Autophagic degradation of cytoplasm (including protein, RNA etc.) is a non-selective bulk process, as indicated by ultrastructural evidence and by the similarity in autophagic sequestration rates of various cytosolic enzymes with different half-lives. The initial autophagic sequestration step, performed by a poorly-characterized organelle called a phagophore, is subject to feedback inhibition by purines and amino acids, the effect of the latter being potentiated by insulin and antagonized by glucagon. Epinephrine and other adrenergic agonists inhibit autophagic sequestration through a prazosin-sensitive alpha 1-adrenergic mechanism. The sequestration is also inhibited by cAMP and by protein phosphorylation as indicated by the effects of cyclic nucleotide analogues, phosphodiesterase inhibitors and okadaic acid. Asparagine specifically inhibits autophagic-lysosomal fusion without having any significant effects on autophagic sequestration, on intralysosomal degradation or on the endocytic pathway. Autophaged material that accumulates in prelysosomal vacuoles in the presence of asparagine is accessible to endocytosed enzymes, revealing the existence of an amphifunctional organelle, the amphisome. Evidence from several cell types suggests that endocytosis may be coupled to autophagy to a variable extent, and that the amphisome may play a central role as a collecting station for material destined for lysosomal degradation. Protein degradation can also take place in a 'salvage compartment' closely associated with the endoplasmic reticulum (ER). In this compartment unassembled protein chains are degraded by uncharacterized proteinases, while resident proteins return to the ER and assembled secretory and membrane proteins proceed through the Golgi apparatus. In the trans-Golgi network some proteins are proteolytically processed by Ca(2+)-dependent proteinases; furthermore, this compartment sorts proteins to lysosomes, various membrane domains, endosomes or secretory vesicles/granules. Processing of both endogenous and exogenous proteins can occur in endosomes, which may play a particularly important role in antigen processing and presentation. Proteins in endosomes or secretory compartments can either be exocytosed, or channeled to lysosomes for degradation. The switch mechanisms which decide between these options are subject to bioregulation by external agents (hormones and growth factors), and may play an important role in the control of protein uptake and secretion.

Abstract. Seven cytosolic enzymes with varying halflives (ornithine decarboxylase, 0.9 h; tyrosine aminotransferase, 3.1 h; tryptophan oxygenase, 3.3 h; serine dehydratase, 10.3 h, glucokinase, 12.7 h; lactate dehydrogenase, 17.0 h; aldolase, 17.4 h) were found to be autophagically sequestered at the same rate (3.5%/h) in isolated rat hepatocytes. Autophagy was measured as the accumulation of enzyme activity in the sedimentable organdies (mostly lysosomes) of electrodisrupted cells in the presence of the proteinase inhibitor leupeprin. Inhibitors of lysosomal fusion processes (vinhlasrine and asparagine) allowed accumulation of catalytically active enzyme (in prelysosomal vacuoles) even in the absence of proteolyric inhibition, showing that no inactivation step took place before lysosomal proteolysis. The completeness of protection by leupeptin indicates, furthermore, that a lysosomal cysteine proteinase is obligatorily required for the initial proteolytic attack upon autophagocytosed proteins. The experiments suggest that sequestration and degradation of normal cytosolic proteins by the autophagic-lysosomal pathway is a nonselective bulk process, and that nonautophagic mechanisms must be invoked to account for differential enzyme turnover.

Proteins sequestered by a non-selective bulk process within the lysosomes turn over with an apparent half-life of about 8 minutes and this rapid lysosomal proteolysis is initiated by endopeptidases, in particular by the cathepsins D and L. We describe also the cathepsins B and H which show mainly exopeptidase and only low endopeptidase activity. Especially cathepsin H is most probably the only lysosomal aminopeptidase in many cell types. Additionally, the properties of other mammalian lysosomal endo- and exopeptidases are compared. Finally, we discuss some of the conditions for the action of lysosomal proteases as the low intralysosomal pH, the high part of lysosomal thiol groups and the absence of intralysosomal proteinase inhibitors.

1. Cathepsin L was purified from rat liver lysosomes by cell fractionation, osmotic disruption of the lysosomes in the lysosomal mitochondria1 pellet, gel filtration of the lysosomal extract and chromatography on CM-Sephadex.2. Cathepsin L is a thiol proteinase and exists in several multiple forms visible on the disc electropherogram. By polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, its molecular weight was found to be 23000-24000. The isoelectric points of the multiple forms of cathepsin L extended from pH 5.8 -6.1 ascertained by analytical isoelectric focusing.3. Using various protein substrates, cathepsin L was found to be the most active endopeptidase from rat liver lysosomes acting at pH 6 -7. In contrast to cathepsin B1, its capability of hydrolyzing N-substituted derivatives of arginine is low and it does not split esters. 4. Greatest activity is obtained close to pH 5.0 with 70-90% of maximal activity at pH 4.0 and pH 6.0 and 30 -40 0 4 at pH 7.0.5. The enzyme is strongly inhibited by leupeptin and the chloromethyl ketone of tosyl-lysine. Leupeptin acts as a pseudo-irreversible inhibitor.6. The enzyme is stable for several months at slightly acid pH values in the presence of thiol compounds in a deep-frozen state.Knowledge of the proteolytic enzymes in the cell is one of the preconditions in studying the molecular mechanism of intracellular protein breakdown. All organelles contain proteolytic activity in various amounts [l]. The lysosomes, in particular, are rich in proteinases. A cell fraction enriched in lysosomes showed at pH 3-4 as well as at pH 6-7 the highest activity hydrolyzing cell-derived proteins in comparison to all other cell organelles [l -61. From this finding it was to be concluded, that the lysosomes certainly play an important role in the overall process of intracellular proteolysis also at physiological pH. A wide variety of lysosomal proteinases has been investigated : cathepsin A (lysosomal carboxypeptidase A) [7-131, cathepsin B1 [7,12-201, cathepsin B2 (lysosomal carboxypeptidase B) [7,12 -14,20-221, cathepsin C (dipeptidyl aminopeptidase I) [7,12,13, Dedicated to Professor Horst Hanson on the occasion of his 65th birthday.Ahbveviation. Leu-CH2C1, 1 -chloro-3-amino-5-methyl-~-hexan-2-one; Tos-Lys-CH2C1, 7-amino-l-chloro-3-tosylamido-~-heptan-2-one ; Tos-Phe-CHzC1, l-chlor0-4-phenyl-3-tosylamido-~-butan-2-one; Bz-L-Arg-NHZ, or-N-benzoyl-L-arginine amide.Enzymes. Cathepsin L (EC 3.4.22.-); cathepsin B1 and B2 (EC 3.4.22.1); cathepsinc (EC 3.4.14.1); cathespin D (EC 3.4.23.5). 18,23,24],cathepsin D [6,7,12,13,23,25-311, cathepsin E [7,13] In studies on the proteolytic activity in a lysosomal extract we succeeded in separating cathepsins BI, C and D by gel filtration on Sephadex G-75. With cytosol proteins as well as with azocasein as substrates at pH 6 and 7, the main part of proteolytic activity was shown to be present in the protein fraction with molecular weights between 20000 and 30000 [2,43]. We could show that in addition to cathepsin B1, the...

1. It has been found that cathepsin L is very susceptible to loss of activity through autolysis. When this is prevented by purification and storage of the enzyme as its mercury derivative, preparations are obtained with higher specific activity than previously. 2. Active-site titration shows, however, that even the new purification method does not give preparations in which the enzyme is 100% active. 3. Benzyloxycarbonylphenylalanylarginine 7-(4-methyl)coumarylamide has been discovered to be a very sensitive substrate for cathepsin L. Like all other known substrates for cathepsin L, however, it is also cleaved by cathepsin B. 4. Cathepsin L degrades insoluble collagen at pH 3.5 over 5-fold faster than at pH 6.0. The specific activity at pH 3.5 is 5-10-fold higher than that of cathepsin B (rat or human) or bovine spleen cathepsin N ('collagenolytic cathepsin'). 5. Qualitatively, the action of cathepsin L on collagen is similar to that of cathepsins B and N, i.e. selective cleavage of terminal peptides leads to conversion of beta- and higher components mainly to alpha-chains.