These authors contributed equally to this work. SummaryA key regulated step in abscisic acid (ABA) biosynthesis in plants is catalyzed by 9-cis epoxycarotenoid dioxygenase (NCED), which cleaves 9-cis xanthophylls to xanthoxin, a precursor of ABA. In Arabidopsis, ABA biosynthesis is controlled by a small family of NCED genes. Nine carotenoid cleavage dioxygenase (CCD) genes have been identi®ed in the complete genome sequence. Of these, ®ve AtNCEDs (2, 3, 5, 6, and 9) have been cloned and studied for expression and subcellular localization. Although all ®ve AtNCEDs are targeted to plastids, they differ in binding activity of the thylakoid membrane. AtNCED2, AtNCED3, and AtNCED6 are found in both stroma and thylakoid membrane-bound compartments. AtNCED5 is exclusively bound to thylakoids, whereas AtNCED9 remains soluble in stroma. A quantitative real-time PCR analysis and histochemical staining of promoter::GUS activity in transgenic Arabidopsis revealed a complex pattern of localized NCED expression in well-watered plants during development. AtNCED2 and AtNCED3 account for the NCED activity in roots, with localized expression in root tips, pericycle, and cortex cells at the base of lateral roots. Localized AtNCED2 and AtNCED3 expression in pericycle cells is an early marker of lateral initiation sites. AtNCED5, AtNCED6, AtNCED3, and AtNCED2 are expressed in¯owers with very high AtN-CED6::GUS activity occurring in pollen. AtNCED5::GUS, and to lesser degrees, AtNCED2::GUS and AtN-CED3::GUS are expressed in developing anthers. AtNCED5, AtNCED6, AtNCED9, and AtNCED3 contribute to expression in developing seeds with high levels of AtNCED6 present at an early stage. GUS analysis indicates that AtNCED3 expression is con®ned to the base of the seed, whereas AtNCED5 and AtNCED6 are expressed throughout the seed. Consistent with the studies conducted by Iuchi and his colleagues in 2001, AtNCED3 is the major stress-induced NCED in leaves. Our results indicate that developmental control of ABA synthesis involves localized patterns of AtNCED gene expression. In addition, differential membrane-binding capacity of AtNCEDs is a potential means of post-translational regulation of NCED activity.

The thylakoid ΔpH-dependent pathway transports folded proteins with twin arginine–containing signal peptides. Identified components of the machinery include cpTatC, Hcf106, and Tha4. The reaction occurs in two steps: precursor binding to the machinery, and transport across the membrane. Here, we show that a cpTatC–Hcf106 complex serves as receptor for specific binding of twin arginine–containing precursors. Antibodies to either Hcf106 or cpTatC, but not Tha4, inhibited precursor binding. Blue native gel electrophoresis and coimmunoprecipitation of digitonin-solubilized thylakoids showed that Hcf106 and cpTatC are members of an ∼700-kD complex that lacks Tha4. Thylakoid-bound precursor proteins were also associated with an ∼700-kD complex and were coimmunoprecipitated with antibodies to cpTatC or Hcf106. Chemical cross-linking revealed that precursors make direct contact with cpTatC and Hcf106 and confirmed that Tha4 is not associated with precursor, cpTatC, or Hcf106 in the membrane. Precursor binding to the cpTatC–Hcf106 complex required both the twin arginine and the hydrophobic core of the signal peptide. Precursors remained bound to the complex when Tha4 was sequestered by antibody, even in the presence of ΔpH. These results indicate that precursor binding to the cpTatC–Hcf106 complex constitutes the recognition event for this pathway and that subsequent participation by Tha4 leads to translocation.

The sequence of the entire genome of citrus tristeza virus (CTV), Florida isolate T36, was completed. The 19,296-nt CTV genome encodes 12 open reading frames (ORFs) potentially coding for at least 17 protein products. The 5'-proximal ORF 1a starts at nucleotide 108 and encodes a large polyprotein with calculated MW of 349 kDa containing domains characteristic of (from 5' to 3') two papain-like proteases (P-PRO), a methyltransferase (MT), and a helicase (HEL). Alignment of the putative P-PRO sequences of CTV with the related proteases of beet yellows closterovirus (BYV) and potyviruses allowed the prediction of catalytic cysteine and histidine residues as well as two cleavage sites, namely Val-Gly/Gly for the 5' proximal P-PRO domain and Met-Gly/Gly for the 5' distal P-PRO domain. The autoproteolytic cleavage of the polyprotein at these sites would release two N-terminal leader proteins of 54 and 55 kDa, respectively, and a 240-kDa C-terminal fragment containing MT and HEL domains. The apparent duplication of the leader domain distinguishes CTV from BYV and accounts for most of the size increase in the ORF 1a product of CTV. The downstream ORF 1b encodes a 57-kDa putative RNA-dependent RNA polymerase (RdRp), which is probably expressed via a +1 ribosomal frameshift. Sequence analysis of the frameshift region suggests that this +1 frameshift probably occurs at a rare arginine codon CGG and that elements of the RNA secondary structure are unlikely to be involved in this process. The complete polyprotein resulting from this frameshift event has a calculated MW of 401 kDa and after cleavage of the two N-terminal leaders would yield a 292-kDa protein containing the MT, HEL, and RdRp domains. Phylogenetic analysis of the three replication-associated domains, MT, HEL, and RdRp, indicates that CTV and BYV form a separate closterovirus lineage within the alpha-like supergroup of positive-strand RNA viruses. Two gene blocks or modules can be easily identified in the CTV genome. The first includes the replicative MT, HEL, and RdRp genes and is conserved throughout the entire alpha-like superfamily. The second block consists of five ORFs, 3 to 7, conserved among closteroviruses, including genes for the CTV homolog of HSP70 proteins and a duplicate of the coat protein gene. The 3'-terminal ORFs 8 to 11 encode a putative RNA-binding protein (ORF 11), and three proteins with unknown functions; this gene array is poorly conserved among closteroviruses.(ABSTRACT TRUNCATED AT 400 WORDS)

The thylakoid ΔpH-dependent/Tat pathway is a novel system with the remarkable ability to transport tightly folded precursor proteins using a transmembrane ΔpH as the sole energy source. Three known components of the transport machinery exist in two distinct subcomplexes. A cpTatC–Hcf106 complex serves as precursor receptor and a Tha4 complex is required after precursor recognition. Here we report that Tha4 assembles with cpTatC–Hcf106 during the translocation step. Interactions among components were examined by chemical cross-linking of intact thylakoids followed by immunoprecipitation and immunoblotting. cpTatC and Hcf106 were consistently associated under all conditions tested. In contrast, Tha4 was only associated with cpTatC and Hcf106 in the presence of a functional precursor and the ΔpH. Interestingly, a synthetic signal peptide could replace intact precursor in triggering assembly. The association of all three components was transient and dissipated upon the completion of protein translocation. Such an assembly–disassembly cycle could explain how the ΔpH/Tat system can assemble translocases to accommodate folded proteins of varied size. It also explains in part how the system can exist in the membrane without compromising its ion and proton permeability barrier.

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 bacterial Sec and signal recognition particle (ffh-dependent) protein translocation mechanisms are conserved between prokaryotes and higher plant chloroplasts. A third translocation mechanism in chloroplasts [the proton concentration difference (⌬pH) pathway] was previously thought to be unique. The hcf106 mutation of maize disrupts the localization of proteins transported through this ⌬pH pathway in isolated chloroplasts. The Hcf106 gene encodes a receptor-like thylakoid membrane protein, which shows homology to open reading frames from all completely sequenced bacterial genomes, which suggests that the ⌬pH pathway has been conserved since the endosymbiotic origin of chloroplasts. Thus, the third protein translocation pathway, of which HCF106 is a component, is found in both bacteria and plants.

ABSTRACISeveral proteases, ie., pronase, a mixture oftrypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum satiium var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and 02 evolution.Methods involving protease treatment have been extensively used for probing the surfaces of cells, organelles, and isolated membranes (5, 13). In general, the exogenously added protease is able to digest proteins exposed on the outer surface of such structures but is prevented from gaining access to proteins on the other side ofthe membrane barrier. Thus, protease treatment has been used for a variety of purposes including: localization of membrane proteins and enzymes (5, 13), determining the orientation of transbilayer polypeptides (5, 13), removing proteins adsorbed or bound to the surface of membrane-bounded structures, etc. (12).The usefulness of protease treatment for such purposes relies on the inability of the protease to penetrate the bounding membrane. In many cases membrane rupture leading to protease penetration is not a problem because the debris from the ruptured material can be excluded from subsequent analysis by repurifying the remaining intact cells, organelles, etc. However, for double membrane-bounded organelles (chloroplasts, mitochondria), such a repurification procedure is not adequate since rupture of the outer membrane is not necessarily accompanied by rupture of the organelle. Consequently, special precautions must be undertaken to demonstrate that the exogenous proteases are limited in effect to the outer membrane.Recently, several studies including one from our own laboratory have utilized proteases as a surface probe of chloroplasts (3,4,6,8,12). Due to the complexity of the chloroplast-bounding membrane, the envelope, and because of certain inconsistencies we found in our own prior experiments using protease treatment (4), we have examined several proteases to determine their usefulness as surface probes of pea chloroplasts. This communication documents the use of thermolysin, a relatively nonspecific metalloprotease, as a probe whose effects are limited to the outer envelope membrane of isolated, intact pea chloroplasts. MATERIALS AND METHODSProteases were obtained from Sigma; radiochemicals were obtained from New England Nuclear. All other chemicals were reagent grade.Intact chloroplasts were purified from homogenates of 2-to 3-week-old pea seedlings (Pisum sativum var Laxton's Progress No. 9) by differential centrifugation followed by Percoll density gradient centrifugation as previously described (4). Chloroplast envelope membranes were isolated and subsequently subfractionated into inner and outer membrane fractions by the procedure described previously (4).For thermolysin treatment, purified intact...

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