Biosynthesis and intracellular transport of enzymes of peroxisomal beta-oxidation.

Abstract

Three peroxisomal enzymes of beta-oxidation from rat liver were synthesized in a cell-free protein-synthesizing system derived from a lysate of rabbit reticulocytes. The in vitro products of acyl-CoA oxidase (EC 1.3.99.3) and a bifunctional protein containing enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities were apparently the same in size and charge as the subunit of the respective mature enzymes; that of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was about 3,000 Da larger and more basic than its mature subunit. The free polysome fraction of rat liver was 3.1-5.7 times more active than the membrane-bound polysome fraction in the synthesis of the three peroxisomal enzymes; these values were similar to those for cytosolic enzymes and differed from that for serum albumin. In isolated rat hepatocytes, radiolabeled acyl-CoA oxidase and bifunctional protein increased with time with no appreciable change in the subunit size. On the other hand, the labeled putative precursor of 3-ketoacyl-CoA thiolase, as well as the mature form of the enzyme, was detected in the hepatocytes. The radioactivity of the putative precursor reached a plateau in 30 min; that of the mature subunit appeared after a lag time of about 5 min and increased with time up to 90 min. In pulse-chase experiments, the putative precursor disappeared with an apparent half-life of several minutes. When the hepatocytes were fractionated into the cytosolic and the particulate fractions, one half of labeled acyl-CoA oxidase and 60% of the bifunctional protein were recovered in the cytosolic fraction after 10 min of labeling, whereas 70-80% of the labeled enzymes were recovered in the particulate fraction after 40-60 min of labeling. These results indicate that the three enzymes of peroxisomal beta-oxidation are synthesized on free polysomes, released into the cytosol, and then transported into peroxisomes. Our findings also indicate that 3-ketoacyl-CoA thiolase undergoes proteolytic processing during maturation. The temporal sequence of the proteolytic cleavage and intracellular transport of the thiolase remains to be determined.