Bohley, P.

Hanson, Horst

Neupert, Walter

Rücker, Axel von

English

Open Access LMU

INTRACELLULAR PROTEIN CATABOLISM Proceedings of the Symposium Reinhardsbrunn Castle German Democratic Republic May 1973 Edited by HORST HANSON and PETER BOHLEY Wissenschaftlich* Beiträge der Martin-Luther-Universität Halle-Wittenberg 1974/6 (R 27) Halle (Saale) Kommissionsverlag: Johann Ambrosius Barth, Leipzig Redaktion: 401 Halle (Saale), August-Bebel-StraBe 13, Deutsche Demokratische Republik, Ruf: 832 541 und 8 81 47. Herausgegeben durch die Martin-Luther-Universltöt Halle-Wittenberg. Für unverlangt eingehende Manuskripte kann keine Haftung übernommen werden. Die Autoren ver-öffentlichen im Dienste der internationalen wissenschaftlichen Verständigung ohne Honorar. Die Verlagsrechte der veröffentlichten Arbelten liegen bei der Martln-Luther-Universltät Halle-Witten-berg. Die Beitröge erscheinen in 18 Reihen in unregelmäßiger Folge. Die Zählung der Beiträge erfolgt jahrgangsweise, die der Reihen unabhängig vom Jahrgang. Bezugsmöglichkeiten: Die Betträge werden Ober den. internationalen Schriftentausch der Biblio-theken verbreitet. Es besteRen beschränkte Bezugsmöglichkelten Ober den Buchhandel. Bestellungen von Buchhändlern nehmen entgegen In der Deutschen Demokratischen Republik: LKG VA 074 701 Leipzig, Postfach 520; in den sozialistischen Ländern: die zuständigen Binnenhandelsunternehmen: in der Bundesrepublik Deutschland: die bekannten Kommissions- und Großbuehhandlungen; In Österreich: Globus-Buchvertrieb, Wien I, Salzgries 10: Im übrigen Ausland: Johann Ambrosius Barth, DDR - 701 Leipzig, SalomonstraBe 18b, Postfach 109, Telefon 2 58 49. Telegramm-Adresse: 25 245 Barth Leipzig. Oesamtherstellung: Druckhaus Freiheit, Hatte Mtau-Nr. 123/13/75 LSV 2015, Bestell-Nr. 793 431 0 EVP 50,- M Contents Preface Properties of lysosomes and their possible role tu protein turnover /B* Poole and C» d8 Duve (New York) Studies on i n t r a c e l l u l a r proteases with r a d i o -iodinated p r o t e i n S u b s t r a t e s J. L. Mego (Alabama) On the relationships between i n t r a c e l l u l a r and secreted proteolytic enzymes V.M. Stepanov (Moscow) Properties of cathepsins from f i s h rauscle Gr. Siebert (Stuttgart) Rates of turnover df cerebral proteins A. Lajtha, D. Dunlop and N. Marks (New York) Studies ön the mechanism of degradation of ce l l u l a r proteins B. Poole and M. IVibo (New York) In vivo h a l f - l i f e times of rat l i v e r proteins M. Miehe (Halle) On the biochemistry of intra c e l l u l a r protein catabolism C. Schwabe (Charleston) Protein turnover and c e l l u l a r environnrent D. J. Millward and P. J. Garlick (London) Degradation of mitochondrial translation produots in Feurospora Crassa A.V. RUcker, R. Michel and W. Neupert (München) Degradation of induced tyrosine aminotransferase F. Auricchio (Naples) Digestion of proteins and Peptides by extracts of rat l i v e r lysosomes J.l3Lfü* Bouma, vi. Huisman and M. Gruber (Groningen CharacterizatioD of a macromolecular protease-inhibi t o r from yeast Takeyöri Saheki, H. Betz and H. Holzer (Freiburg i . Br.) Degradation of rat l i v e r proteins P. Bohley, H. Kirschke, J. Langner, B. Wiederanders, 3. Ansorge «and H. Hanson (Halle) Cathepsin L and proteinases with cathepsin-B 1-like a c t i v i t y from rat l i v e r lysosomes H. Kirschke, J. Langner, B. Wiederanders, S. Ansorge, P. Bohley and H. Hanson (Halle) * Bnzyme inactivation studies as tool for the characterization of lysosomal proteolytic enzymes J. langner, B. Wiederanders, S. Ansorge, P. Bohley, H. Kirschke and H. Hanson (Halle) On cathepsin D of rat l i v e r B. Wiederanders, S. Ansorge, P# Bohley, H. Kirschke, J. Langner and H. Hanson (jHalle) Some properties of human cathepsin B 1, the binding of the enzyme h y ck2^3-07:0glob\xl±n9 and the role of p " i n a c r o g ^ 0 ^ u l i n * D physiological control of proteolytic a c t i v i t y A.J. Barrett (Cambridge) Contribution to Studies on the active s i t e of cathepsin B 1 and D H. Keilova and V. Tomasek (Prague) An improved purification of cathepsins B 1 and B 2 from bovine spieen and on the possible .occurence of isoenzymes of these proteases K. Otto (Bonn) Some properties of cathepsin D isolated from different organs Y. Türk, J. Kregar, F. Gubensek, R. Smith and S. Lapanje (Ljubljana) Leuoocyte proteinases and protein digestion M. Kopitar. V. Türk, V. Cotic and M. Stegnar (Ljubljana) k h e degradation of peptide hormones A.J. Kenny, J.R. Fullarton, S.'G. George and M.A. Kerr t^eeds)' Neurohypophysiol hormones, enzymic inactivation and i t s significance" for the synthesis of metabolically more s table analogues JC. Barth (Prague) Thiol-proteindisulfide oxidoreductase of rat l i v e r -studies in.the breakdown of insulin ^ ß. Ansorjge, P. "Bohley, H. Kirschke, J. Langner, EB. Wiederanders and H. Hanson (Halle) Action pf chymotrypsin om intact Oxytocin" and a linear derivate P. L. Hoffmann and R. Walter (New York) Proteolytic enzymes in Langerhans i s l e t s H. Ztihlke, H. Jalir, S. Schmidt, .D. Gottschling and B. if/ilke (Karlsburg) Insulin breakdown in pancreatic i s l e t s K.B. Kohnert, H.J. Hahn, H. ZUhlke, S. Sahmidt, M. Ziegler and H. Fiedler (Karlsburg) Degradation of mitöchondria in reticulocytes • T. Schewe," W. Krause, S-. Rosenthal and S.M. Rapoport (Berlin) Degradation of ribosomes.in reticulocytes during the maturation process S. Prehn, S. Rosenthal and S.M. Rapoport (Berlin) Studies on "neutral" peptide hydrolases from tissue and blood plasma V.N. Orekhovich (Moscow) Proteolytic Bnzymes in blood ce l l s R.J. Haschen (Halle) Proteolytic enzymes involved in the autolysis of rat skeletal muscle A. Jakubiec-Puka, W. Drabikowski and A. G6recka (Warsaw) 8 Die Wirkung der Denervation auf die proteolytischen Bnzyme des Muskels F. Guba, 0. Takacs, Z. Kiss und T. Pelle (Sseged) T&e role of proteolysis in the protein metabolism "of plants G„ Kühner (Leipzig) Intracellular proteolytic a c t i v i t y in Bacillus megatherium under different physiological conditions J. Chaloupka, V. Obdrzalek and M. Nesmejanova (Prague) Changes of the structure, proteolytic s u s c e p t i b i l i t y and b i o l o g i c a l h a l f - l i f e time of fibrinogen after , treatment of rabbits with cobaltous ions S. Krantz and^Maria Lober (Greifswald) x Occurence and properties of arginine aminopeptidase K.K. Mäkinen (Turku) Investigations into the multiple forms of L-alanyl-peptide hydrolase H. J. HUtter, V / . Gerlach, L. Neef, R.J. Haschen and I. Marquardt (Halle) Arylaminopeptidase N from rat kidney microsomes. -Comparative characterization of the purified eneyme, solubilized by trypsin treatment or acid dissociation of the phosphatase-arylamidase cqmplex R. Kleine, J. Schubert and G. Strauch (Halle) Arylamidase S from gastric juice of the crayfish R. Kleine and Ch. »/agner (Halle) Investigations aboutthe binding of Substrates and inhibitors by bovine eye lens leucine aminopeptidase 3. Fittkau, U. Förster, C. Pascual and äl.-HASchunk (Halle) Protein degrading lysosomal enzyme a c t i v i t i e s i n human l i v e r disease and in experimental animal l i v e r damage U. Stein and H. Heißmeyer (Freiburg i . 3r.) Bvidence £or adsorbed proteolytic a c t i v i t y OD isolated yeast phosphofructokinase: aspeots of subunit composition and kinet.io properties W. Diezel, H.-J. Böhme, K. Nissler and B. Hofmann (Leipzig) 492 Technical applications of proteolytic enzymes B. v. Hofsten (Uppsala) 501 Adaptive behavior of muscle Protease in protein wasting c o n d i t i o n 3 M. Mayer, R. Amin and B. Shafrir (Jerusalem) 510 The lysosomal chain reaction W. KrUger (Dresden) 519 Regulation of protein catabolism in B. c o l i A. Goldberg (Boston) 523 Recent observat'ions on the control of protein degradation in skeletal muscle A. Goldberg (Boston) 548 Some remarks concerning the naming and description of tissue proteinases A.J. Barrett. H. Keilova, H. Kirschke, K. Otto, G. Siebert, v.M. Stepanov and V. Türk 555 Author index 559 165 Degradation of Mitochondrial T r a n s l a t i o n Products i n Neurospora crassa A. v. Rücker, R. Michel and W. Neupert I n s t i t u t für Physiologische Chemie und P h y s i k a l i s c h e Biochemie der Universität München Several mitochondrial membrane proteins (Cytochrome oxidase, Cytochrome b, ATPase) (1) are composed of Polypeptides synthesized a t cytoplasmic and at mitocbonrial ribosomes. The formation of thesfe p r o t e i n s , therefore, requires a coordinated supply of bpth types of translation products. I f one of the translation Systems is blocked, the unblocked system continues to form Polypeptides (2). What happens tö these. precursors, which cannot be assembled to integral complexes of the mito-c h o n d r i a l inner membrane? This question was studied i n the case of -the t r a n s l a t i o n products formed i n the mitochondria. Two experimental procedures are widely used to study the mitochondrial t r a n s l a t i o n process: (a) Incorporation of amino acids i n t o p r o t e i n of i s o l a t e d mitochondria; (b) Incorporation ' of amino a c i d s i n t o mitochondrial p r o t e i n i n whole c e l l s , i n which cytoplasmic t r a n s l a t i o n i s i n h i b i t e d by cycloheximide. In both cases cytoplasmic t r a n s l a t i o n products are e i t h e r t o t a l l y absent, or are.present u n t i l a c e r t a i n pool i s exhausted. 1» T r a n s l a t i o n i n i s o l a t e d mitochondria Incorporation of r a d i o a c t i v e leucitie i n t o TCA p r e c i p i t a b l e p r o t e i n of i s o l a t e d Neurospora mitochondria occurs f o r about 30-40 min. Following t h i s period a decrease of incorporated r a d i o a c t i v i t y i s observed (F i g . 1). A l s o , a f t e r a chase of unlabelled l e u c i n e which e f f e c t s that no more l a b e l l e d Poly-peptide chains are formed, a continuous decrease of TCA p r e c i p i -t a b l e r a d i o a c t i v i t y o r i g i n a l l y present i s noticed. T h i s r e a c t i o n can be t r i g g e r e d regardless whether inc o r p o r a t i o n proceeds or 166 Fig., 1. Incorporation of r a d i o a c t i v e l e u c i n e i n t o i s o l a t e d Neurospora mitochondria. I s o l a t e d mitochondria were incubated i n a medium described by Sebald et a l . (6) with 3-H-L-leucine (32 uM, 70 uCi/ml) at 25°C. Mitochondria were p r e c i p i t a t e d with TCA and washed as described (6). R a d i o a c t i v i t y was determined on glas ^ f i b r e f i l t e r s with a counting e f f i c i e n c y of 20%. At the points i n d i c a t e d e i t h e r u n l a b e l l e d l e u c i n e ( f i n a l concn. 3 mM) or puromyein ( f i n a l conen. 0.4 mM) were added. F i g . 2» Degradation of mitochondrial t r a n s l a t i o n products a f t e r l a b e l i n g of i s o l a t e d mitochondria with r a d i o a c t i v e l e u c i n e . I s o l a t e d mitochondria were incubated with 3-H-L-leucine under the same conditions described f o r F i g . 1. At the time p o i n t i n -di c a t e d a p o r t i o n of the ineubation mixture was cooled to 0°C. The mitochondria were repeatedly washed i n I s o l a t i o n medium (4) and resuspended i n ineubation medium .at 25°C. A l i q u o t s were su c c e s s i v e l y withdrawn. the mitochondria were spun down a f t e r immediate c o o l i n g to 0 C and r a d i o a c t i v i t y was measured i n the supernatant and i n the TCA p r e e i p i t a t e of the mit o c h o n d r i a l p e l l e t . 167 has ceased ( F i g . 1). P r a c t i c a l l y the same e f f e c t i s observed, i f p r o t e i n synthesis i s blocked by adding puromycin ( F i g . 1) or chloramphenicol. S i m i l a r observations were made by Wheeldon and Lehninger with r a t l i v e r mitochondria (3). In a f u r t h e r experiment, i n c o r p o r a t i o n was stopped a f t e r 30 min, free leucine was removed by repeated washing of the mitochondria.. Then the mitochondria were resuspended i n ineu-b a t i o n medium not containing l e u c i n e . üpon f u r t h e r ineubation at 25°C the same decrease of TCA p r e c i p i t a b l e r a d i o a c t i v i t y as described i n F i g . 1 can be seen. The r a d i o a c t i v i t y l o s t from the mitochondria i s found i n the supernatant ( F i g . 2). Double l a b e l l i n g experiments show that only newly formed m a t e r i a l i s r e l e a s e d but not breakdown products of p r e e x i s t i n g p r o t e i n components. These r e s u l t s s t rongly suggest that mitochondrial trans-l a t i o n products are broken down by p r o t e o l y t i c a c t i v i t y . I t appears that i n i s o l a t e d mitochondria a simultaneous formation and breakdown of Polypeptide chains takes place. 2. T r a n s l a t i o n i n whole c e l l s i n the presence of cycloheximide I f mitochondrial t r a n s l a t i o n i s studied i n cycloheximide t r e a t e d c e l l s , i t i s observed that the amount of r a d i o a c t i v e products appearing i n the mitochondrial membrane i s h i g h l y dependent on the time period, f o r which these c e l l s were pre-ineubated with the i n h i b i t o r . A f t e r preineubation of the c e l l s f o r one hr with cycloheximide, the amount of r a d i o a c t i v e l e u c i n e which i s incorporated i n t o TCA p r e c i p i t a b l e m a t e r i a l of the mitochondria i s g r e a t l y diminished ( F i g . 3). This d e c l i n e can have two d i f f e r e n t reasons: (a) Mitochondrial t r a n s l a t i o n i s blocked by some regulatory mechanism, or (b) Mitochondrial t r a n s l a t i o n continues, but the products are not any longer i n t e -grated i n t o the membrane and are degraded by p r o t e o l y t i c enzymes a f t e r release from the ribosomes. The f o l l o w i n g r e s u l t s are i n agreement with the second assumption: (a) Mitochondria i s o l a t e d from c e l l s which were kept i n the presence of cycloheximide f o r 4 hrs, incorporate amino acids to an extent comparable to that of c e l l s not t r e a t e d with the i n h i b i t o r ( F i g . 4). (b) Mitochondrial ribosomes i s o -l a t e d from c e l l s which were kept f o r one hr i n the presence of 168 Tim, period of cycloheximide pcincubotion (rn.nl r > m t o f M ) C u b a | | 0 n { m j n , Fig. 3 F i g . 4 F i g . 3. E f f e c t of the time period of cycloheximide ineubation of Neurospora c e l l s on the i n c o r p o r a t i o n of r a d i o a c t i v e .leucine i n t o mitochondria i n vivo Neurospora c e l l s were preineubated with cycloheximide (0.1 mg/ml) fo r the time periods i n d i c a t e d . Then 3-H-L-leucine was added Q (0.4 uCi/ml). A f t e r 4 min the c e l l s were r a p i d l y cooled to 0 C, mitochondria were i s o l a t e d and the incorporated r a d i o a c t i v i t y was determined. F i g . 4. Incorporation of r a d i o a c t i v e l e u c i n e i n t o i s o l a t e d mitochondria from normal and cycloheximide preineubated Neurospora c e l l s . A c u l t u r e of Neurospora c e l l s was d i v i d e d i n t o two equal po r t i o h s . From one p o r t i o n mitochondria were immediately i s o l a t e d . To the other p o r t i o n cycloheximide was added and a f t e r 4 hrs mitochondria were i s o l a t e d . 3-H-L-leucine was incorporated i n t o mitochondria under the conditions described i n F i g . 1. 169 Time of ineubation (min) F i g . 5. Incorporation of r a d i o a c t i v e leucine i n t o mitochondrial membranes and i n t o t r a n s l a t i o n products on mitochondrial ribosomes i n cycloheximide treated Neurospora c e l l s . To a c u l t u r e of Neurospora c e l l s cycloheximide and a f t e r 2.5 min 3-H-L-leucine (1 uCi/ml) were added. A f t e r the time periods i n d i c a t e d , a l i q u o t s of the c u l t u r e were withdrawn. Mitochondrial membranes and mitochondrial ribosomes were i s o l a t e d and r a d i o a c t i v i t y was measured. 170 cycloheximide and of r a d i o a c t i v e l e u c i n e , s t i l l c ontain about one t h i r d to one h a l f of the nascent r a d i o a c t i v e t r a n s l a t i o n products (4) of the zero time c o n t r o l ( F i g . 5). Since the c e l l u l a r pool of f r e e leucine i s increased to about three times i t s normal s i z e by cycloheximide ineubation (5), i t can be con-cluded that the ribosomes are a c t i v e i n t r a n s l a t i o n to an extent s i m i l a r to that a f t e r Short cycloheximide treatment. These observations suggest that mitochondria contain proteo-l y t i c enzyme a c t i v i t y which may have the f o l l o w i n g funetions: (a) Removal of incomplete or aberrant mitochondrial t r a n s l a t i o n products. (b) A regulatory funetion to remove excess t r a n s l a t i o n products which cannot be integrated i n t o the membrane and assembled to f u n c t i o n a l complexes, because t r a n s l a t i o n counter-parts of cytoplasmic o r i g i n are missing. (1) A. T z a g o l o f f , M.S. Rubin and M.F. S i e r r a (1973) Biochim. Biophy. Acta 301., 71. (2) W. Sebald, H. Weiß and G. J a c k l (1972) Eur. J . Biochem. 30, 413. (3) L.W. Wheeldon and A.L. Lehninger, (1966) Biochemistry 5, 3533. (4) W. Neupert, W. Sebald, H. Schwab, P. Massinger and Th. Bücher (1969) Europ. J . Biochem. 10, 589. (5) A. J . Schwab, W. Sebald and H. Weiß (1972) Eur. J . Biochem. 30, 511. (6) W. Sebald, Th. Bücher, B. O l b r i c h and F. Kaudewitz. (1968) FEBS L e t t . 1, 235. 

Degradation of mitochondrial translation products in Neurospora crassa

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Degradation of mitochondrial translation products in Neurospora crassa

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