Degradation of microinjected methylated and unmethylated proteins in hepatoma tissue culture cells

Abstract

Radioiodinated proteins were introduced into hepatoma tissue culture (HTC) cells by erythrocyte ghost-mediated microinjection, and their degradation was studied. 125I-bovine serum albumin and 125I-lysozyme were degraded with half-lives of about 7 and 11 h, respectively. The process was ATP-dependent. The breakdown of these proteins was not inhibited by the following inhibitors of lysosomal proteolysis: NH4Cl, methylamine, chloroquine, leupeptin, or antipain. Methylation of 94% of the amino groups of bovine serum albumin or 99% of the amino groups of lysozyme had little effect on the rates of their degradation in HTC cells. In contrast, methylation almost completely inhibited the ATP-dependent proteolysis of both proteins in reticulocyte lysates. Methylated bovine serum albumin was not detectably demethylated in HTC cells. It is concluded that in HTC cells, bovine serum albumin and lysozyme are degraded by a nonlysosomal pathway which differs from the ubiquitin-dependent proteolysis system of reticulocytes in that it does not require free amino groups.