a significant percentage of the material hydrolysed in the early chase prriod (Wheatley of al., 1980). Subsequently, as illustrated in Fig. 3, the bulk of proteins which have become stabilized by the proper interaction with substrates and components may be subject to destabilization by one or a combination of factors such as local pH changes, competitors, denaturing agents causing covalent modifications, etc., as extensively illustrated by Grisolia (1964) and Grisolia & Hood (1972).

4. Susceptibility

of Proteins to Degradation

“Metastable”, “stable” and “destabilized” are three terms which have been used in connection with the susceptibility of proteins to proteolysis (Wheatley et al., 1980). Metastable has a similar connotation to nascent, with its implication of vulnerability of the newborn molecules, possibly in their frequent flipping from one conformation to another (Pace, Fisher & Cupo, 1982). Metastable has been used because it relates to “stable”, which is self-explanatory. Destabilized molecules have moved from a state of stability back to one of increased risk to hydrolysis, but it does not necessarily imply that this is a reversion to the same susceptible state(s) as the metastable protein adopted. A protein can become destabilized and highly vulnerable to proteases through some form of denaturation. The risk of proteolysis will not only be decided by the concentration of proteases and the conformational states exhibited by the protein, but by the prevailing conditions. Unlike radioactive decay which is independent of conditions, proteolysis is highly influenced by them. Thus the risk of proteolysis occurring in some species of protein must be seen on a statistical basis which will change with prevailing conditions, especially the availability of sites for integration. The “inherent susceptibility” of protein to hydrolysis measured by 3 simple in vitro assay offers only 3 crude guide to the kinetics of degradation of proteins in duo (discussed in Dean, 1980), and only to their turnover in the second, destabilization phase of gradation. Certain proteins need processing shortly after they have been synthesized. The removal of leader polypeptide sequences occurs in the conversion of proinsulin to insulin, the insertion of mitochondria proteins into the inner membrane, etc., but it would be too difficult at present to offer any suggestion 3s to the extent to which they feature in early proteolysis. The number of proteins presently known to possess leaders is too few to explain the amount of early proteolysis shown in Fig. 1.

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of the Two-phase Proteolytic (A)

Process

HALF-LIVES

The concept of protein “half-lives” may need revision because the half-life estimated after very short pulse-labelling will be far less than that estimated after long pulse-labelling treatment of cells. Two half-lives would be more appropriate, one obtained by extrapolation of early phase of proteolysis after short pulse-labelling, and a second familiar estimate obtained from long-pulse labelling. The half-life of a protein varies according to the requirements of the cell under the prevailing conditions, and cannot be considered a “constitutive” property of the protein. It can only be given as a relatively precise value and a guide to turnover where the exact conditions of a biological system are laid down or in a steady state. For example, the absolute rate of early proteolysis will be highly dependent upon the rate of protein synthesis, which provides its substrates. This would be in keeping with the observations made by, for example, Setlow (1975) on proteolysis in germinating bacteria, and Young, Horn & Noakes (1979) in the foetal lamb, which would otherwise appear paradoxical in showing that degradation accelerates with synthesis in response to growth stimuli in many different organisms. (B)

TURNOVER

OF

UNITS

OF

CYTOPLASM

Although the hypothesis predicts that proteins will turn over at different rates because of the multitude of inherent and environmental factors which variously predispose them to hydrolysis (Bradley et al., 1975; Dice & Goldberg, 1975; Momany, Aguanno & Larabee, 1976; Stellwagen & Wilgus, 1978; Dice, Hess & Goldberg, 1979; Grisolia et al., 1980), an alternative hypothesis suggests that portions of the cytoplasm are engulfed at random by lysosomes, making for a greater uniformity in turnover rates. Uniformity of turnover, however, has been seen under special circumstances not specifically involving lysosomes, e.g. in the proteins of the endoplasmic reticulum in secreting cells (Chiu & Phillips, 1981) and in ribosomal proteins (Lastick & McConkey, 1976)t. (C)

PROTEOLYTIC

CAPACITY

If a cell produces a surplus of proteins which are then reduced to the requirements for growth, maintainance or secretion, a large proteolytic capacity must be present at all times. Grisolia (1964) and Grisolia & Hood t See first

footnote

on p. 292.

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(1972) have already pointed out that the proteolytic potential of a cell must always outstrip its synthetic capacity; in combination with the lysosomes, a whole cell can be autolysed in a comparative short space of time. Exactly how the separate aspects of this large hydrolytic potential are controlled is one of the most important areas for future endeavour in this field, and has been briefly discussed in recent reviews (Dean, 1980; Amenta & Brother, 1981). (D)

GENE

EXPRESSION

IN CYCLING

CELLS

The proposed mechanism for integrating new proteins into biomass makes quantity subservient to quality, i.e. a wide selection of proteins must be produced in order that those which are required can be assimilated on a chance basis. This has important implications for the genetic expression of cells going through division cycles. Contrary to expectation, it has been found that the pattern of proteins synthesized or, more specifically, retained throughout the cell cycle, show remarkably little variation in several cell systems (Al-Bader, Orengo & Rao, 1978; Fink & Zeuthen, 1978; Willard & Anderson, 1979; Ron & Zeuthen, 1980). This lack of distinction in gene expression could be due to the fact that the cell has to make a surplus of all proteins from which it takes those which it can assimilate. A random degradation theory would predict that the pattern of protein synthesis would not change during either a steady state condition or a steady state growth process. However, the overall expression is not inflexible in cycling cells since a perturbation such as a heat shock will suddenly and radically alter the pattern of proteins being synthesized (Ron & Zeuthen, 1980). It is impossible to be more precise because the genetic expression of cells has usually been examined after a considerable period of labelling (e.g. 1 hr in HeLa cells). This might simply reflect the fact that cells retain the same spectrum of proteins under steady state conditions whereas they retain quite different proteins after a shock, a matter which urgently needs to be resolved. In synchronous cells, gene expression should be analysed by electrophoresing labelled proteins immediately after a pulse and at appropriate intervals thereafter to reveal any differences which indicate changes in synthesis as opposed to protein stabilization. (E)

RETENTION

OF

PROTEINS

IN GROWING

CELLS

It follows from (D) that those proteins not retained (stabilized) in the quiescent or growing cell undergo rapid degradationi-. Retention was t See second

footnote

on

p. 292.

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thought initially to be so high in exponentially growing Esherichiu coli (Rotman & Spiegelman, 1954; Mandelstam, 1958), in yeast (Halvorson, 1958) and even in L-strain fibroblasts (King, Bensch & Hill, 1960) that no significant degradation of proteins was detected. Recent data (reviewed in Goldberg & St John, 1976) agrees that “residual protein” turnover rates for these organisms is about l-2% per hour. But does the rate of proteolysis alter retention and therefore affect the rate of growth of proliferating cells? This would give it a positive regulatory role rather than a largely passive one seen so far. Degradation is said to be particularly rapid in quiescent cells, suggesting to some that catabolism might indeed have some ability to regulate growth at more basal levels (Hendil, 1977; Lockwood & Shier, 1977). This observation might be explicable on the two-phase degradation process because the overall rate of degradation must be governed by the availability of protein substrate from both synthesis and destabilization. When synthesis is rapid and the cell is growing fast, degradation will appear to be relatively insignificant unless early proteolysis is included in the estimates. When cells are quiescent, synthesis falls very sharply and degradation becomes more obvious. The apparent increase in degradation of resident, existing proteins in cells moving towards quiescence is not primarily due to catabolism being less involuted than synthesis (Bradley, 1977), but to the fact that since few new proteins are being synthesized, only the destabilizing resident proteins are left at risk to the hydrolytic enzymes. Thus the absence of

significant competition between new and existing (labelled) proteins gives the impression that hydrolysis of the latter is abnormally exaggerated. Preliminary results from work on lymphocytes supports this interpretation (Knecht et al., 1981). (F)

ERROR

AND

ANALOGUE

PROTEIN

DEGRADATION

Early proteolysis helps to explain the marginally greater rate of proteolysis of error and analogue proteins in mammalian cells (Knowles et al., 1975; Wheatley et al., 1979). In the absence of some special system which can aggregate and denature such proteins, as in bacteria (Etlinger and Goldberg, 1977; Giddings & Wheatley, 1979), error proteins will nevertheless have less probability of being assimilated, and therefore run the gaunlet of random hydrolysis for longer. Once assimilated-as occurs with proteins containing one or two p-fluorophenylalanine residues (Wheatley, 1978)they show the same kinetics of turnover as their homologue proteins. Because the rate of the early phase of proteolysis is very high, analogue, error and normal proteins are subject to similar rapid rates of removal. It

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is unlikely that even a substantial number of error proteins will be detected by this means unless one looks specifically at hydrolysis rates of very short pulse-labelled proteins within 1 hr of chase. Since authentic proteins have a greater chance of being assimilated, their removal from the population through integration into the biomass gives the impression that degradation is behaving selectively. The interpretation offered by Knowles & Ballard (1976) is worth comparing in this connection. An extreme example of aberrant proteins being at risk is seen in the hypoxanthine-guanine phosphoribosyl transferase mutants described by Capecchi, Capecchi, Hughes & Wahl (1974). (G)

COMPLEXITY

OF

THE

DEGRADATIVE

SYSTEM

There has been a general change of opinion in recent years (Ballard, 1977) towards a two-tiered system of intracellular protein degradation. One tier is the lysosomal system which deals with exogenous material and autolytic activity. The other is a less well defined (in a structural sense), dispersed proteolytic system in the “cytosol” (Amenta & Brother, 1981), or in mitochondria (e.g. Mori et al., 1980; Grisolia et al., 1980). It is not our intention here to enter into the distinction between their respective roles and provinces of activity, but to comment on the complexity of the overall degradative system. There is no doubt that more than a few enzymes are involved in the cleavage of proteins. Furthermore, the importance of an extensive proteolytic system for turning over proteins required for cell growth and maintenance must be re-emphasized, because of the serious consequences to the cell should it lose its capacity to degrade proteins. This is highly improbable since it would require multiple gene mutations in the same cells, a situtation which naturally protects the cell from this eventuality. Work is only now beginning with mutants deficient in specific proteases, and this may clarify the complexity of the intracellular proteolyti ’ machinery (e.g. Vachova-Philippova & Chaloupka, 1978; Gottesman et al., 1981). 6. Concluding

Remarks

The hypothesis presented in this paper suggests two main episodes in the life of proteins when they are especially vulnerable to proteolytic activity. Their hydrolysis occurs on a random basis in both, giving separate and distinct first order kinetics. At this stage of our understanding there seems no need to postulate that different sets of hydrolytic enzymes are responsible. The proteolytic mechanism appears to be required to ensure

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that the cell can passively assimilate those proteins which are required to maintain, extend or alter the biomass without having to accumulate every one that has been made. The degradation of proteins, particularly nascent proteins, results in a flux through the cell (Fig. 3), which provides a continual stream of all proteins from which quality can be randomly selected at the expense of quantity. This also provides the cell with an eminently flexible system of responding quickly to a change in environmental conditions. In this respect, the protein flux is similar to the amino acid flux described earlier (Wheatley & Inglis, 1980). The rapid hydrolysis of proteins has been seen as a “seemingly wasteful process” (Schimke & Bradley, 1975), but within its limitations this method of maintaining a flux may perhaps be an efficient or optimal method of sustaining an adaptable, autonomous cell which can grow. This is the penalty in energetic costs that the cell must pay for this protective mechanism. Indeed, if the organism had a fixed amount of enzymes which could not be replaced continuously, it would be much harder to induce growth and particularly regeneration since cells and tissues are subject to all sorts of injurious and destructive agents. Schimke & Bradley (1975) concluded in their earlier studies that the proteolytic system in the cell had the task of providing quality control by removing inaccurately synthesized proteins and other anomalies from the sytem. The present hypothesis suggests that this will inevitably occur anyway, as a consequence of a much more important flux, i.e. the bulk of the quality control is concerned with the turnover of all proteins, particularly within the period immediately after their release from the ribosomes. The work was carried out during tenure in Valencia of a short term Fellowship from the European Molecular Biology Organization, for which D.N.W. is most grateful. The authors acknowledge the support of the Commission Asesora a Investigaciones of Spain, and the Fondo de Investigaciones Sanitarias la Seguridad Social. The computation of some of the curves derived in Fig. 1 was done by Mrs Marget Inglis of this Department who also provided excellent assistance in preparing other aspects of the manuscript. REFERENCES AL-BADER, A., ORENGO, A. & RAO, P. N. (1978). ALHANATY,E.,PATINKIN,J.,TAUBER-FINKELSTEIN,M. natn. Acad.

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