The Plastid-encoded ccsA Gene Is Required for Heme Attachment to Chloroplast c-type Cytochromes

Zhiyi Xie,Sabeeha Merchant

doi:10.1074/jbc.271.9.4632

Abstract

A chloroplast gene, ycf5, which displays limited sequence identity to bacterial genes (ccl1/cycK) required for the biogenesis of c-type cytochromes, was tested for its function in chloroplast cytochrome biogenesis in Chlamydomonas reinhardtii. Targeted inactivation of the ycf5 gene results in a non-photosynthetic phenotype attributable to the absence of c-type cytochromes. The cloned ycf5 gene also complements the phototrophic growth deficiency in strain B6 of C. reinhardtii. B6 is unable to synthesize functional forms of cytochromes f and c6 owing to a chloroplast genome mutation that prevents heme attachment. The selected (phototrophic growth) as well as the unselected (holocytochrome c6 accumulation) phenotypes were restored in complemented strains. The complementing gene, renamed ccsA (for c-type cytochrome synthesis), is expressed in wild-type and B6 cells but is non-functional in B6 owing to an early frameshift mutation. Antibodies raised against the ccsA gene product recognize a 29-kDa protein in C. reinhardtii. The 29-kDa protein is absent in strain B6 but is restored in a spontaneous revertant (B6R) isolated from a culture of B6. Sequence analysis of the ccsA gene in strain B6R indicates that it is a true revertant. We conclude that the ccsA gene is expressed and that it encodes a protein required for heme attachment to c-type cytochromes.

Highlights

We have assigned a function to the chloroplast gene, ycf5, shown that it is expressed in C. reinhardtii, and identified a protein product of the gene
This protein product, which migrates with a mobility corresponding to a 29-kDa polypeptide, is essential for the biosynthesis of both membrane and soluble c-type cytochromes in the chloroplast, at the step of heme attachment
We hope to test the functional importance of the Trp-rich sequence motif in the chloroplast heme attachment reaction

Summary

EXPERIMENTAL PROCEDURES

Strain and Culture Conditions—C. reinhardtii strains CC503 (cw mtϩ), CC425 (cw arg mtϩ), and CC125 (wt) were obtained from the Chlamydomonas Genetics Center at Duke University, Durham, NC. Cells were collected by centrifugation, washed once in TAP medium, and resuspended in 8 ml of a solution containing 20 mM Tris-Cl (pH 8.0), 0.1 M NaCl, 50 mM EDTA. Southern Hybridization for Localization of ycf5—Purified chloroplast DNA (1.5 ␮g) was digested (3 h) with BamHI (4 units), BglII (3 units), EcoRI (4 units), and PstI (1 unit), and the resulting fragments were separated by electrophoresis (60 V, 20 h) through 0.8% agarose in TBE (89 mM Tris, 89 mM boric acid, 2 mM Na2EDTA (pH 8.3)). With the help of the restriction map of C. reinhardtii chloroplast DNA (24), the position of the ycf homologue was localized to an ϳ1.6-kb BamHI-EcoRI fragment contained in Bam and Eco (26). Cloning and Sequence Analysis of ycf5/ccsA—C. reinhardtii chloroplast DNA fragment, Bam, and a 5-kb XhoI-BamHI subfragment of Bam, were obtained as inserts in plasmids p15 and p207, respectively, from Dr Elizabeth Harris (Chlamydomonas Genetics Center).

Cytochrome Biogenesis in Chloroplasts

RESULTS

DISCUSSION

Zhiyi Xie and Sabeeha Merchant