Mechanisms of protein degradation in growing and non-growing L-cell cultures

Abstract

L-cells [mouse fibroblasts] prelabeled with [14C]leucine and [3H]thymidine were placed in either fresh growth medium (minimal essential medium with 10% serum) or stepdown medium (minimal essential medium) for 3 days. The 14C/3H ratio remained constant in the growing cultures and decreased in the stationary-phase cultures, indicating no protein turnover in growing cultures and a degradative rate of 0.6%/h in the stationary-phase cultures. Media analysis indicated that 14C-labeled proteins were being degraded at .apprx. 1.2%/h in growing cultures and 1.7%/h in stationary-phase cultures. Additional studies indicated that a subpopulation of L-cells in the monolayer, comprising .apprx. 20-30% of the total, were lost in the original processing procedure. Experiments where recoveries approached 100% by fixation of the monolayer in situ indicated that a protein-degrading subpopulation accounted for all the observed proteolysis in the growing cultures. Proteolysis in these cultures was only partially inhibited with NH4Cl, indicating that only a small part of the protein degradation was occurring in an activated lysosomal-autophagic system. NaF produced a more effective inhibition of proteolysis, but whether this effect was on an ATP-requiring basal-turnover mechanism or a direct effect on unregulated activity of proteinases in the cell hyaloplasm was not determined. NH4Cl inhibited the proteolysis induced when cells were placed in stepdown medium, suggesting that the induced proteolysis was occurring via the autophagic system. L-cells apparently exist in at least 2 states with respect to protein degradation; a subpopulation that is actively replicating and does not degrade cellular proteins and a 2nd subpopulation of cells, derived from the preceding one, which degraded most of its labeled proteins, incapable of further replication, and not sedimented in an iso-osmotic EDTA buffer solution. Proliferating L-cells, when placed in stepdown medium, begin to degrade cell protein through a mechanism involving autophagolysosomes.