Biomedical subjects

Jürgen Soll

Publications and source records attributed to Jürgen Soll.

At least 19 recordsLinked to original sources

PubMed2007

Chloroplast biogenesis: the use of mutants to study the etioplast-chloroplast transition.

In angiosperm plants, the etioplast-chloroplast transition is light-dependent. A key factor in this process is the protochlorophyllide oxidoreductase A (PORA), which catalyzes the light-induced reduction of protochlorophyllide to chlorophyllide. The import pathway of the precursor protein prePORA into chloroplasts was analyzed in vivo and in vitro by using homozygous loss-of-function mutants in genes coding for chlorophyllide a oxygenase (CAO) or for members of the outer-envelope solute-channel protein family of 16 kDa (OEP16), both of which have been implied to be key factors for the import of prePORA. Our in vivo analyses show that cao or oep16 mutants contain a normally structured prolamellar body that contains the protochlorophyllide holochrome. Furthermore, etioplasts from cao and oep16 mutants contain PORA protein as found by mass spectrometry. Our data demonstrate that both CAO and OEP16 are dispensable for chloroplast biogenesis and play no central role in the import of prePORA in vivo and in vitro as further indicated by protein import studies.

Arabidopsis↗

PubMed2006

Characterization of the preprotein and amino acid transporter gene family in Arabidopsis.

Seventeen loci encode proteins of the preprotein and amino acid transporter family in Arabidopsis (Arabidopsis thaliana). Some of these genes have arisen from recent duplications and are not in annotated duplicated regions of the Arabidopsis genome. In comparison to a number of other eukaryotic organisms, this family of proteins has greatly expanded in plants, with 24 loci in rice (Oryza sativa). Most of the Arabidopsis and rice genes are orthologous, indicating expansion of this family before monocot and dicot divergence. In vitro protein uptake assays, in vivo green fluorescent protein tagging, and immunological analyses of selected proteins determined either mitochondrial or plastidic localization for 10 and six proteins, respectively. The protein encoded by At5g24650 is targeted to both mitochondria and chloroplasts and, to our knowledge, is the first membrane protein reported to be targeted to mitochondria and chloroplasts. Three genes encoded translocase of the inner mitochondrial membrane (TIM)17-like proteins, three TIM23-like proteins, and three outer envelope protein16-like proteins in Arabidopsis. The identity of Arabidopsis TIM22-like proteins is most likely a protein encoded by At3g10110/At1g18320, based on phylogenetic analysis, subcellular localization, and complementation of a yeast (Saccharomyces cerevisiae) mutant and coexpression analysis. The lack of a preprotein and amino acid transporter domain in some proteins, localization in mitochondria, plastids, or both, variation in gene structure, and the differences in expression profiles indicate that the function of this family has diverged in plants beyond roles in protein translocation.

Amino Acid Sequence↗

PubMed2006

A protein kinase family in Arabidopsis phosphorylates chloroplast precursor proteins.

A serine/threonine protein kinase that is able to phosphorylate chloroplast-destined precursor proteins was purified from leaf extract of Arabidopsis thaliana and was identified by mass spectrometry. The protein kinase, encoded by AT2G17700, belongs to a small protein family comprising in addition AT4G35780 and AT4G38470. All three proteins were expressed heterologously in Escherichia coli and characterized with regard to their properties in precursor protein phosphorylation. They were able to phosphorylate several chloroplast-destined precursor proteins within their cleavable presequences. In contrast, a mitochondria-destined precursor protein was not a substrate for these kinases. For all three enzymes, the phosphorylation reaction was specific for ATP with apparent K(m) values between 14 and 67 microM. They did not utilize other NTPs nor were those able to compete for ATP in the reaction. An excess of ADP was able to inhibit ATP-dependent phosphorylation. Furthermore, all three kinases exhibited autophosphorylation. The protein kinases described here could represent subunits of a regulatory network involved in the cytosolic events of chloroplast protein import.

Amino Acid Sequence↗

PubMed2006

Calcium regulation of chloroplast protein translocation is mediated by calmodulin binding to Tic32.

The import of nuclear-encoded proteins into chloroplasts is tightly controlled on both sides of the envelope membranes. Regulatory circuits include redox-control as well as calcium-regulation, with calmodulin being the likely mediator of the latter. Using affinity-chromatography on calmodulin-agarose, we could identify the inner envelope translocon component Tic32 as the predominant calmodulin-binding protein of this membrane. Calmodulin-binding assays corroborate the interaction for heterologously expressed as well as native Tic32. The interaction is calcium-dependent and is mediated by a calmodulin-binding domain between Leu-296 and Leu-314 close to the C-proximal end of the pea Tic32. We furthermore could establish Tic32 as a bona fide NADPH-dependent dehydrogenase. NADPH but not NADH or NADP(+) affects the interaction of Tic110 with Tic32 as well as Tic62. At the same time, dehydrogenase activity of Tic32 is affected by calmodulin. In particular, binding of NADPH and calmodulin to Tic32 appear to be mutually exclusive. These results suggest that redox modulation and calcium regulation of chloroplast protein import convene at the Tic translocon and that both could be mediated by Tic32.

Amino Acid Sequence↗

PubMed2006

The synechocystis sp PCC 6803 oxa1 homolog is essential for membrane integration of reaction center precursor protein pD1.

Synechocystis sp PCC 6803 Slr1471p, an Oxa1p/Alb3/YidC homolog, is an essential protein for cell viability for which functions in thylakoid membrane biogenesis and cell division have been proposed. Using a fusion of green fluorescent protein to the C terminus of Slr1471p, we found that the mutant slr1471-gfp is photochemically inhibited when light intensities increase to 80 micromol x m(-2) x s(-1). We show that photoinhibition correlates with an increased redox potential of the reaction center quinone Q(A)(-) and a decreased redox potential of Q(B)(-). Analysis reveals that membrane integration of the D1 precursor protein is affected, leading to the accumulation of pD1 in the membrane phase. We show that Slr1471p interacts directly with the D1 protein and discuss why the accumulation of pD1 in two reaction center assembly intermediates is dependent on Slr1471p.

Bacterial Proteins↗

PubMed2006

OEP37 is a new member of the chloroplast outer membrane ion channels.

The chloroplast outer envelope protein OEP37 is a member of the growing beta-barrel protein family of the outer chloroplast membrane. The reconstituted recombinant protein OEP37 from pea forms a rectifying high conductance channel with a main conductance (lambda) of Lambda= 500 picosiemens (symmetrical 250 mm KCl). The OEP37 channel is cation-selective (P(K+)/P(K-) = 14:1) with a voltage-dependent open probability maximal at V(mem) = 0 mV. The channel pore reveals an hourglass-shaped form with different diameters for the vestibule and restriction zone. The diameters of the vestibule at the high conductance side were estimated by d = 3.0 nm and the restriction zone by d = 1.5 nm. The OEP37 channel displayed a nanomolar affinity for the precursor of the chloroplast inner membrane protein Tic32, which is imported into the chloroplast through a yet unknown pathway. Pre-proteins imported through the usual Toc pathway and synthetic control peptides, however, did not show a comparable block of the OEP37 channel. In addition to the electrophysiological characterization, we studied the gene expression of OEP37 in the model plant Arabidopsis thaliana. Here, transcripts of AtOEP37 are ubiquitously expressed throughout plant development and accumulate in early germinating seedlings as well as in late embryogenesis. The plastid intrinsic protein could be detected in isolated chloroplasts of cotyledons and rosette leaves. However, the knock-out mutant oep37-1 shows that the proper function of this single copy gene is not essential for development of the mature plant. Moreover, import of Tic32 into chloroplasts of oep37-1 was not impaired when compared with wild type. Thus, OEP37 may constitute a novel peptide-sensitive ion channel in the outer envelope of plastids with function during embryogenesis and germination.

Arabidopsis↗

PubMed2006

The molecular chaperone Hsp90 delivers precursor proteins to the chloroplast import receptor Toc64.

Precursor protein targeting toward organellar surfaces is assisted by different cytosolic chaperones. We demonstrate that the chloroplast protein translocon subunit Toc64 is the docking site for Hsp90 affiliated preproteins. Thereby, Hsp90 is recognised by the clamp type TPR domain of Toc64. The subsequent transfer of the preprotein from Toc64 to the major receptor of the Toc complex, namely Toc34, is affinity driven and nucleotide dependent. We propose that Toc64 acts as an initial docking site for Hsp90 associated precursor proteins. We outline a mechanism in which chaperones are recruited for a specific targeting event by a membrane-inserted receptor.

Amino Acid Sequence↗

PubMed2006

A second thylakoid membrane-localized Alb3/OxaI/YidC homologue is involved in proper chloroplast biogenesis in Arabidopsis thaliana.

The integral membrane proteins Alb3, OxaI, and YidC belong to an evolutionary conserved protein family mediating protein insertion into the thylakoid membrane of chloroplasts, the inner membrane of mitochondria, and bacteria, respectively. Whereas OxaI and YidC are involved in the insertion of a wide range of membrane proteins, the function of Alb3 seems to be limited to the insertion of a subset of the light-harvesting chlorophyll-binding proteins. In this study, we identified a second chloroplast homologue of the Alb3/OxaI/YidC family, named Alb4. Alb4 is almost identical to the Alb3/OxaI/YidC domain of the previously described 110-kDa inner envelope protein Artemis. We show that Alb4 is expressed as a separate 55-kDa protein and that Artemis was identified mistakenly. Alb4 is located in the thylakoid membrane of Arabidopsis thaliana chloroplasts. Analysis of an Arabidopsis mutant (Salk_136199) and RNA interference lines with a reduced level of Alb4 revealed chloroplasts with an altered ultrastructure. Mutant plastids are larger and more spherical in appearance, and the grana stacks within the mutant lines are less appressed than in the wild-type chloroplasts. These data indicate that Alb4 is required for proper chloroplast biogenesis.

Amino Acid Sequence↗

PubMed2006

Molecular properties of Oep21, an ATP-regulated anion-selective solute channel from the outer chloroplast membrane.

The flux of phosphorylated carbohydrates, the major export products of chloroplasts, is regulated at the level of the inner and presumably also at the level of the outer membrane. This is achieved through modulation of the outer membrane Oep21 channel currents and tuning of its ion selectivity. Refined analysis of the Oep21 channel properties by biochemical and electrophysiological methods revealed a channel formed by eight beta-strands with a wider pore vestibule of dvest approximately 2.4 nm at the intermembrane site and a narrower filter pore of drestr approximately 1 nm. The Oep21 pore contains two high affinity sites for ATP, one located at a relative transmembrane electrical distance delta = 0.56 and the second close to the vestibule at the intermembrane site. The ATP-dependent current block and reduction in anion selectivity of the Oep21 channel is relieved by the competitive binding of phosphorylated metabolic intermediates like 3-phosphoglycerate and glycerinaldehyde 3-phosphate. Deletion of a C-terminal putative FX4K binding motif in Oep21 decreased the capability of the channel to tune its ion selectivity by about 50%, whereas current block remained unchanged.

Adenosine Triphosphate↗

PubMed2006

A chloroplastic inner envelope membrane protease is essential for plant development.

Regulated intramembrane proteolysis (RIP) is a fundamental mechanism for controlling a wide range of cellular functions. Cleavage of membrane embedded proteins results in soluble fragments exerting their function, e.g., as transcription factors and thereby regulating gene expression. This process is highly conserved throughout all kingdom of life as are the involved proteases. RIP has been described in eukaryotes, bacteria and archea though until recently not in plant organelles. Here we describe a chloroplastic membrane protease which belongs to the conserved S2P family of membrane metallo proteases. We show that this protease is localized in the inner envelope membrane and is essential for plant development. It could function in a RIP like process regulating the concordant action in the plant cytosol, nucleus and plastids.

Amino Acid Sequence↗

PubMed2005

Chloroplast membrane transport: interplay of prokaryotic and eukaryotic traits.

Chloroplasts are specific plant organelles of prokaryotic origin. They are separated from the surrounding cell by a double membrane, which represents an effective barrier for the transport of metabolites and proteins. Specific transporters in the inner envelope membrane have been described, which facilitate the exchange of metabolites. In contrast, the outer envelope has been viewed for a long time as a molecular sieve that offers a mere size constriction to the passage of molecules. This view has been challenged lately, and a number of specific and regulated pore proteins of the outer envelope (OEPs) have been identified. These pores seem to have originated by adaptation of outer membrane proteins of the cyanobacterial ancestor of the chloroplast. In a similar fashion, the transport of proteins across the two envelope membranes is achieved by two hetero-oligomeric protein complexes called Toc (translocon in the outer envelope of chloroplasts) and Tic (translocon in the inner envelope of chloroplasts). The phylogenetic provenance of the translocon components is less clear, but at least the channel protein of the Toc translocon is of cyanobacterial origin. Characteristic of cyanobacteria and chloroplasts is furthermore a specialized internal membrane system, the thylakoids, on which the components of the photosynthetic machinery are located. Despite the importance of this membrane, very little is known about its phylogenetic origin or the manner of its synthesis. Vipp1 appears to be a ubiquitous component of thylakoid formation, while in chloroplasts of land plants, additionally a vesicle transport system of eukaryotic origin might be involved in this process.

Biological Evolution↗

PubMed2005

Membrane protein insertion: mixing eukaryotic and prokaryotic concepts.

Proteins are translocated across or inserted into membranes by machines that are composed of soluble and membrane-anchored subunits. The molecular action of these machines and their evolutionary origin are at present the focus of intense research. For instance, our understanding of the mode of insertion of beta-barrel membrane proteins into the outer membrane of endosymbiotically derived organelles has increased rapidly during the past few years. In particular, the identification of the Omp85/YaeT-involving pathways in Neisseria meningitidis, Escherichia coli and cyanobacteria, and homologues of Omp85/YaeT in chloroplasts and mitochondria, has provided new clues about the ancestral beta-barrel protein insertion pathway. This review focuses on recent advances in the elucidation of the evolutionarily conserved concepts that underlie the translocation and insertion of beta-barrel membrane proteins.

Animals↗

PubMed2005

Calcium regulation of chloroplast protein import.

The majority of chloroplast proteins is nuclear-encoded and therefore synthesized on cytosolic ribosomes. In order to enter the chloroplast, these proteins have to cross the double-membrane surrounding the organelle. This is achieved by means of two hetero-oligomeric protein complexes in the outer and inner envelope, the Toc and Tic translocon. The process of chloroplast import is highly regulated on both sides of the envelope membranes. Our studies indicate the existence of an undescribed mode of control for this process so far, at the same time providing further evidence that the chloroplast is integrated into the calcium-signalling network of the cell. In pea chloroplasts, the calmodulin inhibitor Ophiobolin A as well as the calcium ionophores A23187 and Ionomycin affect the translocation of those chloroplast proteins that are imported with an N-terminal cleavable presequence. Import of these proteins is inhibited in a concentration-dependent manner. Addition of external calmodulin or calcium can counter the effect of these inhibitors. Translocation of chloroplast proteins that do not possess a cleavable transit peptide, that is outer envelope proteins or the inner envelope protein Tic32, is not affected. These results suggest that the import of a certain subset of chloroplast proteins is regulated by calcium. Our studies furthermore indicate that this regulation occurs downstream of the Toc translocon either within the intermembrane space or at the inner envelope translocon. A potential promoter of the calcium regulation is calmodulin, a protein well known as part of the plant's calcium signalling system.

Calcimycin↗

PubMed2005

Conserved pore-forming regions in polypeptide-transporting proteins.

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the beta-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the beta-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.

Amino Acid Sequence↗

PubMed2004

Toc12, a novel subunit of the intermembrane space preprotein translocon of chloroplasts.

Translocation of proteins across membranes is essential for the biogenesis of each cell and is achieved by proteinaceous complexes. We analyzed the translocation complex of the intermembrane space from chloroplasts and identified a 12-kDa protein associated with the Toc machinery. Toc12 is an outer envelope protein exposing a soluble domain into the intermembrane space. Toc12 contains a J-domain and stimulates the ATPase activity of DnaK. The conformational stability and the ability to stimulate Hsp70 are dependent on a disulfide bridge within the loop region of the J-domain, suggesting a redox-regulated activation of the chaperone. Toc12 is associated with Toc64 and Tic22. Its J-domain recruits the Hsp70 of outer envelope membrane to the intermembrane space translocon and facilitates its interaction to the preprotein.

Adenosine Triphosphate↗

PubMed2004

Inner envelope protein 32 is imported into chloroplasts by a novel pathway.

The 32 kDa chloroplast inner envelope protein (IEP32) is imported into the organelle in the absence of a cleavable N-terminal pre-sequence. The ten N-terminal amino acids form an essential portion of this targeting information as deduced from deletion mutants. Recognition and translocation of IEP32 is not catalysed by the general chloroplast outer envelope translocon subunits Toc159, Toc75III and Toc34, because IEP32 import is neither inhibited by proteolytic removal of Toc34 and Toc159 nor by inhibition of the Toc75 import channel by CuCl(2) or spermine. Import of IEP32 only requires ATP concentrations of below 20 microM indicating that stromal chaperones are not involved in the process, but that IEP32 might be directly inserted from the intermembrane space into the inner envelope by a so far unidentified pathway. IEP32 may require the assistance of Tic22, an intermembrane space translocon subunit for import as indicated by the presence of a chemical crosslinked product between both polypeptides.

Adenosine Triphosphate↗

PubMed2004

Intracellular localization of VDAC proteins in plants.

Voltage-dependent anion channels (VDACs) are porin-type beta-barrel diffusion pores. They are prominent in the outer membrane of mitochondria and facilitate metabolite exchange between the organelle and the cytosol. Here we studied the subcellular distribution of a plant VDAC-like protein between plastids and mitochondria in green and non-green tissue. Using in vitro studies of dual-import into mitochondria and chloroplasts as well as transient expression of fluorescence-labeled polypeptides, it could be clearly demonstrated that this VDAC isoform targets exclusively to mitochondria and not to plastids. Our results support the idea that plastids evolved a concept of solute exchange with the cytosol different from that of mitochondria.

Amino Acid Sequence↗

PubMed2004

The Arabidopsis thaliana chloroplast inner envelope protein ARTEMIS is a functional member of the Alb3/Oxa1/YidC family of proteins.

The Arabidopsis thaliana protein ARTEMIS is an integral component of the chloroplast inner envelope required for chloroplast division. It contains a domain of significant homology to members of the Alb3/Oxa1/YidC protein family. Here, we show that upon expression in yeast mitochondria, ARTEMIS can partially take over the function of yeast Oxa1 in the insertion and assembly of mitochondrial membrane proteins. This identifies ARTEMIS as a functional member of the Alb3/Oxa1/YidC protein family and suggests the existence of a novel protein sorting pathway in chloroplasts which integrates polypeptides from the stroma into the inner envelope by an evolutionary conserved process.

Arabidopsis↗