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Acta Biotechnol. 6 (1985)2, 129-13G

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Autolysis in Yeasts

BABAYAN,T. L.l and BEZRUEOV,M. G.a

1

Academy of Sciences of the U.S.S.R. A. N. Nesmeyanov Institute of Elementorganic Compounds

28

Vavilov Street, 117813 Moscow, U.S.S.R.

2

Allunion Research Institute for Biosynthesis of Protein Substances

27 Bolshaya Kommunisticheskaya Street, 109004 Moscow, U.S.S.R.

Summary

The term “autolysis” was introduced into biological literature by SALROVSRY[l]. Ever since it began to be used to designate self-digestion of cells under the action of their own intracellular enzymes. This definition is sufficiently satisfactorywith regard to bacteria, and there are published lots of original and review papers dealing specifically with bacteria. Considerably less material has been accumulated on eukaryotes, apparently due to the absence of the fact of the cell self- digestion in most yeasts.

Autolytic Processes and the Term ‘‘Autolysis”

According to STOLPEand STARR[2] “autolysis should be understood as a lythic event in the cells caused by the action of their own intracellular mureinase” (the enzymes that degrade peptidoglycans of the cell wall). Yet, the two limitations contained in this definition prevent it from being universal : (1) the assumed mureic organisation of the cell wall and (2) the action of solely mureinases as the cause of the process. The mureic type of cell wall is inherent in far from all bacteria, and in yeast and fungi its structure is totally different. The yeast cell envelope constitutes up to 30 percent of the cell dry weight [3]. It in-

cludesthe thick external cellwall(100 pm) and the thin internal cytoplasmaticmembrane

- CPM (10 pm). The external wall consists of some layers, containing glucan, mannan, protein, chitin etc. The CPM is chiefly of lipoprotein nature and, like the external wall, heterogeneous:two protein layers separated by a lipid phase are distinguished therein.

ROGERS[a] also associates autolysis with digestion of cell walls,

and STARRthe importance of the functional integrity of the CPM and other cell orga- nelles. It should be recalled here some bacteria forming autoplasts (functioning proto- plasts that had lost their walls) under the action of their own depolymerases or exo- ferments. If, in this case, the CPM remains undamaged, the cell is still able to restore its wall [5, 61, i.e. the cell is still not irreversibly damaged. The development of autolysis in microorganism cells may take place on two ways. The first one, beginning with loss of the cell wall under the action of its own hydro- lases is characteristic of “exo-type” autolysis, which is observed in most bacteria. The exo-type autolysis may terminate at this stage and not affect other cell components. The second way begins with disturbance of lipoprotein structures in the cell membranes

overlooking like STOLPE

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Act&Biotechnol. 5 (1985)2

(“endo-type” autolysis, to which fungi, yeasts, and certain kinds of bacteria are sub- ject). If denaturation of intracellular hydrolases takes place during disturbance in the cell, the process of autolysis is absent, albeit the cell dies [7]. Hydrolysis of the cell components may begin only following activation of hydrolytic endoenzymes. However, if hydrolase activation takes place within a limited scope of the cell and is genetically controlled, the cell does not die. At various stages of the life cycle, in definite cell sites, the need arises for degradation processes to prevail over synthesis. The regulatory role in living organisms is connected with so-called limited proteolysis (selective hydrolysis of specific peptide bonds) [S]. The latter underlies the various physiological processes that take place inside the living cells. New data on proteolytic system of yeast certify the free citosol peptidases to participate in cell regulation [9]. The mechanism of cell degradation in yeast during budding was found to involve the autolytic complex, which activates the chitin synthase precursor to lead to synthesis of chitin, forming a mem- brane and gemmating a daughter cell [lo]. LAHOSEand coworkers report on partici- pation of the autolytic complex enzymes in the process of wall growth in fungi [ll]. Besides, products released under the action of chitinase in fungi were shown to be used in morphogeneticprocesses.in fruit-bearingfungi, chitinaseplays a role in cap maturation to lead to spore release. In Neurospwu crassa, the proteases play the role of “softeners” in the cell wall before it branches [12]. In all these cases the balance relationships are moved towards degradation of cell biopolymers in specific loci. Yet, the cell does not die, since this violation is of reversible nature. It involves autolytic processes, but auto- lysis aa such does not occur. Hence, in our view, MATILE’S[13] definition, namely that autolysis should be seen as an “irreversible process entailed with cell death occurring as a result of disturbed regulation of the equilibrium work of enzymic systems towards prevalence of a hydrolytic system” may be recognised as the most accurate one. Yet, even this definition needs certain additions. We believe that hydrolysis of intracellular biopolymers under the action of endohydrolases entailed with cell death and the for- mation of low-molecular products releasing from the intracellular space, should be regarded as autolysis in yeasts. Apart from separating the process into the exo- and endo-types, autolysis is also clas- sified into “natural” and “induced” types, both of which are inherent in absolutely all microorganisms, irrespective of their taxonomy. In periodic cultivation of micro- organisms, cell autolysis at the end of the stationary phase is caused by natural ageing of the culture. Such autolysis is termed natural, and it is difficult to reveal any single basic cause of it’s development. The so-called “induced autolysis” of microorganism cells of any age, may be artificially caused by means of various physical, chemical, or biological agents.

Inductors of Microorganism Autolysis

Any extreme disturbance of physiological conditions specific to any organism, viz. the so-called “corresponding conditions”, may be the cause of autolysis.

Physical Inductors

(1) Temperature conditions, e.g. extreme rise in temperature [14], and also alternate freezing and melting [15]. In the first case, the development of autolysis is said to be caused by changes in membrane structure. In the second case, moreover, ice crystals form to result in mechanical violation of organell’s struct.ura1integrity. The autolysis rate depends on temperature, and the most optimal temperature interval for yeast was observed at 45-60°C [la, 161.

BABAYAN,T. L., BEZRUKOV,M. G.: Autolysis in Yeasts

131

(2)Osmotic pressure of the medium, e.g. increased NaCl content [171. Changed osmotic

conditions may have two consequences, i.e.

mation [18]. A third version of the process is also possible, when in certain NaCl and saccharose concentrations it is preceded by plasmolysis, i.e. by inner rearrangements unaccompanied by hydrolysis [191. (3) Irradiation by ultraviolet or X-rays which causes deep changes in regulatory and biosyntheticsl cell processes, but does not impair their autolytic capability [20]. (4) Mechanical disintegration of microorganism cells that leads to the liberation of the autolytic complex enzymes contained in lysosomes, and also those connected with the

complete cell degradation or autoplast for-

cell membranes and walls [21].

Chemical Inductors of autolysis

(1) Changes in solution ionic strength, its pH [22] and ion composition of the medium [23], since these factors can produce multiple effects leading to disturbed cell meta- bolic activity. The qualitative ion composition in the incubation medium is highly significant for the autolysis rate 1243. The influence of metal cations (group 11 metals) on yeast autolysis is used in invention [25] and discussed in [26]. (2) Various membranotropic compounds, including detergents, proteins, peptides and amino acids [27-321, that violate the structural entity and functional abihty of mem- branes. A mixture of the products of biomass autolysis [33] and casein proteolysis [34] are fine inductors of yeast autolysis. The degrading effect of these agents upon membranes is caused by their ability to soluhilize in the membranes, or to change the energetics of hydrophobic interactions at the expense of changed water structure, this leading to changed membrane permeabili- ty and leakage of the lysing enzymes. Conversely, some amino acids are inhibitors of autolysis [82], which inhibitory effect is dependent upon their hydrophobicity. (3) Antibiotics induce autolysis as a result of disbalance between synthetases and de- polymerases. Besides, whereas previously the thereby-caused disbalance was associated only with cell wall synthesis, [35] at present antibiotics have been proven to affect the membrane [36].

Biological Inductors

(1) Extent of aeration [37, 141. Removal of oxygen from an actively growing culture of aerobic microorganisms causes impaired energy supply to the cells. When a medium with anaerobic microorganisms is aerated, oxygen causes a toxic effect on the cells. (2) Partial [88, 391 or complete deprivation of the medium of sources of nutrition and energy, for example transfer of cells into buffer solutions [40,41]. Nitrogen limitation of Asperg illus nidulans results in activation of lisosomal proteases and inactivation of citoplasmatic inhibitors [42]. WELSH[43] formulated a hypothesis, according to which any disturbance in cell energy supply, irrespective of the cause, induces autolysis. However, it was shown [44] that inhibition of the cell energy-supply systems for example limited ATP supply, does not always results in autolysis, but support anabiosis. An analysis of the few examples of inductors of eukaryotic cell autolysis cited here permits to conclude that inductors of varying nature may cause similar cell disturbances both functional and structural. E.g., the temperature effect and the action of deter- gents are reduced to changing membrane structural organisation. Removal of oxygen from the growing aerobic culture leads to disbalance between the synthetic and auto- lytic enzyme systems, i.e. to the same effect produced by lactamic antibiotics.

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Characteristics of the Process of Autolysis

Autolysis in fungi and yeasts is accompaniedby characteristic visually-observedchanges, including changes in their rheology and colour [161. The presence of at least two stages of autolysis was exemplified by changed morpholo- gy of a yeast cell studied by electron microscopy [45]. The first stage of autolysis at a submicroscopic level manifests itself in degradation

of ceH endostructures : turgor is

fragments are discernible; all the cell content is uniformly distributed over a space restricted by the cell wall; the cell diameter reduces almost 1.5-fold. Compared with the starting cell, the cell wall is considerablythickened, but its integrity is not destroyed. The second stage of autolysis involves hydrolysis of intracellular biopolymers, which

absent ; neither the organelles themselves nor their

leads to diffusion of the hydrolysis products into the extra-cellular medium. However, at pH 5-6, the lipids aggregate in the form of a large drop and do not pass beyond the cell bounds. The autolytic changes in the S. cerevisiae cells induced by starvation [40, 461 were shown to be distinguished at a submicroscopic level much later, than by additives- induced one (after 2-3 days instead of 2-3 h, respectively). The authors note the in- tactness of mitochondria up to the very latest stages of autolysis, this being in contra- diction with the evidence of [45, 471, who observed these organelles to digest during autolysis. The number of autolysing cells in a unit of medium volume did not change; however “Many of them assumed an irregular form” [40]. It signifies that yeast autolysis does not involve clarification of the yeast suspension, whose optical density remains vir- tually unchanged. The rate and depth of autolysis in bacteria is known to be assessed by a decrease in the optical density of the cell suspension [19]. Since this criterion is unacceptable for yeast, investigators resort to characterising protein hydrolysis pro- ducts; the degradation products of other biopolymers may also be measured, for exam- ple those of nucleic acids [48]. In the process of autolysis the cell wall in niost yeasts undergoes only certain structural modifications ; however, its completeness is retained, this being observed also at t.he *endof the process, and the cytoplasmatic material gradually diffuses into the extra- cellular space [49, 16, 451. This kind of autolysis (“endo-type”) in yeast is similar to that in Bac. subtilis described by KOGAand KUSAKA[50], who observed hollow cells (shadows). In both cases, autolysis begins with CPM degradation. However, autolysis in certain fungi, e.g. in Schizophyllum commune, is found to involve both CPM and the cell wall degradation, explained by the highly active endo- and exo- glucanases in these organisms [51]. Autolysis depends on the culture age and the physiological conditions in the cell [52]. Proliferating culture cells autolyse quicker and to a higher degree than cells of the statio- nary growth phase [53, 541, for autolytic enzymes are synthesizing most intensively precisely in the cells of the exponential growth phase. Hence, autolysis occurs when induction by means of a given factor not only fails to lead to degradation of autolytic enzymes, but when there are optimal conditions for their action. In natural autolysis at the end of the stationary phase, when culture growth is hindered and the culture is considered aged, the cell physiological conditions are such that all together they promote autolysis [55-571. Natural autolysis caused by ageing of the microorganism culture has been studied well in fungi [58-611. RIEMEYand TROGER [59] note that autolysis in yeast differs little from that in other fungi and called it micolysis.

BABAYAN,T. L., BEZBIJKOV,M. G. : Autolysis in Yeasts

133

Enzymes of the Autolytic Complex

Hydrolytic enzymes are responsible for autolytic degradation of most cell polymers, the main role in this process in yeast belongs to proteases, nucleases, and glucanases. Since yeast autolysis is said to be associated with initial degradation of the CPM con- taining protein-lipid complexes, it would be logical to assume the involvement in the process of phospholipases [62]. A relevant work [63] indicates the possibility of mito- chondria degradation at the expense of phospholipases A and D during glucose repres- sion of the yeast s.cerevisiae. Works performed on fungi and yeasts [51,30] are indicative of the major role of gluca- nases in the autolytic complex. Ten enzynies were revealed in the autolysate extra-

cellular fluid of A. niger, including

neither mycodextranases, nor proteinases were detected. In fungus Neurospora crussa, the investigators [61] succeeded in establishing the de- pendence of the autolytic complex composition upon cultivation conditions, i.e. air flow, mixing rate, and temperature. Earlier, five intracellular proteinaaes were isolated from the same fungus and purified [MI. A cell-destroying enzyme complex was studied in Schizophyllum commune [51]. Fluc- tuations in the activities of 1,3-a-glucanase (exoglucanases),1,3-(4)-,9-glucanase(endo- glucanase), wamylase, and invertase were observed in the extracellular solution and mycelium of that organism. During autolysis, the activity of these eneynies increases to fall by the end of autolysis (with the exception of that of &-amylase,whose activity continues to be high). Proteases, nucleases and esterases were isolated from S. cerevisiue vacuoles. Similar

Subsequently, aminopeptidases and other

vacuoles were permitted to be lysosomes [65].

enzymes of the lytic complex were identified in the enzyme composition of yeast lyso- sonies [66, 671 and citosol[41]. A distinctive feature of the lysosome complex is its sets of enzymes, which differ in substrate specificity and ensure complete hydrolysis of the respective substrates. At least seven proteases and peptidases take part in protein hydrolysis. The following should be considered as important principles in the action of lysosome enzymic groups :

(1) consecutiveness, (2) absence of narrow specificity and resultant deep splitting of biopolymers to low-molecular products. The resistance of lysosomal enzymes to the action of hydrolases is explained by the linkage with carbohydrate prosthetic groups

chitinases and (1,3),9-glucanases; in this case,

[681.

MATILEsuggested that the resistance of proteolytic enzyniesto self-digestionis allegedly caused by the spatial separation of the proteolytic enzymes and their substrates in the

cell. Autolytic protein degradation becomes possible only after this situation is violated and biosynthesis is delayed; yet, in this case too, a systeni of inhibitors may prevent

protease, and inhibition is of competitive

nature. Inhibitors represent the proteins pepstatin, chimostatin, and antipapain, the first one inhibiting only proteinase A, and the other two, - proteaae B. In S. cerevisiae, proteases A, B and C were found to be present in active form in the vacuole, and the respective inhibitors in the cytosol. During the mechanical or other disturbance the proteases are released from the vacuoles to combine with corresponding inhibitors. The proteases break away from the inhibitors after the latter are digested to subse- quently perform non-selective proteolysis of other yeast proteins. Inasmuch as the inhi- bitors are separated from their proteases, it would seem that they cannot be regulators of protease activity [70]. The available data in extracellular enzymes in yeasts are controversial: some of them indicate the presence of proteolytic activity in yeast autolysates 1711, and others the

autolysis [69]. Every inhibitor is specific to its

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Acto Biotechnol. 5 (1985) 2

complete absence of proteolytic, nucleolytic and amylolytic activity [45]. The extra- cellular lysing enzymes found in numerous microorganisms are assumed to be evolutio- nally related with autolysins [72]. The review papers [73, 741describe in detail the bio- chemical characteristics of the endo-proteases and endo-peptidases and their inhibitors detected in yeast.

Localisation and Action Mechanism of Autolytic Complex

ARNOLD[27] found the autolytic enzymic system of living yeast cell to be localised on the inner side of the cytoplasma membrane. The others reported that hydrolases are localised in the periplasma [59] and the cell wall [75]. Yeast cell walls were used to iso- late the enzymes fixed therein; most of these enzymes proved to belong to the class of hydrolases [76, 771. The majority of investigators do not refute that hydrolases are localised in the cell walls, both in prokaryotes and eukaryotes. There is evidence on existence of free peptidases in yeast citosol[9], and proteases with pH optimum 8.0-8.5 in yea& nuclei [77]. And still the largest number of hydrolases in yeast are concentrated in the lysosomes [41,78]. At present there is no exact understanding of the mechanism of autolysis in yeast, whereas in bacteria there are several logically grounded relevant hypotheses 179-811. The analysis of available materials permits to conclude the presence of at least four stages of the autolytic process in yeast [82,83].

1. The first step involves a disturbance of supramolecular intracellular structures that violates the spatial apartment of hydrolytic enzymes and of their substrates at, the ex- pense of any effect upon the lipoprotein membranes of lysosomes, CPM and other organelles. 2. The second step involves interaction of released enzymes with the cytosol inhibi- tors. Interacting digestion begins through cross proteolysis of inhibitors that specifi- cally inhibit a corresponding enzyme. Inhibitor hydrolysis results in activation of the hydrolytic enzymes themselves. 3. In the third step, activated enzymes interact with intracellular polymer components (substrates), resulting in accumulation of their hydrolysis products in the space re- stricted by the cell wall.

4. In step four, as the molecular mass of the hydrolysis products decreases to an extent commensurable with the size of the cell wall pores, the said products diffuse into the extracellular medium.

The main autolysis products found outside the cell are oligopeptides, amino acids, nucleic components, and sugars. At pH physiological. value (5-6), lipids aggregate to remain inside the cell wall. Throughout autolysis, the cell wall substantially loosens, but maintains the properties of semipermeable membrane and retains about 50 per cent of initial total nitrogen [46]. The process terminates with exhaustion of hydrolytic activity resulting from enzyme selfdigestion. As was noted above, at present there are still many unstudied and unresolved issues with regard to the regulation and physiological role of autolytic enzymes in eukaryotes. The comprehension of these processes will ensure to control such physiological pro- cesses as biomass growth, prevention of premature cell death, quickened germination of producent spores, etc. All these tasks are on the agenda of present-day biotech- nology.

Received June 4, 1984

BABAYAX,T. L., REZRUKOV,M. G. : Autolysis in Yeasts

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