Many thylakoid proteins are cytosolically synthesized and have to cross the two chloroplast envelope membranes as well as the thylakoid membrane en route to their functional locations. In order to investigate the localization pathways of these proteins, we over‐expressed precursor proteins in Escherichia coli and used them in competition studies. Competition was conducted for import into the chloroplast and for transport into or across isolated thylakoids. We also developed a novel in organello method whereby competition for thylakoid transport occurred within intact chloroplasts. Import of all precursors into chloroplasts was similarly inhibited by saturating concentrations of the precursor to the OE23 protein. In contrast, competition for thylakoid transport revealed three distinct precursor specificity groups. Lumen‐resident proteins OE23 and OE17 constitute one group, lumenal proteins plastocyanin and OE33 a second, and the membrane protein LHCP a third. The specificity determined by competition correlates with previously determined protein‐specific energy requirements for thylakoid transport. Taken together, these results suggest that thylakoid precursor proteins are imported into chloroplasts on a common import apparatus, whereupon they enter one of several precursor‐specific thylakoid transport pathways.

The two deep traps responsible for current collapse in AlGaN/GaN high electron mobility transistors grown by metalorganic vapor-phase epitaxy have been studied by photoionization spectroscopy. Varying the growth pressure of the high resistivity GaN buffer layer results in a change in the deep trap incorporation that is reflected in the observed current collapse. Variations in the measured trap concentrations with growth pressure and carbon incorporation indicate that the deepest trap is a carbon-related defect, while the mid-gap trap may be associated with grain boundaries or dislocations.

Most chloroplast proteins are nuclear encoded, synthesized as larger precursor proteins in the cytosol, posttranslationally imported into the organelle, and routed to one of six different compartments. Import across the outer and inner envelope membranes into the stroma is the major means for entry of proteins destined for the stroma, the thylakoid membrane, and the thylakoid lumen. Recent investigations have identified several unique protein components of the envelope translocation machinery. These include two GTP-binding proteins that appear to participate in the early events of import and probably regulate precursor recognition and advancement into the translocon. Localization of imported precursor proteins to the thylakoid membrane and thylakoid lumen is accomplished by four distinct mechanisms; two are homologous to bacterial and endoplasmic reticulum protein transport systems, one appears unique, and the last may be a spontaneous mechanism. Thus chloroplast protein targeting is a unique and surprisingly complex process. The presence of GTP-binding proteins in the envelope translocation machinery indicates a different precursor recognition process than is present in mitochondria. Mechanisms for thylakoid protein localization are in part derived from the prokaryotic endosymbiont, but are more unusual and diverse than expected.

Multiple sorting pathways operate in chloroplasts to localize proteins to the thylakoid membrane. The signal recognition particle (SRP) pathway in chloroplasts employs the function of a signal recognition particle (cpSRP) to target light harvesting chlorophyll-binding protein (LHCP) to the thylakoid membrane. In assays that reconstitute stroma-dependent LHCP integration in vitro, the stroma is replaceable by the addition of GTP, cpSRP, and an SRP receptor homolog, cpFtsY. Still lacking is an understanding of events that take place at the thylakoid membrane including the identification of membrane proteins that may function at the level of cpFtsY binding or LHCP integration. The identification of Oxa1p in mitochondria, an inner membrane translocase component homologous to predicted proteins in bacteria and to the albino3 (ALB3) protein in thylakoids, led us to investigate the potential role of ALB3 in LHCP integration. Antibody raised against a 50-amino acid region of ALB3 (ALB3-50aa) identified a single 45-kDa thylakoid protein. Treatment of thylakoids with antibody to ALB3-50aa inhibited LHCP integration, whereas the same antibody treatment performed in the presence of antigen reversed the inhibition. In contrast, transport by the thylakoid Sec or Delta pH pathways was unaffected. These data support a model whereby a distinct translocase containing ALB3 is used to integrate LHCP into thylakoid membranes.

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...

The mechanisms involved in the integration of proteins into the thylakoid membrane are largely unknown. (3); the N-terminal portion governs transport across the envelope, whereas the C-terminal portion directs translocation across the thylakoid membrane (4). In the case of LHCP, the mature apoprotein contains sufficient information for thylakoid targeting (5), although the specific signal has not yet been identified (6, 7). Studies of the early events of LHCP trafficking using isolated chloroplasts showed that LHCP is present as a soluble form in the stroma prior to integration into thylakoids (8). When integration was inhibited, this intermediate LHCP accumulated in the stroma as a larger complex (9). Formation of this complex, which we have designated the "transit complex," maintains LHCP solubility and integration competence (9). Both transit complex formation and LHCP integrationThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.into thylakoids have been reconstituted in vitro; both require a proteinaceous component(s) of the stroma (9, 10).The chaperone nature of this "stromal factor" led to the suggestions that heat shock proteins Cpn6O (11) and Hsp7O (12) were involved in LHCP integration. These molecular chaperones are abundant in the stroma (13,14), are able to bind polypeptides and release them upon ATP hydrolysis, and have been shown to be involved in mitochondrial protein import (15). However, immunoprecipitation experiments with antibodies against the two proteins failed to reveal the presence of Cpn6O and Hsp7O in the transit complex (9, 14). Furthermore, stromal extract depleted of Hsp7O still supported transit complex formation and LHCP integration, indicating that neither process requires this chaperone (14). The recent demonstration of a stromal GTP requirement (16) for LHCP integration now suggests the involvement of a guanine nucleotide-binding protein in this process. One likely candidate is 54CP, a chloroplast homologue of the 54-kDa protein of the mammalian signal recognition particle (SRP) (17). Protein transport across or integration into the endoplasmic reticulum (ER) is mediated by a GTP-dependent mechanism involving SRP (18). The 54-kDa polypeptide subunit of SRP (SRP54) plays a major role in SRP-dependent targeting. It binds guanine nucleotides as well as nascent polypeptides that are destined for ER transport or integration (19). Furthermore, SRP54 appears to be required for docking to the SRP receptor (20) and hydrolyzes the GTP required for dissociation of SRP from the receptor (21). In this report, we show that 54CP is bound to LHCP in the transit complex and is essential for LHCP integration into the thylakoid membrane. (p) were prepared by in vitro transcription and translation as described (7). Unlabeled pLHCP and pOE33 (33-kDa subunit of the oxygenevolving complex) were expressed in Escherichia coli and pur...

The SecA protein is an essential, azide-sensitive component of the bacterial protein translocation machinery. A SecA protein homolog (CPSecA) now identified in pea chloroplasts was purified to homogeneity. CPSecA supported protein transport into thylakoids, the chloroplast internal membrane network, in an azide-sensitive fashion. Only one of three pathways for protein transport into thylakoids uses the CPSecA mechanism. The use of a bacteria-homologous mechanism in intrachloroplast protein transport provides evidence for conservative sorting of proteins within chloroplasts.

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...