Selective release of peptides from lysosomes.

We demonstrate a selective release of peptides by lysosomes in vitro. A lysosomal fraction from human fibroblasts that had previously endocytosed [3H]ribonuclease A was incubated for 2 h, and radioactivity released into the medium and radioactivity retained within lysosomes were analyzed. A variety of radiolabeled molecules including peptides of an appropriate size to serve as antigens for T cell-mediated immunity were released. One small peptide was predominantly released, while others, as well as intact ribonuclease A, were predominantly retained. A 4-5-fold range was also evident in the relative release of three 3H-labeled tripeptide probes of similar charge derived from the sequence of ribonuclease A. This selectivity and the fact that similar peptide degradation fragments were also released and retained by intact cells after endocytosis of [3H]ribonuclease A argues strongly that the release observed in vitro is physiological and not due to damaged lysosomal membranes.

A selective pathway for degradation of cytosolic proteins by lysosomes.

A lysosomal pathway of proteolysis is selective for cellular proteins containing peptide sequences biochemically related to Lys-Phe-Glu-Arg-Gln (KFERQ). This pathway is activated in confluent cultured cells that are deprived of serum growth factors and in certain tissues of fasted animals. We have reconstituted this lysosomal degradation pathway in vitro. Transport into lysosomes requires a KFERQ-like sequence in the substrate protein and uptake and/or degradation is stimulated by ATP. A member of the heat shock 70 kDa protein family, the 73 kDa constitutive heat shock protein, binds to KFERQ-like peptide regions within proteins and, in some as yet unidentified manner, facilitates transfer of the proteins into lysosomes. Several possible mechanisms of selective protein transport into lysosomes are discussed.

Secretion of intact proteins and peptide fragments by lysosomal pathways of protein degradation.

We report that degradation of proteins microinjected into human fibroblasts is accompanied by release into the culture medium of peptide fragments and intact proteins as well as single amino acids. For the nine proteins and polypeptides microinjected, acid-precipitable radioactivity, i.e. peptide fragments and/or intact proteins, ranged from 10 to 67% of the total released radioactivity. Peptide fragments and/or intact protein accounted for 60% of the radioactivity released into the medium by cells microinjected with ribonuclease A. Two major radiolabeled peptide fragments were found, and one was of an appropriate size to function as an antigen in antigen-presenting cells. The peptides released from microinjected ribonuclease A were derived from lysosomal pathways of proteolysis based on several lines of evidence. Previous studies have shown that microinjected ribonuclease A is degraded to single amino acids entirely within lysosomes (McElligott, M. A., Miao, P., and Dice, J. F. (1985) J. Biol. Chem. 260, 11986-11993). We show that release of free amino acids and peptide fragments and/or intact protein was equivalently stimulated by serum deprivation and equivalently inhibited by NH4Cl. We also show that lysosomal degradation of endocytosed [3H]ribonuclease A was accompanied by the release of two peptide fragments similar in size and charge to those from microinjected [3H]ribonuclease A. These findings demonstrate that degradation within lysosomes occurs in a manner that spares specific peptides; they also suggest a previously unsuspected pathway by which cells can secrete cytosol-derived polypeptides.

Transpancreatic transport of digestive enzyme.

When porcine alpha-amylase or bovine chymotrypsinogen A was added to the medium bathing the rabbit pancreas in short-term organ culture, the secretion of these enzymes collected via the duct system increased greatly. To determine if it was indeed the amylase added to the bath that was recovered in secretion, endogenous enzyme stores were prelabeled during a 4 h incubation with [3H]-leucine and the specific radioactivity of amylase in secretion followed. The addition of unlabeled exogenous amylase to the bathing medium reduced the specific radioactivity of secreted amylase by about 90% suggesting that the response was due to the transpancreatic transport of the added enzyme. This inhibition was maintained over time, and was a result, not only of the increased secretion of unlabeled enzyme, but also of a 72% steady-state inhibition in the secretion of endogenous (labeled) amylase. This latter observation indicates that the exogenous enzyme crosses the acinar cell and mixes with endogenous cellular stores. A cellular route is also suggested by the observation that the addition of amylase to the bath increased the amylase concentration in ductal fluid relative to that in the bath by about 20 times; it did not reduce it as would be expected if paracellular shunts were involved. In addition, a cellular pathway is suggested by the observation that a 2 h prior incubation in bovine chymotrypsinogen resulted in a greatly augmented chymotrypsinogen response to a maximal cholinergic stimulus. In all, the data support the prediction of the equilibrium theory of digestive enzyme secretion that enzyme secretion should be responsive to mass action, and the prediction of the enteropancreatic circulation hypothesis that a capacity exists for a substantial transpancreatic flux of digestive enzyme.

Diffusion-like process can account for protein secretion by the pancreas.

When fluid secretion by the pancreas was mechanically blocked, amylase secretion into the duct ceased. When flow was reduced in a graded fashion by the application of a back pressure, amylase output was reduced proportionately and amylase concentration in secretion was maintained constant. Thus, the secretion of digestive enzyme from the cell into the duct appears to be dependent upon the concentration of enzyme in the duct system. This behavior is most simply explained by diffusion-like (concentration dependent, bidirectional) fluxes of digestive enzyme across the plasma membrane. A unidirectional process, such as exocytosis, whose rate should be unaffected by fluid flow, cannot readily explain these results.

Transport of alpha-amylase across the basolateral membrane of the pancreatic acinar cell.

The flux of alpha-amylase (1,4-alpha-D-glucan glucanohydrolase; EC 3.2.1.1) across the basolateral membrane of the acinar cell was measured in the cell-to-bath direction using the whole rabbit pancreas in organ culture. This in vitro preparation is polarized so that apical and basolateral secretions can be collected separately. The unstimulated amylase flux from cell to bath was substantial at the initial rate (approximately three times the concurrent apical flux). With time, bath amylase approached a steady-state concentration, suggesting an equilbrating process. During the same time interval, ductal amylase secretion remained constant. At the steady state, the amylase concentration in the bath was at least an order of magnitude less than its ductal concentration. Hourly replacement of bathing medium reproduced the initial rate of amylase release into the bath for five consecutive hours. Pancreozymin (cholecystokinin), a peptide hormone, did not alter the steady-state bath amylase content, although it greatly augmented ductal amylase secretion. In contrast, a cholinergic agonist greatly increased both the flux from the cell to bath and the ductal secretion of amylase. Taken together, these results indicate a natural bidirectional permeability of the basolateral membrane to digestive enzyme and support evidence previously obtained suggesting that such a permeability might exist.