We have characterized the biochemical nature and the function of PsbZ, the protein product of a ubiquitous open reading frame, which is known as ycf9 in Chlamydomonas and ORF 62 in tobacco, that is present in chloroplast and cyanobacterial genomes. After raising specific antibodies to PsbZ from Chlamydomonas and tobacco, we demonstrated that it is a bona fide photosystem II (PSII) subunit. PsbZ copurifies with PSII cores in Chlamydomonas as well as in tobacco. Accordingly, PSII mutants from Chlamydomonas and tobacco are deficient in PsbZ. Using psbZ -targeted gene inactivation in tobacco and Chlamydomonas, we show that this protein controls the interaction of PSII cores with the light-harvesting antenna; in particular, PSII-LHCII supercomplexes no longer could be isolated from PsbZ-deficient tobacco plants. The content of the minor chlorophyll binding protein CP26, and to a lesser extent that of CP29, also was altered substantially under most growth conditions in the tobacco mutant and in Chlamydomonas mutant cells grown under photoautotrophic conditions. These PsbZ-dependent changes in the supramolecular organization of the PSII cores with their peripheral antennas cause two distinct phenotypes in tobacco and are accompanied by considerable modifications in (1) the pattern of protein phosphorylation within PSII units, (2) the deepoxidation of xanthophylls, and (3) the kinetics and amplitude of nonphotochemical quenching. The role of PsbZ in excitation energy dissipation within PSII is discussed in light of its proximity to CP43, in agreement with the most recent structural data on PSII. INTRODUCTIONWith the completion of an ever-increasing number of chloroplast genome sequences (Quigley and Weil, 1985; Bookjans et al., 1986;Ohyama et al., 1986;Shinozaki et al., 1986; Hiratsuka et al., 1989; Evrard et al., 1990; Hallick et al., 1993; Wakasugi et al., 1994 Wakasugi et al., , 1997Kowallik et al., 1995;Maier et al., 1995;Reith and Munholland, 1995; Douglas and Penny, 1999;Sato et al., 1999; Turmel et al., 1999;Hupfer et al., 2000;Lemieux et al., 2000), the functions of most of the major open reading frames of chloroplast DNA have now been studied. The ability to perform reverse genetics with tobacco and Chlamydomonas chloroplasts has contributed greatly to this process. However, this strategy, which consists mainly of the phenotypic characterization of mutants inactivated for the gene under study, can be unsuccessful in two cases: (1) when the gene product is required for cell viability, in which case homoplasmic transformation cannot be obtained, and (2) when homoplasmic transformants do not display any clear-cut mutant phenotype under laboratory conditions. In both cases, this may preclude a reliable functional characterization of the gene and its product.ycf9 , which is predicted to encode a 6.5-kD protein with two putative membrane-spanning segments, is an interesting case because attempts to inactivate the gene have yielded widely different results ranging from potential lethality to 1 These authors cont...