Arabidopsis plants were transformed with a multi-gene construct for expression of the polyhydroxybutyrate (PHB) biosynthetic pathway containing a gene switch that can be activated by commercially available non-steroidal ecdysone analogs approved for use on some crops as pesticides. T(1) progeny of transgenic Arabidopsis plants were isolated and screened for PHB production in the presence of ecdysone analogs. T(2) progeny derived from selected T(1) lines were subjected to further analysis by comparing PHB production levels prior to treatment with inducing agent and 21 days after initiation of induction. Significant PHB production was delayed in many of the engineered plants until after induction. PHB levels of up to 14.3% PHB per unit dry weight were observed in young leaves harvested from engineered T(2) plants after applications of the commercial ecdysone analog Mimic. PHB in older leaves reached levels of up to 7% PHB per unit dry weight. This study represents a first step towards engineering a chemically inducible gene switch for PHB production in plants using inducing agents that are approved for field use.

Many of the proteins in the chloroplast envelope play an important role in facilitating the biochemical and transport processes of the compartment. For the transport of proteins into the chloroplast, we have recently identified at least three different envelope proteins (Com44/Cim44, Com70, and Cim97) in close physical proximity to a partially translocated chimeric precursor protein (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264 -32271). In this study we report the characterization of a cDNA clone encoding a member of the Com44/Cim44 envelope proteins. The combined data from nucleotide sequencing, and RNA and protein blot analyses indicate the existence of multiple forms of the 44-kDa envelope protein. Depending on the plant species examined, immunologically-related protein bands with molecular masses of 42 to 46 kDa were observed. Organelle subfractionation, protease treatment, and immunomicroscopic studies together provide an indication that the immunologically-related proteins may be present in both the outer and inner envelope membranes. Co-migration of the product synthesized from the cDNA insert with a 44-kDa immunoreactive band of the chloroplast envelope, and the in vitro import results, together suggest that the in vitro synthesized 44-kDa protein is targeted to the envelope membrane without any further processing.Chloroplast envelope proteins play a major role in modulating the vectorial flow of molecules across the membrane, including large proteinaceous entities. The import of proteins into the chloroplast is a complex process requiring the close collaboration of both the outer envelope and the inner envelope membranes. Evidence for the possible existence of two distinct protein import complexes, one in each envelope membrane, is beginning to emerge from a number of recent investigations (Waegemann and Soll, 1991;Soll and Waegemann, 1992;Schnell and Blobel, 1993;Alefson et al., 1994; Schell et al., 1994;Kessler et al., 1994;Wu et al., 1994). An important step in the characterization of the protein translocating complexes is the identification of the components involved. The identification of outer and inner envelope polypeptides of these protein translocating complexes has been achieved using a variety of strategies (Cornwall and Keegstra, 1987;Kaderbhai et al., 1988;Pain et al., 1988; Schnell et al., 1990aSchnell et al., , 1994Hinz and Flugge, 1988;Soll and Waegemann, 1992;Waegemann et al., 1990;Perry and Keegstra, 1994;Alefson et al., 1994;Wu et al., 1994;Hirsch et al., 1994;Gray and Row, 1995). So far these studies collectively indicate that envelope proteins with molecular masses of 30, 34, 36, 44, 45, 51, 66, 70, 75, 86, 97, and 100 kDa may be possible constituents of the chloroplast protein import apparatus; however, it is not obvious from the existing data whether some of the predicted similar sized components are identical to each other.The complex nature of protein translocation mechanisms observed in other membranous systems, such as the mitochondrion and the endoplas...

SummaryThe production of polyhydroxybutyrate (PHB) involves a multigene pathway consisting of thiolase, reductase and synthase genes. In order to simplify this pathway for plant-based expression, a library of thiolase and reductase gene fusions was generated by randomly ligating a short core linker DNA sequence to create in-frame fusions between the thiolase and reductase genes. The resulting fusion constructs were screened for PHB formation in Escherichia coli . This screen identified a polymer-producing candidate in which the thiolase and reductase genes were fused via a 26-amino-acid linker. This gene fusion, designated phaA-phaB , represents an active gene fusion of two homotetrameric enzymes. Expression of phaA-phaB in E. coli and Arabidopsis yielded a fusion protein observed to be the expected size by Western blotting techniques. The fusion protein exhibited thiolase and reductase enzyme activities in crude extracts of recombinant E. coli that were three-fold and nine-fold less than those of the individually expressed thiolase and reductase enzymes, respectively. When targeted to the plastid, and coexpressed with a plastid-targeted polyhydroxyalkanoate (PHA) synthase, the fusion protein enabled PHB formation in Arabidopsis , yielding roughly half the PHB formed in plants expressing individual thiolase, reductase and synthase enzymes. This work represents a first step towards simplifying the expression of the PHB biosynthetic pathway in plants.

SecA is an essential ATP‐dependent motor protein that interacts with the preprotein and translocon to drive protein translocation across the eubacterial plasma membrane. A region containing residues 267–340 has been proposed to comprise the preprotein binding site of Escherichia coli SecA. To elucidate the function of this region further, we isolated mutants using a combination of region‐specific polymerase chain reaction (PCR) mutagenesis and a genetic and biochemical screening procedure. Although this region displayed considerable plasticity based on phylogenetic and genetic analysis, Tyr‐326 was found to be critical for SecA function. secA mutants with non‐conservative substitutions at Tyr‐326 showed strong protein secretion defects in vivo and were completely defective for SecA‐dependent translocation ATPase activity in vitro. The SecA‐Y326 mutant proteins were normal in their membrane, SecYE and nucleotide‐binding properties. However, they exhibited a reduced affinity for preprotein and were defective in preprotein release, as assessed by several biochemical assays. Our results indicate that the region containing Tyr‐326 functions as a conformational response element to regulate the preprotein binding and release cycle of SecA.

The chloroplast envelope protein Com70 is a hsp70 homolog identified recently as a component of the protein translocation apparatus. The stage of protein import involving Com70 was determined by examining the nature of the association of Com70 with the envelope and its interaction with translocating proteins. Com70 is accessible to thermolysin, but its association with the envelope could not be disrupted by stringent washes. In light of the external membrane-bound location, the involvement of Com70 at the early stage of protein translocation was investigated using a combination of in vitro binding assays, chemical cross-linking, and coimmunoprecipitation. The results provide evidence that Com70 is in close physical proximity to different types of chloroplast protein precursors under conditions supporting binding rather than complete translocation. The formation of cross-linked complexes is dependent on the presence of a typical plastid transit signal and proteaseaccessible outer envelope components. The close proximity of Com70 and the translocating protein occurs while the protein is still exposed to the cytosol.The mechanism for importing chloroplast protein precursors into the compartment requires energy and is facilitated by specific envelope proteins (1). Recent studies revealed that envelope proteins with sizes of 34, 36, 44, 70 (at least two such related proteins), 75, 86, and 97 kDa are in close physical proximity to translocating proteins (1, 2). Although the role of each component remains to be elucidated, the outer envelope location of the 34-, 70-, 75-, and 86-kDa proteins places their involvement in the earlier part of the import pathway. The combination of GTP-binding domains in the 34-and 86-kDa proteins and the inhibition of precursor binding with nonhydrolyzable GTP analogs suggests that these two components may be involved in the energy-requiring aspect of the binding step (3-5). The 75-kDa protein is hypothesized, based on its predicted characteristics, to be a candidate for the import channel (6, 7). The 70-kDa components are related forms of the heat shock proteins (hsp70s) and may thus act similarly as chaperones/unfoldases (6,8,9).The importance of hsp70 in protein transport is exemplified by their widespread involvement in translocation mechanisms of the cytosol and/or the interior of different organelles (10 -12). Chloroplast protein import also appears to involve multiple hsp70-containing sites (cytosol, stroma, and envelope) (6,(13)(14)(15)(16). Since the chloroplast protein import machinery contains hsp70-related components at different locales within the outer envelope, external (Com70) versus internal (Iap70 and Hsc70) (6, 14, 17) it would be important to assess the individual contribution of each different hsp70-containing locale and the stage of its involvement. In this study we assessed further the role of Com70 in protein import by first examining the nature of the Com70 association with the outer envelope. In light of the strong association of Com70 with the cytosolic face of t...