Role of heme in the biosynthesis of cytochrome c6.

Abstract

Cytochrome c6, a nuclear-encoded protein, is synthesized in the cytoplasm in a precursor form (pre-apocytochrome c6). Time course radiolabeling experiments support the model that the pre-protein is the substrate for a lumen-targeting post-translational pathway which includes two proteolytic cleavage events and the covalent attachment of heme to cysteinyl thiols on the polypeptide. Cell fractionation studies indicate that the fully processed protein fractionates with other soluble proteins, whereas the precursor and intermediate forms appear to be membrane-associated. To determine whether either of the processing steps is influenced by heme attachment, the biosynthetic pathway was examined 1) in a Chlamydomonas reinhardtii mutant (B6) that is defective in the heme attachment step and 2) in wild-type cells treated with gabaculine, an inhibitor of heme synthesis. In vivo radiolabeling experiments with the B6 mutant showed that a defect in heme attachment affects neither the synthesis of the pre-apoprotein nor its processing to the mature apoform to any significant extent, supporting the notion that heme attachment and processing are not obligatorily coupled in this pathway. A similar conclusion is reached from examination of cytochrome c6 synthesis in gabaculine-treated cells where inhibition of heme attachment did not prevent either of the two processing steps. The fully processed apoprotein is a suitable substrate for heme attachment in vivo, since the apoprotein was indeed converted to holoprotein in gabaculine-treated cells, albeit more slowly than in untreated cells. Despite the lack of effect of gabaculine treatment on the accumulation of cytochrome c6-encoding messages, the amount of holocytochrome c6 precursors synthesized during a brief labeling period is 4-7-fold less than in untreated cells, suggesting that synthesis of the polypeptide may be coupled to heme availability by a control mechanism operating at the translational level.