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Autolysis in Yeasts
BABAYAN, L.l and BEZRUEOV, G.a T. M.
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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. 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 Ever since it [l]. 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 selfdigestion in most yeasts.
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(endo-type autolysis, to which fungi, yeasts, and certain kinds of bacteria are subject). I denaturation of intracellular hydrolases takes place during disturbance in f the cell, the process of autolysis is absent, albeit the cell dies [ ] Hydrolysis of the cell 7. 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 membrane and gemmating a daughter cell [lo]. LAHOSE coworkers report on particiand 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 autolysis aa such does not occur. Hence, in our view, MATILES [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 formation 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 classified into natural and induced types, both of which are inherent in absolutely all microorganisms, irrespective of their taxonomy. In periodic cultivation of microorganisms, cell autolysis a t 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 its 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.
(1) Temperature conditions, e.g. extreme rise in temperature [14], and also alternate freezing and melting [15]. I n the first case, the development of autolysis is said to be caused by changes in membrane structure. I n the second case, moreover, ice crystals form to result in mechanical violation of organells struct.ura1integrity. The autolysis rate depends on temperature, and the most optimal temperature interval for yeast was observed at 45-60C [la, 161.
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( 2 ) Osmotic pressure of the medium, e.g. increased NaCl content [171. Changed osmotic conditions may have two consequences, i.e. complete cell degradation or autoplast formation [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 cell membranes and walls [21].
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 detergents are reduced to changing membrane structural organisation. Removal of oxygen from the growing aerobic culture leads to disbalance between the synthetic and autolytic enzyme systems, i.e. to the same effect produced by lactamic antibiotics.
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complete absence of proteolytic, nucleolytic and amylolytic activity [45]. The extracellular lysing enzymes found in numerous microorganisms are assumed to be evolutionally related with autolysins [72]. The review papers [73, 741 describe in detail the biochemical 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 isolate 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 expense 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 inhibitors. Interacting digestion begins through cross proteolysis of inhibitors that specifically inhibit a corresponding enzyme. Inhibitor hydrolysis results in activation of the hydrolytic enzymes themselves. 3. I n the third step, activated enzymes interact with intracellular polymer components (substrates), resulting in accumulation of their hydrolysis products in the space restricted by the cell wall. 4. I n 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 processes as biomass growth, prevention of premature cell death, quickened germination of producent spores, etc. All these tasks are on the agenda of present-day biotechnology. Received June 4, 1984
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