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J Soll

Publications and source records attributed to J Soll.

At least 55 records · Page 3Linked to original sources
PubMed1997

A nuclear-coded chloroplastic inner envelope membrane protein uses a soluble sorting intermediate upon import into the organelle.

The chloroplastic inner envelope protein of 110 kD (IEP110) is part of the protein import machinery in the pea. Different hybrid proteins were constructed to assess the import and sorting pathway of IEP110. The IEP110 precursor (pIEP110) uses the general import pathway into chloroplasts, as shown by the mutual exchange of presequences with the precursor of the small subunit of ribulose-1,5-bisphosphate carboxylase (pSSU). Sorting information to the chloroplastic inner envelope is contained in an NH2-proximal part of mature IEP110 (110N). The NH2-terminus serves to anchor the protein into the membrane. Large COOH-terminal portions of this protein (80-90 kD) are exposed to the intermembrane space in situ. Successful sorting and integration of IEP110 and the derived constructs into the inner envelope are demonstrated by the inaccessability of processed mature protein to the protease thermolysin but accessibility to trypsin, i.e., the imported protein is exposed to the intermembrane space. A hybrid protein consisting of the transit sequence of SSU, the NH2-proximal part of mature IEP110, and mature SSU (tpSSU-110N-mSSU) is completely imported into the chloroplast stroma, from which it can be recovered as soluble, terminally processed 110NmSSU. The soluble 110N-mSSU then enters a reexport pathway, which results not only in the insertion of 110N-mSSU into the inner envelope membrane, but also in the extrusion of large portions of the protein into the intermembrane space. We conclude that chloroplasts possess a protein reexport machinery for IEPs in which soluble stromal components interact with a membrane-localized translocation machinery.

Biological Transport↗
PubMed1997

Postimport methylation of the small subunit of ribulose-1,5-bisphosphate carboxylase in chloroplasts.

Electron impact mass spectronomy analysis of the amino-terminal amino acid of the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase (Rubisco) showed that the amino-terminal methionine residue is post-translationally modified to N-methyl-methionine. Modification of the amino-terminal methionine residue was found in mature SSU proteins from the dicotyledonous plants pea and spinach as well as the monocotyledonous plants barley and corn. SSU methyltransferase is a soluble protein in the chloroplast stroma and accepts heterologously expressed non-methylated SSU as a substrate using S-adenosylmethionine as methyl-group donor. We show that this modification occurs after post-translational uptake of the precursor form of SSU into chloroplasts and processing to its mature size. This reaction represents a new step in the import and assembly pathway of Rubisco holoenzyme.

Chloroplasts↗
PubMed1997

Chloroplastic isoforms of DnaJ and GrpE in pea.

The folding and assembly of proteins in cells often requires the assistance of molecular chaperones such as the Hsp70 and Hsp60 heat shock proteins. Hsp70 chaperones cooperate with DnaJ and GrpE homologues to ensure a productive folding cycle. In this study we describe the gene for the first chloroplast localized DnaJ homologue and present evidence that the gene product is at least partially associated with the inner envelope membrane. Immunoblot analysis also provides evidence for the presence of a GrpE homologue in plastids.

Amino Acid Sequence↗
PubMed1996

A protein import receptor of chloroplasts is inserted into the outer envelope membrane by a novel pathway.

The outer envelope protein OEP86 functions as a receptor for precursor proteins in the chloroplastic import machinery. In contrast to most other organellar outer membrane proteins it is synthesized as a precursor polypeptide (preOEP86) in the cytosol and is post-translationally targeted to the organelles. PreOEP86 is targeted to and productively inserted into the chloroplastic outer envelope mediated by a bipartite signal consisting of the presequence and the COOH terminus of the precursor protein. The cleavable presequence alone does not seem to contain sufficient information to target preOEP86 without the COOH terminus or a hybrid protein consisting of the presequence of preOEP86 and the mature form of the small subunit of ribulose bisphosphate carboxylase to intact chloroplasts. The presequence seems to be required to maintain preOEP86 in an integration competent state, whereas interaction of preOEP86 with chloroplasts is accomplished by a short sequence of amino acids in the COOH-terminal portion of the mature protein. The COOH-terminal portion of preOEP86 contains enough information to also direct mature OEP86 into the outer envelope membrane of pea chloroplasts. However, mature OEP86 enters the productive folding pathway much less efficiently than preOEP86. The COOH terminus of preOEP86 not only serves as a membrane anchor but seems to be required for a productive translocation through an interaction with other outer envelope proteins. Although the binding was ATP-dependent, productive folding was not. PreOEP86 seems to follow a unique road into the chloroplastic outer envelope.

Adenosine Triphosphate↗
PubMed1996

Topology of IEP110, a component of the chloroplastic protein import machinery present in the inner envelope membrane.

Proteins from both the inner and outer envelope membranes are engaged in the recognition and translocation of precursor proteins into chloroplasts. A 110 kDa protein of the chloroplastic inner envelope membrane was identified as a component of the protein import apparatus by two methods. First, this protein was part of a 600 kDa complex generated by cross-linking of precursors trapped in the translocation process. Second, solubilization with detergents of chloroplasts containing trapped precursors resulted in the identification of a complex containing both radiolabeled precursor and IEP110. Trypsin treatment of intact purified chloroplasts was used to study the topology of IEP110. The protease treatment left the inner membrane intact while simultaneously degrading domains of inner envelope proteins exposed to the intermembrane space. About 90 kDa of IEP110 was proteolitically removed, indicating that large portions protrude into the intermembrane space. Hydropathy analysis of the protein sequence deduced from the isolated cDNA clone in addition to Western blot analysis using an antiserum of IEP110 specific to the N-terminal 20 kDa, suggests that the N-terminus serves to anchor the protein in the membrane. We speculate that IEP110 could be involved in the formation of translocation contact sites due to its specific topology.

Amino Acid Sequence↗
PubMed1996

Phosphorylation of the transit sequence of chloroplast precursor proteins.

A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step, which could lead to a specific sorting and productive translocation in plant cells.

Base Sequence↗
PubMed1996

Molecular chaperones are present in the thylakoid lumen of pea chloroplasts.

A soluble protein fraction was obtained from pea chloroplast thylakoids, which represents highly enriched lumenal components. Using antisera against chaperonin 60 (cpn60), chaperonin 10 (cpn10) and the heat shock cognate protein of 70 kDa (hsc70) we are able to demonstrate, that the thylakoid lumen contains a separate set of molecular chaperones, which is distinct from the stromal one. In contrast to the alpha and beta subunits of cpn60 present in the stroma the lumen contains only one cpn60 isoform of distinct isoelectric point. Furthermore the lumenal cpn10 is of 'normal' size and not like its stromal counterpart of a double-domain tandem architecture. The immunoreactive hsc70 isoforms in the lumen seem also to be different from the stromal ones. Thus, chloroplasts seem to contain the largest number of molecular chaperone isoforms present in one organelle.

Chaperonin 10↗
PubMed1996

CP12: a small nuclear-encoded chloroplast protein provides novel insights into higher-plant GAPDH evolution.

Higher-plant chloroplast NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (NAD(P)-GAPDH; EC 1.2.1.13) is composed of two different nuclear-encoded subunits, GAPA and GAPB, forming the highly active heterotetrameric A2B2 enzyme. The main difference between these two subunits is a C-terminal extension of about 30 amino acid residues of GAPB. We present cDNA clones for a nuclear-encoded chloroplast protein from pea, spinach and tobacco, which we have named CP12. The mature protein consists of only 74, 75 and 76 amino acid residues, respectively and contains two domains with significant homology to the C-terminal extension of GAPB. Affinity chromatography approaches reveal also a specific interaction between CP12 and chloroplast GAPDH. Northern blot analysis indicates that CP12 is, like plastid GAPDH, expressed in green and also in etiolated leaves. Further homology is observed between CP12 and ORF3, an open reading frame located in the hox gene cluster of Anabaena variabilis. This gene cluster encodes the subunits of the bidirectional NADP(+)-dependent [NiFeS] dehydrogenase. We propose therefore a common evolutionary origin of CP12 and higher-plant chloroplast GAPDH subunit GAPB from the cyanobacterial ORF3.

Amino Acid Sequence↗
PubMed1996

The pea chloroplast membrane-associated protein, IEP96, is a subunit of acetyl-CoA carboxylase.

Two forms of acetyl-CoA carboxylase (ACCase) have been characterized in pea (Pisum sativum L.) leaves; a heteromeric chloroplast enzyme and a homomeric, presumably cytosolic enzyme. The biotin carboxylase (BC), biotin carboxyl carrier protein (BCCP), and beta-carboxyltransferase (CT) subunits of the plastidial-ACCase have recently been characterized and cloned. To further characterize the carboxyltransferase, an improved assay for CT was developed and used to follow its partial purification. CT activity co-purifies with ACCase activity during gel permeation chromatography. However, upon anion-exchange chromatography or native PAGE, CT separates from the BC and BCCP subunits of plastidial-ACCase and ACCase activity is lost. In addition, it is demonstrated that a previously sequenced pea chloroplast cDNA of unknown function (IEP96) with a predicted molecular weight of 91 kDa encodes the alpha-CT subunit of the MS-ACCase. Antibodies raised against the first 404 amino acids of IEP96 protein detected a polypeptide with molecular weight of 91 kDa that co-eluted during gel permeation chromatography with plastidial CT and ACCase activities. These antibodies also immunoprecipitated the activities of both ACCase and CT with the concomitant precipitation of the beta-CT subunit. Furthermore, antibodies against beta-CT immunoprecipitated the IEP96 protein. Two-dimensional PAGE and DEAE purification of ACCase protein demonstrated that the beta-CT forms a tight association with the IEP96 protein. Pea leaf was fractionated into soluble and membrane fractions and the alpha-CT subunit was primarily associated with the membrane fraction. Together, these data demonstrate that IEP96 is the alpha-CT subunit of pea chloroplast ACCase.

Acetyl-CoA Carboxylase↗
PubMed1995

Functional characterization of the higher plant chloroplast chaperonins.

The higher plant chloroplast chaperonins (ch-cpn60 and ch-cpn10) have been purified and their structural/functional properties examined. In all plants surveyed, both proteins were constitutively expressed, and only modest increases in their levels were detected upon heat shock. Like GroEL and GroES of Escherichia coli, the chloroplast chaperonins can physically interact with each other. The asymmetric complexes that form in the presence of ADP are "bullet-shaped" particles that likely consist of 1 mol each of ch-cpn60 and ch-cpn10. The purified ch-cpn60 is a functional molecular chaperone. Under "nonpermissive" conditions, where spontaneous folding was not observed, it was able to assist in the refolding of two different target proteins. In both cases, successful partitioning to the native state also required ATP hydrolysis and chaperonin 10. Surprisingly, however, the "double-domain" ch-cpn10, comprised of unique 21-kDa subunits, was not an obligatory co-chaperonin. Both GroES and a mammalian mitochondrial homolog were equally compatible with the ch-cpn60. Finally, the assisted-folding reaction mediated by the chloroplast chaperonins does not require K+ ions. Thus, the K(+)-dependent ATPase activity that is observed with other known groEL homologs is not a universal property of all chaperonin 60s.

Adenosine Diphosphate↗
PubMed1995

Copper chloride, an inhibitor of protein import into chloroplasts.

We have used the oxidant CuCl2 to study its effect on precursor protein import into chloroplasts and on the components involved. CuCl2 reversibly oxidizes thiol groups, which in turn, can form disulfide bridges. Concentrations of 40 microM CuCl2 almost completely inhibit precursor protein binding and subsequent translocation into chloroplasts. This inhibitory effect is reversible by a dithiothreitol treatment. Disulfide bridges, which form upon oxidation by CuCl2, are build up intramolecular and intermolecular, if the thiol groups are in close vicinity to each other. CuCl2 can thus be used as a thiol cleavable crosslinker without an additional spacer distance between the two targets. When purified outer envelope membranes were treated with CuCl2, a hetero oligomeric complex is detected, consisting of OEP86, OEP75 and OEP34, indicating the close vicinity and protein-protein interaction between polypeptides in situ, which are involved in protein translocation into chloroplasts.

Biological Transport↗
PubMed1995

Spinach chloroplast cpn21 co-chaperonin possesses two functional domains fused together in a toroidal structure and exhibits nucleotide-dependent binding to plastid chaperonin 60.

Chloroplasts contain a 21-kDa co-chaperonin polypeptide (cpn21) formed by two GroES-like domains fused together in tandem. Expression of a double-domain spinach cpn21 in Escherichia coli groES mutant strains supports growth of bacteriophages lambda and T5, and will also suppress a temperature-sensitive growth phenotype of a groES619 strain. Each domain of cpn21 expressed separately can function independently to support bacteriophage lambda growth, and the N-terminal domain will additionally suppress the temperature-sensitive growth phenotype. These results indicate that chloroplast cpn21 has two functional domains, either of which can interact with GroEL in vivo to facilitate bacteriophage morphogenesis. Purified spinach cpn21 has a ring-like toroidal structure and forms a stable complex with E. coli GroEL in the presence of ADP and is functionally interchangeable with bacterial GroES in the chaperonin-facilitated refolding of denatured ribulose-1,5-bisphosphate carboxylase. Cpn21 also inhibits the ATPase activity of GroEL. Cpn21 binds with similar efficiency to both the alpha and beta subunits of spinach cpn60 in the presence of adenine nucleotides, with ATP being more effective than ADP. The tandemly fused domains of cpn21 evolved early and are present in a wide range of photosynthetic eukaryotes examined, indicating a high degree of conservation of this structure in chloroplasts.

Adenosine Triphosphatases↗
PubMed1995

Functional analysis of isolated cpn10 domains and conserved amino acid residues in spinach chloroplast co-chaperonin by site-directed mutagenesis.

The possibilities of independent function of the two chaperonin 10 (cpn10) domains of the cpn10 homologue from spinach chloroplasts and the role of five conserved amino acid residues in the N-terminal cpn10 unit were investigated. Recombinant single domain proteins and complete chloroplast cpn10 proteins carrying amino acid exchanges of conserved residues in their N-terminal cpn10 domain were expressed in Escherichia coli and partially purified. The function of the recombinant proteins was tested using GroEL as chaperonin 60 (cpn60) partner for in vitro refolding of denatured ribulose-1,5-bisphosphate carboxylase (Rubisco). Interaction with cpn60 was also monitored by the ability to inhibit GroEL ATPase activity. In vitro both isolated cpn10 domains were found to be incapable of co-chaperonin function. All mutants were also severely impaired in cpn10 function. The results are interpreted in terms of an essential role of the exchanged amino acid residues for the interaction between co-chaperonin and cpn60 partner and in terms of a functional coupling of both cpn10 domains. To test the function of mutant chloroplast cpn10 proteins in vivo the cpn10 deficiency of E. coli strain CG712 resulting in an inability to assemble lambda-phage was exploited in a complementation assay. Transformation with plasmids directing the expression of mutant chloroplas cpn10 proteins in two cases restored lambda-phage assembly in this bacterial strain to the same extent as did transformation with a plasmid encoding wild-type cpn10 protein. In contrast a plasmid encoded third mutant and truncated forms of chloroplast cpn10 showed significantly reduced complementation efficiencies.

Adenosine Triphosphatases↗
PubMed1995

Import of a new chloroplast inner envelope protein is greatly stimulated by potassium phosphate.

A cDNA clone encoding a major chloroplast inner envelope membrane protein of 96 kDa (IEP96) was isolated and characterized. The protein is synthesized as a larger-molecular-weight precursor (pIEP96) which contains a cleavable N-terminal transit sequence of 50 amino acids. The transit peptide exhibits typical stromal targeting information. It is cleaved in vitro by the stromal processing peptidase, though the mature protein is clearly localized in the inner envelope membrane. Translocation of pIEP96 into chloroplasts is greatly stimulated in the presence of 80 mM potassium phosphate which results in an import efficiency of about 90%. This effect is specific for potassium and phosphate, but cannot be ascribed to a membrane potential across the inner envelope membrane. Protein sequence analysis reveals five stretches of repeats of 26 amino acids in length. The N-terminal 300 amino acids are 45% identical (76% similarity) to the 35 kDa alpha-subunit of acetyl-CoA carboxyl-transferase from Escherichia coli. The C-terminal 500 amino acids share significant similarity (69%) with USOI, a component of the cytoskeleton in yeast.

Amino Acid Sequence↗
PubMed1995

A constituent of the chloroplast import complex represents a new type of GTP-binding protein.

The 34 kDa polypeptide of the outer envelope membranes from pea chloroplasts (OEP 34) is a major constituent of this membrane. OEP 34 is detected on polyacrylamide gels under non-reducing condition in association with OEP 75, the putative protein translocation pore. An antiserum against OEP 34 is able to co-immunoprecipitate the precursor of Rubisco small subunit from a partially purified import complex of chloroplast outer envelope membranes. A full-length cDNA clone coding for pea OEP 34 has been isolated. Analysis of the deduced amino acid sequence revealed typical and conserved sequence motifs found in GTP-binding proteins, making it a new and unique member of this superfamily. OEP 34 behaves as an integral constituent of the outer chloroplast envelope, which is anchored by its C-terminus into the membrane, while the majority of the protein projects into the cytoplasm. OEP 34 does not possess a cleavable N-terminal transit sequence but it is targeted to the chloroplasts and integrated into the outer membranes by internal sequence information which seems to be present in the C-terminal membrane anchor region. Productive integration of OEP 34 into the outer envelope requires, in contrast to other OEPs, protease-sensitive chloroplast surface components and is stimulated by ATP. The GTP binding specificity of OEP 34 is demonstrated by photo-affinity labelling in the presence of [alpha-32P]GTP. Overexpressed and purified OEP 34 possesses endogenous GTPase activity. These results indicate a possible regulatory function of OEP 34 in protein translocation into chloroplasts.

Amino Acid Sequence↗
PubMed1995

Nucleoside diphosphate kinase from pea chloroplasts: purification, cDNA cloning and import into chloroplasts.

Nucleoside diphosphate kinase (NDPK; EC 2.7.4.6) was enriched 1900-fold from purified pea (Pisum sativum L. cv. Golf.) chloroplasts. The active enzyme preparation contained two polypeptides of apparent molecular weight 18.5 kDa and 17.4 kDa. Both proteins were enzymatically active and were recognized by an antiserum raised against NDPK from spinach chloroplasts, suggesting the existence of two isoforms in pea chloroplasts. The N-terminal protein sequence data were obtained for both polypeptides and compared with the nucleotide sequence of a cDNA clone isolated from a pea cDNA library. The analysis revealed that the two NDPK forms are encoded for by one mRNA, indicating that the lower-molecular-weight form could represent a proteolytic breakdown product of the 18.5-kDa NDPK. The pea chloroplastic NDPK is made as a larger precursor protein which is imported into chloroplasts. The NDPK precursor is then processed by the stromal processing peptidase to yield the 18.5-kDa form.

Amino Acid Sequence↗
PubMed1994

A receptor component of the chloroplast protein translocation machinery.

The chloroplast outer envelope protein OEP86 functions as a receptor in precursor protein translocation into chloroplasts. Sequence analysis suggests that the precursor of OEP86 is directed to the chloroplast outer envelope by a cleavable, negatively charged, and unusually long amino-terminal peptide. This presequence is unlike other potential targeting signals and suggests the existence of another membrane insertion pathway. Insertion of precursor OEP86 required the hydrolysis of adenosine triphosphate and the existence of surface exposed chloroplast membrane components, and it was not competed by another precursor protein destined for the internal plastid compartments.

Adenosine Triphosphate↗