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J Soll

Publications and source records attributed to J Soll.

At least 19 recordsLinked to original sources
PubMed2002

Structural and guanosine triphosphate/diphosphate requirements for transit peptide recognition by the cytosolic domain of the chloroplast outer envelope receptor, Toc34.

Toc34 is a transmembrane protein located in the outer envelope membrane of chloroplasts and involved in transit peptide recognition. The cytosolic region of Toc34 reveals 34% alpha-helical and 26% beta-strand structure and is stabilized by intramolecular electrostatic interaction. Toc34 binds both chloroplast preproteins and isolated transit peptides in a guanosine triphosphate- (GTP-) dependent mechanism. In this study we demonstrate that the soluble, cytosolic domain of Toc34 (Toc34deltaTM) functions as receptor in vitro and is capable to compete with the import of the preprotein of the small subunit (preSSU) of ribulose-1,5-bisphosphate carboxylase-oxygenase into chloroplasts in a GTP-dependent manner. We have developed a biosensor assay to study the interaction of Toc34deltaTM with purified preproteins and transit peptides. The results are compared with the interactions of both a full-size preprotein and the transit peptide of preSSU with the translocon of the outer envelope of chloroplasts (Toc complex) in situ. Several mutants of the transit peptide of preSSU were evaluated to identify amino acid segments that are specifically recognized by Toc34. We present a model of how Toc34 may recognize the transit peptide and discuss how this interaction may facilitate interaction and translocation of preproteins via the Toc complex in vivo.

Amino Acid Sequence↗
PubMed2002

Chloroplast quest: a journey from the cytosol into the chloroplast and beyond.

Chloroplasts are characteristic organelles of plants and algae and the site of oxygenic photosynthesis. They are surrounded by a double membrane and possess an internal membrane system, the thylakoids, on which the photosynthetic machinery is located. They originated more than 1.2 billion years ago from an endosymbiotic event between an already photosynthetic ancestor of present day cyanobacteria and a mitochondriate host cell. During the transformation of the internalized cyanobacterium into a cell organelle most of the genetic information of the endosymbiot got lost or was transferred into the nucleus of the host. Chloroplast proteins encoded by nuclear genes are synthesized on cytoplasmic ribosomes and have to be relocated into the organelle. This is achieved by a proteinaceous import machinery in the outer and inner envelope of the chloroplasts. Proteins destined for the thylakoid membrane and the thylakoid lumen are further translocated by several different pathways into or across this membrane. The subject of this review is the quest of nuclear encoded chloroplast proteins into the organelle and to their final suborganellar location.

Arabidopsis↗
PubMed2001

Toc, Tic, and chloroplast protein import.

The vast majority of chloroplast proteins are synthesized in precursor form on cytosolic ribosomes. Chloroplast precursor proteins have cleavable, N-terminal targeting signals called transit peptides. Transit peptides direct precursor proteins to the chloroplast in an organelle-specific way. They can be phosphorylated by a cytosolic protein kinase, and this leads to the formation of a cytosolic guidance complex. The guidance complex--comprising precursor, hsp70 and 14-3-3 proteins, as well as several unidentified components--docks at the outer envelope membrane. Translocation of precursor proteins across the envelope is achieved by the joint action of molecular machines called Toc (translocon at the outer envelope membrane of chloroplasts) and Tic (translocon at the inner envelope membrane of chloroplasts), respectively. The action of the Toc/Tic apparatus requires the hydrolysis of ATP and GTP at different levels, indicating energetic requirements and regulatory properties of the import process. The main subunits of the Toc and Tic complexes have been identified and characterized in vivo, in organello and in vitro. Phylogenetic evidence suggests that several translocon subunits are of cyanobacterial origin, indicating that today's import machinery was built around a prokaryotic core.

Arabidopsis↗
PubMed2001

A vesicle transport system inside chloroplasts.

Intracellular transport via membrane vesicle traffic is a well known feature of eukaryotic cells. Yet, no vesicle transport system has been described for prokaryotes or organelles of prokaryotic origin, such as chloroplasts and mitochondria. Here we show that chloroplasts possess a vesicle transport system with features similar to vesicle traffic in homotypic membrane fusion. Vesicle formation and fusion is affected by specific inhibitors, e.g. nucleotide analogues, protein phosphatase inhibitors and Ca2+ antagonists. This vesicle transfer is an ongoing process in mature chloroplasts indicating that it represents an important new pathway in the formation and maintenance of the thylakoid membranes.

Biological Transport↗
PubMed2001

VIPP1, a nuclear gene of Arabidopsis thaliana essential for thylakoid membrane formation.

The conversion of light to chemical energy by the process of photosynthesis is localized to the thylakoid membrane network in plant chloroplasts. Although several pathways have been described that target proteins into and across the thylakoids, little is known about the origin of this membrane system or how the lipid backbone of the thylakoids is transported and fused with the target membrane. Thylakoid biogenesis and maintenance seem to involve the flow of membrane elements via vesicular transport. Here we show by mutational analysis that deletion of a single gene called VIPP1 (vesicle-inducing protein in plastids 1) is deleterious to thylakoid membrane formation. Although VIPP1 is a hydrophilic protein it is found in both the inner envelope and the thylakoid membranes. In VIPP1 deletion mutants vesicle formation is abolished. We propose that VIPP1 is essential for the maintenance of thylakoids by a transport pathway not previously recognized.

Amino Acid Sequence↗
PubMed2001

Vipp1 deletion mutant of Synechocystis: a connection between bacterial phage shock and thylakoid biogenesis?

Plant chloroplasts originated from an endosymbiotic event by which an ancestor of contemporary cyanobacteria was engulfed by an early eukaryotic cell and then transformed into an organelle. Oxygenic photosynthesis is the specific feature of cyanobacteria and chloroplasts, and the photosynthetic machinery resides in an internal membrane system, the thylakoids. The origin and genesis of thylakoid membranes, which are essential for oxygenic photosynthesis, are still an enigma. Vipp1 (vesicle-inducing protein in plastids 1) is a protein located in both the inner envelope and the thylakoids of Pisum sativum and Arabidopsis thaliana. In Arabidopsis disruption of the VIPP1 gene severely affects the plant's ability to form properly structured thylakoids and as a consequence to carry out photosynthesis. In contrast, Vipp1 in Synechocystis appears to be located exclusively in the plasma membrane. Yet, as in higher plants, disruption of the VIPP1 gene locus leads to the complete loss of thylakoid formation. So far VIPP1 genes are found only in organisms carrying out oxygenic photosynthesis. They share sequence homology with a subunit encoded by the bacterial phage shock operon (PspA) but differ from PspA by a C-terminal extension of about 30 amino acids. In two cyanobacteria, Synechocystis and Anabaena, both a VIPP1 and a pspA gene are present, and phylogenetic analysis indicates that VIPP1 originated from a gene duplication of the latter and thereafter acquired its new function. It also appears that the C-terminal extension that discriminates VIPP1 proteins from PspA is important for its function in thylakoid formation.

Amino Acid Sequence↗
PubMed2001

Lipid composition of outer leaflet of chloroplast outer envelope determines topology of OEP7.

OEP7, a 6.7-kDa outer envelope protein of spinach chloroplasts inserts into the outer envelope of the organelle independent of a classical cleavable targeting signal. The insertion of OEP7 was studied to describe the determinants for association with, integration into, and orientation of the protein in the outer envelope of chloroplasts. The insertion of OEP7 into the membrane is independent of outer membrane channel proteins and can be reconstituted with the use of protein-free liposomes. In situ, the binding of OEP7 to the membrane surface is not driven by electrostatic interaction because reduction of phosphatidylglycerol or phosphatidylinositol did not reduce the association with the liposomes. The positively charged amino acids flanking the transmembrane domain at the C terminus are essential to retain the native N(in)-C(out) orientation during insertion into chloroplasts. OEP7 inserts with reversed orientation into liposomes containing the average lipid composition of the outer envelopes. The native like N(in)-C(out) orientation is achieved by reduction of the phoshpatidylglycerol concentration mimicking the composition of the outer leaflet of the outer envelope of chloroplasts. We conclude that the unique lipid composition of the outer leaflet due to lipid asymmetry of the outer envelope is essential for the correct topology of OEP7.

Chloroplasts↗
PubMed2000

In vitro reconstitution and biophysical characterization of OEP16, an outer envelope pore protein of pea chloroplasts.

More than 30% of all proteins in the living cell are membrane proteins; most of them occur in the native membranes only in very low amounts, which hinders their functional and structural investigation. Here we describe the in vitro reconstitution of overexpressed Outer Envelope Protein 16 (OEP16) from pea chloroplasts, a cation-selective channel, which has been purified from E. coli inclusion bodies. Reconstitution in detergent micelles was monitored by CD and fluorescence spectroscopy. Electron microscopy showed a homogeneous size distribution of the reconstituted protein, and differential scanning calorimetry gave an estimate of the enthalpy of protein folding. First protein crystals were obtained that have to be further refined for X-ray structural analysis. The described methods of membrane protein reconstitution and biophysical analysis might prove helpful in the study of other membrane proteins.

Calorimetry, Differential Scanning↗
PubMed2000

Toc34 is a preprotein receptor regulated by GTP and phosphorylation.

Most proteins present in chloroplasts are synthesized in the cytosol and are posttranslationally translocated into the organelle. A multicomponent translocation machinery located in both the outer and the inner envelope of chloroplasts was identified, but the mode of action of many subunits remains unclear. Here, we describe the regulation of an early step of translocation occurring at the outer envelope. The outer envelope translocon subunit Toc34 can be phosphorylated, and GTP binding is regulated by phosphorylation. In vitro, Toc34 acts as a receptor for proteins containing a chloroplast-targeting signal. Interaction of Toc34 with the transit peptide is highly regulated and depends on GTP binding to Toc34 and on phosphorylation of the transit peptide of the preprotein.

Chloroplasts↗
PubMed2000

Identification of the pore-forming region of the outer chloroplast envelope protein OEP16.

The chloroplast outer envelope protein OEP16 forms a cation-selective high conductance channel with permeability to amines and amino acids. The region of OEP16 directly involved in channel formation has been identified by electrophysiological analysis of a selection of reconstituted OEP16 mutants. Because analysis of these mutants depended on the use of recombinant protein, we evaluated the electrophysiological properties of OEP16 isolated directly from pea chloroplasts and of the recombinant protein produced in Escherichia coli. The results show that the basic properties like conductance, selectivity, and open probability of the channel formed by native pea OEP16 are comparable with the channel activity formed by the recombinant source of the protein. Following electrophysiological analysis of OEP16 mutants we found that point mutations and insertion of additional amino acid residues in the region of the putative helix 1 (Glu(73) to Val(91)) did not change the properties of the OEP16 channel. The only exception was a Cys(71)-->Ser mutation, which led to a loss of the CuCl(2) sensitivity of the channel. Analysis of N- and C-terminal deletion mutants of OEP16 and mutants containing defined shuffled domains indicated that the minimal continuous region of OEP16, which is able to form a channel in liposomes, lies in the first half of the protein between amino acid residues 21 and 93.

Amines↗
PubMed2000

Toc64, a new component of the protein translocon of chloroplasts.

A subunit of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) of 64 kD is described, Toc64. Toc64 copurifies on sucrose density gradients with the isolated Toc complex. Furthermore, it can be cross-linked in intact chloroplasts to a high molecular weight complex containing both Toc and Tic subunits and a precursor protein. The 0 A cross-linker CuCl(2) yields the reversible formation of disulfide bridge(s) between Toc64 and the established Toc complex subunits in purified outer envelope membranes. Toc64 contains three tetratricopeptide repeat motifs that are exposed at the chloroplast cytosol interface. We propose that Toc64 functions early in preprotein translocation, maybe as a docking protein for cytosolic cofactors of the protein import into chloroplasts.

Amino Acid Sequence↗
PubMed2000

Travelling of proteins through membranes: translocation into chloroplasts.

Most proteins involved in plastid biogenesis are encoded by the nuclear genome. They are synthesised in the cytosol and have to be transported toward and subsequently translocated into the organelle. This targeting and import process is initiated by a specific chloroplast-targeting signal. The targeting signal of the preprotein is recognised and modified by cytosolic proteins which function in transport toward the chloroplast and in maintaining the import-competent state of the preprotein. The precursor is transferred onto a multi-component complex in the outer envelope of the chloroplasts, which is formed by receptor proteins and the translocation channel. Some proteins, not containing transit sequences, are directly sorted into the outer membrane whereas the majority, containing transit sequences, will be translocated into the stroma. This involves the joint action of a protein complex in the outer envelope, one complex in the inner envelope, and soluble proteins in the intermembrane space and the stroma. The origin of this translocation complex following the endosymbiotic events is an unsolved question. Recent identification of homologous proteins to some members of this machinery in the cyanobacterium Synechocystis PCC6803 gives an initial insight into the origin of the translocation complex.

Cell Membrane↗
PubMed2000

14-3-3 proteins form a guidance complex with chloroplast precursor proteins in plants.

Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins. Plant 14-3-3 proteins interact specifically with wheat germ lysate-synthesized chloroplast precursor proteins and require an intact phosphorylation motif within the transit sequence. Chloroplast precursor proteins do not interact with 14-3-3 when synthesized in the heterologous reticulocyte lysate. In contrast, a precursor protein destined for plant mitochondria was found to be associated with 14-3-3 proteins present in the reticulocyte lysate but not with 14-3-3 from wheat germ lysate. This indicates an unrecognized selectivity of 14-3-3 proteins for precursors from mitochondria and plastids in plants in comparison to fungi and animals. The heterooligomeric complex has an apparent size of 200 kD. In addition to the precursor protein, it contains 14-3-3 (probably as a dimer) and a heat shock protein Hsp70 isoform. Dissociation of the precursor complex requires ATP. Protein import experiments of precursor from the oligomeric complex into intact pea chloroplasts reveal three- to fourfold higher translocation rates compared with the free precursor, which is not complexed. We conclude that the 14-3-3-Hsp70-precursor protein complex is a bona fide intermediate in the in vivo protein import pathway in plants.

14-3-3 Proteins↗
PubMed2000

Topology studies of the chloroplast protein import channel Toc75.

A major goal in understanding protein transport across membranes is the investigation of the structure and regulation of the translocon subunits. We analysed Toc75, a pore-forming subunit of the translocon of the outer envelope of chloroplasts. Toc75 was overexpressed and reconstituted into liposomes. Immunoprecipitation of liposome-reconstituted Toc75 indicates an N(in)-C(in) orientation of Toc75. Limited proteolytic digestion of Toc75 present in outer envelope vesicles with specific proteases combined with amino acid sequencing was used to study the topology of Toc75. Finally, computer modelling based on known protein structures indicates that Toc75 traverses the outer envelope with 16 amphiphilic beta sheets and the topology model is presented.

Algorithms↗
PubMed2000

NADPH:Protochlorophyllide oxidoreductase uses the general import route into chloroplasts.

Chloroplast differentiation in angiosperm plants depends on the light-dependent conversion of protochlorophyllide to chlorophyllide by NADPH:protochlorophyllide oxidoreductase (PORA; EC 1.6.99.1), a nuclearly encoded protein. The protein import of the precursor form of PORA into plastids was shown previously to strictly depend on the presence of its substrate protochlorophyllide. PORA seemed to follow a novel, posttranslationally regulated import route. Here we demonstrate that the precursor of PORA from barley is imported into isolated barley plastids independently of protochlorophyllide. PORA as well as PORB import is competed for by the precursor of the small subunit of Rubisco. The data demonstrate that the PORA precursor uses the general import pathway into plastids. Furthermore, en route into chloroplasts the pea POR precursor can be cross-linked to the protein import channel in the outer envelope Toc75 from pea.

Aminolevulinic Acid↗
PubMed1999

Tic40, a new "old" subunit of the chloroplast protein import translocon.

The protein import translocon at the inner envelope of chloroplasts (Tic complex) is a heteroligomeric multisubunit complex. Here, we describe Tic40 from pea as a new component of this complex. Tic40 from pea is a homologue of a protein described earlier from Brassica napus as Cim/Com44 or the Toc36 subunit of the translocon at the outer envelope of chloroplasts, respectively (Wu, C., Seibert, F. S., and Ko, K. (1994) J. Biol. Chem. 269, 32264-32271; Ko, K., Budd, D., Wu, C., Seibert, F., Kourtz, L., and Ko, Z. W. (1995) J. Biol. Chem. 270, 28601-28608; Pang, P., Meathrel, K., and Ko, K. (1997) J. Biol. Chem. 272, 25623-25627). Tic40 can be covalently connected to Tic110 by the formation of a disulfide bridge under oxidizing conditions, indicating its close physical proximity to an established translocon component. The Tic40 protein is synthesized in the cytosol as a precursor with an N-terminal cleavable chloroplast targeting signal and imported into the organelle via the general import pathway. Immunoblotting and immunogold-labeling studies exclusively confine Tic40 to the chloroplastic inner envelope, in which it is anchored by a single putative transmembrane span.

Amino Acid Sequence↗