Development of thylakoid membranes depends upon the transport of membrane vesicles from the chloroplast inner envelope and subsequent fusion of vesicles within the interior of the plastid. The Arabidopsis (Arabidopsis thaliana) Thylakoid formation1 (Thf1) gene product is shown here to control an important step required for the normal organization of these vesicles into mature thylakoid stacks and ultimately for leaf development. The Arabidopsis Thf1 gene encodes an imported chloroplast protein, as shown by in vitro import and localization of a Thf1-green fluorescent protein fusion product in transgenic plants. This gene is conserved in oxygenic photoautotrophs ranging from cyanobacteria to flowering land plants. Transcript levels for Thf1 are induced in the light and decrease under dark conditions, paralleling profiles of light-regulated nuclear genes involved in chloroplast function. Disruption of the Thf1 gene via T-DNA insertion results in plants that are severely stunted with variegated leaf patterns. Nongreen sectors of variegated leaves lacking Thf1 expression contain plastids that accumulate membrane vesicles on the interior and lack organized thylakoid structures. Green sectors of Thf1-disrupted leaves contain some chloroplasts that form organized thylakoid membranes, indicating that an inefficient compensatory mechanism supports thylakoid formation in the absence of Thf1. Genetic complementation of a Thf1 knockout line confirms the role of this gene in chloroplast and leaf development. Transgenic plants expressing the Thf1 gene in antisense orientation are stunted with altered thylakoid organization, especially in young seedlings. The data indicate that the Thf1 gene product plays a crucial role in a dynamic process of vesicle-mediated thylakoid membrane biogenesis.The development of normal chloroplasts is a crucial step in the survival of photosynthetic eukaryotes. Maturation and activity of the chloroplast is highly dependent on its relationship with the nucleus, as the bulk of proteins that function in the chloroplast are encoded in the nucleus and are posttranslationally imported from the cytoplasm (Keegstra and Cline, 1999). Lesions in genes encoding plastid-localized proteins can have severe effects on the many metabolic processes that occur there and ultimately can prevent normal plant growth and development. Demonstrating the overriding importance of chloroplast proteins, the majority of mutations in a screen of mutagenized Arabidopsis (Arabidopsis thaliana), designed to identify genes important for seedling viability, appeared to affect chloroplast formation or function (Budziszewski et al., 2001). Environmental conditions, especially light, also play important roles in triggering chloroplast development and orchestrating gene expression required for plastid function (Anderson, 1986). In spite of the dependence of plants on the photosynthesis occurring on chloroplast thylakoid membranes, there is much to be learned regarding the mechanisms by which these internal membranes originate and how th...

Signal recognition particles (SRPs) in the cytosols of prokaryotes and eukaryotes are used to target proteins to cytoplasmic membranes and the endoplasmic reticulum, respectively. The mechanism of targeting relies on cotranslational SRP binding to hydrophobic signal sequences. An organellar SRP identified in chloroplasts (cpSRP) is unusual in that it functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins. In assays that reconstitute thylakoid integration of the light harvesting chlorophyll-binding protein (LHCP), stromal cpSRP binds LHCP posttranslationally to form a cpSRP͞LHCP transit complex, which is believed to represent the LHCP form targeted to thylakoids. In this investigation, we have identified an 18-aa sequence motif in LHCP (L18) that, along with a hydrophobic domain, is required for transit complex formation. Fusion of L18 to the amino terminus of an endoplasmic reticulum-targeted protein, preprolactin, led to transit complex formation whereas wild-type preprolactin exhibited no ability to form a transit complex. In addition, a synthetic L18 peptide, which competed with LHCP for transit complex formation, caused a parallel inhibition of LHCP integration. Translocation of proteins by the thylakoid Sec and Delta pH transport systems was unaffected by the highest concentration of L18 peptide examined. Our data indicate that a motif contained in L18 functions in precursor recruitment to the posttranslational SRP pathway, one of at least four different thylakoid sorting pathways used by chloroplasts. Signal recognition particle (SRP) and its receptor comprise essential components of a signal peptide-based protein targeting mechanism that is conserved across evolutionary boundaries (1-3). SRPs in the cytosols of eukaryotes and Escherichia coli target proteins cotranslationally to the endoplasmic reticulum and cytoplasmic membrane, respectively. Targeting is initiated as a result of SRP binding to the hydrophobic domain of amino-terminal signal peptides or signal anchors as they emerge from the ribosome. The entire ribosome͞nascent polypeptide chain complex (RNC) then is piloted by SRP to an SRP receptor that functions at the membrane. GTP binding and hydrolysis by SRP and its receptor result in both the release of SRP from its receptor and the release of SRP from the RNC, whereupon the nascent chain enters a translocation pore that directs the translating polypeptide into or across the lipid bilayer.An organellar SRP, which exhibits striking structural and functional differences from cytosolic SRPs, also has been identified in chloroplasts (4, 5). Chloroplast SRP (cpSRP) is a soluble Ϸ200-kDa stromal particle that contains an evolutionary conserved 54-kDa subunit (cpSRP54) as well as a unique 43-kDa polypeptide (cpSRP43) (6). Unlike cytosolic SRPs, an RNA moiety is conspicuously lacking in cpSRP. Biochemical and genetic evidence have demonstrated that cpSRP functions posttranslationally to localize a subset of nuclear-encoded thylakoid proteins belonging to the chlorophyll...

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The signal recognition particle (SRP) and its receptor (FtsY in prokaryotes) are essential for cotranslational protein targeting to the endoplasmic reticulum in eukaryotes and the cytoplasmic membrane in prokaryotes. An SRP/FtsY-like protein targeting/integration pathway in chloroplasts mediates the posttranslational integration of the light-harvesting chlorophyll a/b-binding protein (LHCP) into thylakoid membranes. GTP, chloroplast SRP (cpSRP), and chloroplast FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes. Here, we report the reconstitution of the LHCP integration reaction with purified recombinant proteins and salt-washed thylakoids. Our data demonstrate that cpSRP and cpFtsY are the only soluble protein components required for LHCP integration. In addition, our studies reveal that ATP, though not absolutely required, remarkably stimulates LHCP integration into saltwashed thylakoids. ATP stimulates LHCP integration by a mechanism independent of the thylakoidal pH gradient (⌬pH) and exerts no detectable effect on the formation of the soluble LHCP-cpSRP-targeting complex. Taken together, our results indicate the participation of a thylakoid ATP-binding protein in LHCP integration.Chloroplasts are structurally complex organelles with multiple membranes and aqueous compartments. The innermost chloroplast membrane, the thylakoid membrane, separates the stroma from the thylakoid lumen and is the site of light-driven photophosphorylation. Although chloroplasts possess their own genome and protein synthesis machinery, the majority of thylakoid proteins are encoded in the nucleus, synthesized in the cytoplasm, and imported into the stroma by a general protein translocase in the envelope. Imported precursor proteins are processed to pathway intermediates in the stroma and then enter one of four different targeting pathways for localization to the thylakoids (1, 2). The cpSRP-dependent pathway appears to specifically target the light-harvesting chlorophyllbinding proteins that use an Albino3-containing translocase for integration into the thylakoid membrane (3, 4).The major light-harvesting chlorophyll-binding protein (LHCP) 1 is an integral thylakoid membrane protein with three transmembrane helices. Following import into the stroma, LHCP binds cpSRP to form a soluble targeting complex (5-7). Whereas the 54-kDa subunit of cpSRP (cpSRP54) is thought to bind one of three hydrophobic domains, the 43-kDa subunit of cpSRP (cpSRP43) binds an 18-residue hydrophilic domain (L18) located between the second and the third transmembrane helices of LHCP (8,9). Interaction between cpSRP43 and L18 is required for LHCP-targeting complex formation with cpSRP and confers cpSRP54 with its unique posttranslational binding activity. Both cpSRP and a chloroplast homologue of the bacterial SRP receptor FtsY (cpFtsY) are required for LHCP integration into thylakoid membranes (6,7,10,11). As previous studies on reconstituting LHCP integration have never been conducted in the absence of wheat germ extract or reticulo...