Protein ACSepharose 4 Fast Flow beads (Amersham Biosciences) were prewashed three times with water and three times with IP buffer. mutant etioplasts appeared normal. These results demonstrate the essentiality of cpFtsY for the biogenesis not only of the LHCPs but also for the assembly of the other membrane-bound components of the photosynthetic apparatus. INTRODUCTION The biogenesis of chloroplast thylakoid membranes requires the coordinated expression and assembly of proteins encoded by both the nuclear and chloroplast genomes. Nucleus-encoded components are synthesized in the cytosol, imported into the chloroplast stroma, and subsequently targeted by one of four mechanistically distinct pathways to the thylakoid membrane. The four pathways engage different substrates and can be distinguished by their protein factor and energy requirements as follows. (1) The Sec pathway Mouse monoclonal to ESR1 requires ATP and cpSecA and has been shown to be involved in the transport of plastocyanin, the 33-kD subunit of the oxygen-evolving complex (OE33), PSI-F (photosystem I-F), and the plastid-encoded cytochrome binding proteins (LHCPs) that make up the antennae of photosystems I and II. (3) A pH-dependent, or Tat, pathway uses a transthylakoidal pH gradient as its single energy source and does not require the participation of stromal protein factors; this pathway transports OE23, OE17, PSII-T, and PSI-N. (4) A spontaneous pathway that seems not to require soluble factors or energy is responsible for the insertion of CFoII, PSII-W, PSII-X, and ELIP. In general, proteins destined for the thylakoid lumen are transported by either the Sec or the pH pathway, whereas integral membrane proteins are targeted by the SRP or the spontaneous pathway. Components of the Sec, pH, and cpSRP pathways all have homologs in extant bacteria and presumably were derived from the cyanobacteria-like ancestor of the chloroplast (for reviews, see Robinson et al., 1998; Keegstra and Cline, 1999). The SRP consists of Ffh (54 homolog) and 4.5S RNA and plays a central role by interacting with signal sequences as membrane-destined proteins emerge from the ribosome (for reviews, see Walter and Johnson, 1994; Rapoport et al., 1996; Fekkes and Driessen, 1999). The cpSRP in higher herb chloroplasts consists of the SRP54/Ffh homolog cpSRP54 and cpSRP43, AZM475271 which is unique for the chloroplast SRP system, but contains no RNA components (Schuenemann et al., 1998; Klimyuk et al., 1999; Tu et al., 1999; Groves et al., 2001). The LHCPs are the best characterized substrates of the cpSRP pathway. They are the most abundant thylakoid membrane proteins and bind almost half of the chlorophyll (for review, see Wollman et al., 1999). LHCPs form a superfamily of related nucleus-encoded proteins with three or four transmembrane helices. After their import into the chloroplast stroma, they are bound by cpSRP to form a soluble transit complex, which prevents the aggregation of these hydrophobic proteins and maintains their competence for thylakoid insertion (Payan and Cline, 1991; Li et al., 1995; Schuenemann et al., 1998). The details of the binding interactions between LHCP, cpSRP43, and cpSRP54 were elucidated recently (DeLille et al., 2000; Tu et al., 2000; Jonas-Straube et al., 2001). In addition to AZM475271 soluble cpSRP in the stroma, targeting of LHCP to the thylakoid membrane also requires a membrane-bound protein called cpFtsY, which is a homolog of the bacterial SRP AZM475271 receptor FtsY (Kogata et al., 1999; Tu et al., 1999). Finally, integration of LHCP into the thylakoid membrane is usually mediated by the integral membrane protein ALB3, which is a homolog of the mitochondrial Oxa1p and bacterial YidC proteins (Moore et al., 2000; Woolhead et al., 2001). Both cpSRP54 and cpFtsY contain a GTPase domain name, and LHCP integration into the thylakoid membrane was shown to require GTP hydrolysis (Hoffman and Franklin, 1994; Kogata et al., 1999; Tu et al., 1999). Several Arabidopsis mutants related AZM475271 to the cpSRP pathway have been isolated and characterized. Interestingly, these mutants seemed to vary in their AZM475271 phenotypes. A null mutant of cpSRP43, ratio, and a specific reduction in 7 of the 11.