Turnover of mitochondrial matrix polypeptides in hepatoma monolayer cultures

Abstract

The degradation rates of mitochondrial matrix polypeptides were examined in nonproliferating hepatoma monolayers. The cultures were first pulsed with [3H]methionine, and after chasing for 41 h in the presence of excess methionine, the cultures were pulsed with [35S]methionine. Sonic extracts from the mitochondrial fraction of the double-labeled cells were then resolved on two-dimensional isoelectric focussing-electrophoresis gels in the presence of excess matrix proteins from digitonin-fractionated rat liver mitochondria. Thirty-three of 80 spots appearing upon staining and destaining contained radioactivity significantly above background, indicating that these polypeptides were present in hepatoma as well as liver mitochondria. The half-lives of isolated polypeptides were then determined from their 3H/35S, the 3H/35S of the isolated mitochondria fraction, and the half-life of the mitochondria fraction determined independently from a decay experiment. The 3H/35S of these resolved polypeptides ranged from 0.32 to 1.93, corresponding to calculated half-lives of 17 to 100+ h. The 3H/35S of these same polypeptides from mixed control cultures given [3H]- and [35S]methionine pulses, respectively, at the beginning and end of confluent maintenance in the absence of chase were nearly identical (1.78 +/- 0.17), thus assuring unchanging rates of protein synthesis during the chase experiment. The results show that mitochondrial matrix polypeptides are degraded at heterogeneous rates in these nonproliferating cells.