SummaryWe have used fusions of gibberellin biosynthesis enzymes to green¯uorescent protein (GFP) to determine the subcellular localization of the early steps of the pathway. Gibberellin biosynthesis from geranylgeranyl diphosphate is catalysed by enzymes of the terpene cyclase, cytochrome P450 monooxygenase and 2-oxoglutarate-dependent dioxygenase classes. We show that the N-terminal presequences of the Arabidopsis thaliana terpene cyclases copalyl diphosphate synthase (AtCPS1) and entkaurene synthase (AtKS1) direct GFP to chloroplasts in transient assays following microprojectile bombardment of tobacco leaves. The AtKS1±GFP fusion is also imported by isolated pea chloroplasts. The N-terminal portion of the cytochrome P450 protein ent-kaurene oxidase (AtKO1) directs GFP to chloroplasts in tobacco leaf transient assays. Chloroplast import assays with 35 S-labelled AtKO1 protein show that it is targeted to the outer face of the chloroplast envelope. The leader sequences of the two ent-kaurenoic acid oxidases (AtKAO1 and AtKAO2) from Arabidopsis direct GFP to the endoplasmic reticulum. These data suggest that the AtKO1 protein links the plastid-and endoplasmic reticulumlocated steps of the gibberellin biosynthesis pathway by association with the outer envelope of the plastid.

Summary β‐Amylase is one of the most abundant starch degrading activities found in leaves and other plant organs. Despite its abundance, most if not all of this activity has been reported to be extrachloroplastic and for this reason, it has been assumed that β‐amylases are not involved in the metabolism of chloroplast‐localized transitory leaf starch. However, we have identified a novel β‐amylase gene, designated ct‐Bmy, which is located on chromosome IV of Arabidopsis thaliana. Ct‐Bmy encodes a precursor protein which contains a typical N‐terminal chloroplast import signal and is highly similar at the amino acid level to extrachloroplastic β‐amylases of higher plants. Expression of the ct‐Bmy cDNA in E. coli confirmed that the encoded protein possesses β‐amylase activity. CT‐BMY protein, synthesized in vitro, was efficiently imported by isolated pea chloroplasts and shown to be located in the stroma. In addition, fusions between the predicted CT‐BMY transit peptide and jellyfish green fluorescent protein (GFP) or the entire CT‐BMY protein and GFP showed accumulation in vivo in chloroplasts of Arabidopsis. Expression of the GUS gene fused to ct‐Bmy promoter sequences was investigated in transgenic tobacco plants. GUS activity was most strongly expressed in the palisade cell layer in the leaf blade and in chlorenchyma cells associated with the vascular strands in petioles and stems. Histochemical staining of whole seedlings showed that GUS activity was largely confined to the cotyledons during the first 2 weeks of growth and appeared in the first true leaves at approximately 4 weeks.

Immunophilins are intracellular receptors of the immunosuppressants cyclosporin A, FK506, and rapamycin. Although all immunophilins possess peptidyl-prolyl isomerase activity and are identified from a wide range of organisms, little is known about their cellular functions. We report the characterization and functional analysis of an FK506 and rapamycin-binding protein (AtFKBP13) from Arabidopsis. The AtFKBP13 protein is synthesized as a precursor that is imported into chloroplasts and processed to the mature form located in the thylakoid lumen, as shown by chloroplast import assays and Western blot analysis. Experiments show that AtFKBP13 is translocated across the thylakoid membrane by the ⌬pH-dependent pathway. Yeast twohybrid screening identified Rieske FeS protein, a subunit of the cytochrome bf complex in the photosynthetic electron transport chain, as an interacting partner for AtFKBP13. Both yeast two-hybrid and in vitro protein-protein interaction assays showed that the precursor, but not the mature form, of AtFKBP13 interacted with Rieske protein, suggesting that interaction between the two proteins occurs along the import pathway. When AtFKBP13 expression was suppressed by RNA interference method, the level of Rieske protein was significantly increased in the transgenic plants.

Signal recognition particles (SRPs) have been identified in organisms as diverse as mycoplasma and mammals; in several cases these SRPs have been shown to play a key role in protein targeting. In each case the recognition of appropriate targeting signals is mediated by SRP subunits related to the 54-kDa protein of mammalian SRP (SRP54). In this study we have characterized the specificity of 54CP, a chloroplast homologue of SRP54 which is located in the chloroplast stroma. We have used a nascent chain cross-linking approach to detect the interactions of 54CP with heterologous endoplasmic reticulum-targeting signals. 54CP functions as a bona fide signal recognition factor which can discriminate between functional and non-functional targeting signals. Using a range of authentic thylakoid precursor proteins we found that 54CP discriminates between thylakoid-targeting signals, interacting with only a subset of protein precursors. Thus, the light-harvesting chlorophyll a/b-binding protein, cytochrome f, and the Rieske FeS protein all showed strong cross-linking products with 54CP. In contrast, no cross-linking to the 23-and 33-kDa proteins of the oxygen-evolving complex were detected. The selectivity of 54CP correlates with the hydrophobicity of the thylakoid-targeting signal and, in the case of light-harvesting chlorophyll a/b-binding protein, with previously determined transport/integration requirements. We propose that 54CP mediates the targeting of a specific subset of precursors to the thylakoid membrane, i.e. those with particularly hydrophobic signal sequences. The signal recognition particle (SRP)1 of mammalian cells is a ribonucleoprotein complex which promotes the signal sequence-dependent targeting of nascent precursor proteins to the endoplasmic reticulum (1-3). Mammalian SRP is composed of six polypeptides and a 7 S RNA, although only one of the polypeptides, the 54-kDa subunit (SRP54), binds to the hydrophobic endoplasmic reticulum (ER)-targeting signals (1-2). Functional homologues of SRP54 have been identified in many organisms and appear relatively conserved during evolution (4, 5). These proteins are usually found complexed with a 7 S-like RNA and the minimum requirement for SRP-dependent protein targeting seems to be a ribonucleoprotein particle composed of an SRP54-like protein and a 7 S-like RNA (6 -8), together with a cognate receptor for the SRP-precursor protein complex (3, 9, 10). Perturbation of SRP-dependent targeting pathways often leads to the accumulation of secretory proteins. However, in many cases only a subset of precursors accumulate while other proteins continue to be secreted normally (9,(11)(12)(13). This suggests that a discrete population of precursors preferentially utilize an SRP-dependent targeting pathway (1, 4, 13).The delivery of precursor proteins to the thylakoid membrane of chloroplasts is governed by thylakoid-targeting signals. These signals are clearly related to those which target proteins to the ER membrane of eukaroytes and the cytoplasmic membrane of prokaryotes....