| The expression of the chloroplast gene psbD, encoding the D2 protein of photosystem II, requires nuclear encoded factors. The nonphotosynthetic mutants of Chlamydomonas reinhardtii, ac-115 and nac1-18, have nuclear mutations that affect the expression of the D2 protein. Two approaches were used in an attempt to clone the AC115 and NAC1 genes. The AC115 gene was isolated through genomic complementation using an indexed library of C. reinhardtii DNA. AC115 is an intronless gene encoding a novel 113 amino acid polypeptide. This polypeptide contains a putative chloroplast transit peptide at the N-terminus, and a transmembrane domain with a cysteine rich region at the C-terminus. The possible roles of this novel polypeptide are discussed. This polypeptide may be a component of photosystem II required for the proper folding or the stabilization of the nascent D2 protein within the thylakoid membrane. Alternatively, this nuclear factor may use the cysteine rich region to recruit the non-heme iron of photosystem II and facilitate the binding of this cofactor to the nascent D2 protein which would allow for complete expression of the D2 protein. DNA markers closely linked to the AC115 and NAC1 genes were identified by random amplified polymorphic DNA (RAPD) analysis. The use of these molecular markers to isolate the genes of interest is discussed. These molecular markers can be used as initial probes in chromosome walking experiments to get to the genes of interest. These RAPD markers can also be used to screen genomic libraries with larger inserts (e.g. a YAC library). Finally, the level of plastoquinone A was determined in wild type, ac-115, nac1-18, FUD47 (a mutant with a primary effect in the D2 protein synthesis), and F35 (a mutant with a primary effect in the synthesis of D1 protein). All of these mutants appear to be deficient in plastoquinone A. The results from this study provide an understanding of the relationship between nuclear encoded factors and the synthesis of a chloroplast encoded protein. |
