The delta pH‐driven and Sec‐related thylakoidal protein translocases recognise distinct types of thylakoid transfer signal, yet all transfer signals resemble bacterial signal peptides in structural terms. Comparison of known transfer signals reveals a single concrete difference: signals for the delta pH‐dependent system contain a common twin‐arginine motif immediately before the hydrophobic region. We show that this motif is critical for the delta pH‐driven translocation process; substitution of the arg‐arg by gln‐gln or even arg‐lys totally blocks translocation across the thylakoid membrane, and replacement by lys‐arg reduces the rate of translocation by > 100‐fold. The targeting information in this type of signal thus differs fundamentally from that of bacterial signal peptides, where the required positive charge can be supplied by any basic amino acid. Insertion of a twin‐arg motif into a Sec‐dependent substrate does not alter the pathway followed but reduces translocation efficiency, suggesting that the motif may also repel the Sec‐type system. Other information must help to specify the choice of translocation mechanism, but this information is unlikely to reside in the hydrophobic region because substitution by a hydrophobic section from an integral membrane protein does not affect the translocation pathway.

Isoamylases are debranching enzymes that hydrolyze ␣ -1,6 linkages in ␣ -1,4/ ␣ -1,6-linked glucan polymers. In plants, they have been shown to be required for the normal synthesis of amylopectin, although the precise manner in which they influence starch synthesis is still debated. cDNA clones encoding three distinct isoamylase isoforms (Stisa1, Stisa2, and Stisa3) have been identified from potato. The expression patterns of the genes are consistent with the possibility that they all play roles in starch synthesis. Analysis of the predicted sequences of the proteins suggested that only Stisa1 and Stisa3 are likely to have hydrolytic activity and that there probably are differences in substrate specificity between these two isoforms. This was confirmed by the expression of each isoamylase in Escherichia coli and characterization of its activity. Partial purification of isoamylase activity from potato tubers showed that Stisa1 and Stisa2 are associated as a multimeric enzyme but that Stisa3 is not associated with this enzyme complex. Our data suggest that Stisa1 and Stisa2 act together to debranch soluble glucan during starch synthesis. The catalytic specificity of Stisa3 is distinct from that of the multimeric enzyme, indicating that it may play a different role in starch metabolism.

The recently discovered potato tuber (Solanum tuberosum) ␣-glucan, water dikinase (GWD) (formerly known as R1) catalyzes the phosphorylation of starch by a dikinase-type reaction mechanism in which the ␤-phosphate of ATP is transferred to either the C-6 or the C-3 position of the glucosyl residue of starch. In the present study, we found that the GWD enzyme is inactive in the oxidized form, which is accompanied by the formation of a specific intramolecular disulfide bond as determined by disulfide-linked peptide mapping. The regulatory properties of this disulfide linkage were confirmed by site-directed mutagenesis studies. Both reduced thioredoxin (Trx) f and Trx m from spinach leaves reduced and activated oxidized GWD at very low concentrations, with Trx f being the more efficient, yielding an S0.5 value of 0.4 M. Interestingly, GWD displays a reversible and selective binding to starch granules depending on the illumination state of the plant. Here we show that starch granule-bound GWD isolated from dark-adapted plants exists in the inactive, oxidized form, which is capable of reactivation upon treatment with reduced Trx. Furthermore, the soluble form of GWD was found in its fully reduced state, providing evidence of a Trx-controlled regulation mechanism linking enzymatic activity and specific binding affinities of a protein to an intracellular surface. The regulatory site sequence, CFATC, of potato GWD is conserved in chloroplast-targeted GWDs from other species, suggesting an overall redox regulation of the GWD enzyme.starch ͉ redox regulation ͉ thioredoxin

The Bundle sheath defective2 ( Bsd2 ) gene is required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2 -conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript ( rbcL ) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2 -conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize. INTRODUCTIONIn most plants, the primary function of leaves is to fix carbon through photosynthesis. The development of photosynthetic competence within the leaf requires the coordinated expression of both nucleus-and chloroplast-encoded genes. In particular, the accumulation of the most abundant photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco), requires direct contributions from both genomes. Higher plant Rubisco is a hexadecameric enzyme composed of eight large subunits (LSUs) encoded by a single chloroplast gene, rbcL , and eight small subunits (SSUs) encoded by a small nuclear RbcS gene family (reviewed in Gutteridge and Gatenby, 1995). As chloroplast differentiation in the leaf begins, light and plastidic signals induce the accumulation of the SSU protein (Tobin and Silverthorne, 1985;Mullet, 1988;Taylor, 1989). Concomitantly, the LSU is synthesized in the stroma of the chloroplast where both LSU and SSU complexes are assembled through a chaperonin-mediated process. This intricate assembly process requires the precise coordination of nuclear and chloroplast gene activities in response to both developmental and environmental signals (Mullet, 1988;Mayfield et al., 1995).As the name implies, Rubisco is also capable of oxygenating ribulose-1,5-bisphosphate without any net fixation of carbon. In many C 4 plants, including maize, Rubisco is restrict...

The homologous proteins Oxa1, YidC, and Alb3 mediate the insertion of membrane proteins in mitochondria, bacteria, and chloroplast thylakoids, respectively. Depletion of YidC in Escherichia coli affects the integration of every membrane protein studied, and Alb3 has been shown previously to be required for the insertion of a signal recognition particle (SRP)-dependent protein, Lhcb1, in thylakoids. In this study we have analyzed the "global" role of Alb3 in the insertion of thylakoid membrane proteins. We show that insertion of two chlorophyll-binding proteins, Lhcb4.1 and Lhcb5, is almost totally blocked by preincubation of thylakoids with anti-Alb3 antibodies, indicating a requirement for Alb3 in the insertion pathway. Insertion of the related PsbS protein, on the other hand, is unaffected by Alb3 antibodies, and insertion of a group of SRP-independent, signal peptide-bearing proteins, PsbX, PsbW, and PsbY, is likewise completely unaffected. Proteinase K is furthermore able to completely degrade Alb3, but this treatment does not affect the insertion of these proteins. Among the thylakoid proteins studied here, Alb3 requirement correlates strictly with a requirement for stromal factors and nucleoside triphosphates. However, the majority of proteins tested do not require Alb3 or any other known form of translocation apparatus.The post-translational insertion of proteins into their target membranes has attracted a great deal of experimental attention in recent years in an effort to determine how hydrophobic regions are transferred from an aqueous environment into the membrane bilayer, and how the correct topology is achieved during this process. In bacteria, a complex "assisted" pathway (reviewed in Refs. 1 and 2) has been characterized in which newly synthesized membrane proteins interact with signal recognition particle (SRP), 1 FtsY and membrane-bound components of the secretory (Sec) apparatus (3-8). SRP appears to be involved in membrane protein biogenesis by virtue of its tendency to interact with particularly hydrophobic protein segments (6, 9). A broadly similar assisted pathway operates in plant thylakoids for the targeting of the major light-harvesting chlorophyll-binding (LHC) protein, Lhcb1, after import of this protein from the cytosol. Insertion of Lhcb1 into thylakoids requires nucleoside triphosphates (NTPs), stromal SRP, FtsY, and a thylakoid translocase minimally composed of Albino3 (Alb3) (10 -13). Post-translational formation of a SRP/Lhcb1 targeting complex requires a hydrophobic domain along with a novel SRP-binding element in Lhcb1, termed the L18 domain, which is found only in members of the LHC protein family (14, 15). These data along with studies on chloroplast-synthesized D1 (16) suggest that SRP is again used primarily to direct membrane proteins to the thylakoid membrane.For many years it was believed that other membrane proteins, in both bacteria and chloroplasts, were targeted by unassisted or "spontaneous" insertion pathways, in which the protein inserted directly into the bilayer...

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma.Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.

Two distinct mechanisms have been previously identified for the transport of proteins across the chloroplast thylakoid membrane, one of which is unusual in that neither soluble factors nor ATP are required; the system requires only the transthylakoidal delta pH. We have examined this mechanism by studying the properties of one of its substrates: the extrinsic 23-kDa protein (23K) of photosystem II. Previous work has shown that this protein can be transported into isolated thylakoids as the full-length precursor protein; we show that the stromal import intermediate form of this protein is similarly translocation-competent. Gel filtration tests indicate that the stromal intermediate is probably monomeric. Protease sensitivity tests on both the initial in vitro translation product and the stromal import intermediate show that the presequence is highly susceptible to digestion whereas the mature protein is resistant to high concentrations of trypsin. The mature protein becomes very sensitive to digestion if unfolded in urea, or after heating, and we therefore propose that the natural substrate for this translocation system consists of a relatively unfolded presequence together with a tightly folded passenger protein. The ability of thylakoids to import pre-23K is destroyed by prior treatment of the thylakoids with low concentrations of trypsin, demonstrating the involvement of surface-exposed proteins in the import process. However, we can find no evidence for the binding of pre-23K or i23K to the thylakoid surface, and we therefore propose that the initial interaction of these substrates with the thylakoidal translocase is weak, reversible, and probably delta pH-independent. In the second phase of the translocation mechanism, the delta pH drives either the translocation and unfolding of proteins, or the translocation of a fully folded protein.

The Bundle sheath defective2 ( Bsd2 ) gene is required for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) accumulation in maize. Using a Mutator transposable element as a molecular probe, we identified a tightly linked restriction fragment length polymorphism that cosegregated with the bsd2 -conferred phenotype. This fragment was cloned, and sequences flanking the Mutator insertion were used to screen a maize leaf cDNA library. Using a full-length cDNA clone isolated in this screen, we show that an abundant 0.6-kb transcript could be detected in wild-type plants but not in bsd2-m1 plants. This 0.6-kb transcript accumulated to low levels in plants carrying an allele derived from bsd2-m1 that conditions a less severe mutant phenotype. Taken together, these data strongly suggest that we have cloned the Bsd2 gene. Sequence analysis of the full-length cDNA clone revealed a chloroplast targeting sequence and a region of homology shared between BSD2 and the DnaJ class of molecular chaperones. This region of homology is limited to a cysteine-rich Zn binding domain in DnaJ believed to play a role in protein-protein interactions. We show that BSD2 is targeted to the chloroplast but is not involved in general photosynthetic complex assembly or protein import. In bsd2 mutants, we could not detect the Rubisco protein, but the chloroplast-encoded Rubisco large subunit transcript ( rbcL ) was abundant and associated with polysomes in both bundle sheath and mesophyll cells. By characterizing Bsd2 expression patterns and analyzing the bsd2 -conferred phenotype, we propose a model for BSD2 in the post-translational regulation of rbcL in maize. INTRODUCTIONIn most plants, the primary function of leaves is to fix carbon through photosynthesis. The development of photosynthetic competence within the leaf requires the coordinated expression of both nucleus-and chloroplast-encoded genes. In particular, the accumulation of the most abundant photosynthetic enzyme, ribulose-1,5-bisphosphate carboxylase/ oxygenase (Rubisco), requires direct contributions from both genomes. Higher plant Rubisco is a hexadecameric enzyme composed of eight large subunits (LSUs) encoded by a single chloroplast gene, rbcL , and eight small subunits (SSUs) encoded by a small nuclear RbcS gene family (reviewed in Gutteridge and Gatenby, 1995). As chloroplast differentiation in the leaf begins, light and plastidic signals induce the accumulation of the SSU protein (Tobin and Silverthorne, 1985;Mullet, 1988;Taylor, 1989). Concomitantly, the LSU is synthesized in the stroma of the chloroplast where both LSU and SSU complexes are assembled through a chaperonin-mediated process. This intricate assembly process requires the precise coordination of nuclear and chloroplast gene activities in response to both developmental and environmental signals (Mullet, 1988;Mayfield et al., 1995).As the name implies, Rubisco is also capable of oxygenating ribulose-1,5-bisphosphate without any net fixation of carbon. In many C 4 plants, including maize, Rubisco is restrict...