BIOSYNTHESIS OF CYTOCHROME-F IN CHLAMYDOMONAS-REINHARDTII - ANALYSIS OF THE PATHWAY IN GABACULINE-TREATED CELLS AND IN THE HEME ATTACHMENT MUTANT-B6

Chlamydomonas reinhardtii uses two c-type cytochromes for photosynthet ic electron transfer: the thylakoid membrane-bound cytochrome f of the cytochrome b6f complex and the soluble cytochrome c6. Previously, a c lass of photosynthesis-minus, acetate-requiring mutants was identified which were deficient in both c-type cytochromes, and biochemical anal yses of cytochrome c6 biosynthesis in these strains indicated that the y were each blocked at the step of heme attachment to apocytochrome c6 . In order to demonstrate that the deficiency in cytochrome f results from the same biochemical and genetic defect, cytochrome f biosynthesi s was examined in the B6 mutant (a representative of this phenotypic c lass) and in spontaneous suppressor strains derived from B6. Pulse-rad iolabeling experiments show that B6 synthesizes a form of cytochrome f that is rapidly degraded in vivo. This polypeptide is membrane associ ated and migrates with an electrophoretic mobility identical to that o f standard apocytochrome f produced in vitro but slightly greater than that of standard holocytochrome f produced in vivo by wild-type cells . These findings suggest that the B6 strain is unable to convert apocy tochrome f to holocytochrome f and that apocytochrome f is unstable in vivo. In the suppressed strains, accumulation of both holocytochrome f and holocytochrome c6 is restored. One suppressor mutation (strain B 6R) displays uniparental inheritance whereas another (B6T3) displays M endelian inheritance. In both cases, the three phenotypes, photosynthe sis-plus, b6f(+) and cyt c6(+) co-segregate in genetic crosses. This s tudy therefore confirms that the dual cyt b6f(-)/cytc6(-) deficiency i n B6 results from a single mutation that affects a step in holocytochr ome formation that is common to the biosynthetic pathways of both plas tidic c-type cytochromes. The study also confirms that pre-apocytochro me f synthesis, processing and association with the membrane is not de pendent on heme attachment. Synthesis of cytochrome f does, however, a ppear to be dependent on heme availability. In cells depleted of tetra pyrrole pathway intermediates by gabaculine treatment, cytochrome f sy nthesis was significantly reduced. Since gabaculine treatment did not affect the stability of cytochrome f nor the accumulation of cytochrom e f-encoding transcripts, the reduction is attributed to post-transcri ptional regulation of pre-apocytochrome f synthesis via a pathway that is sensitive to the availability of heme or a tetrapyrrole pathway in termediate.