The outer membrane Omp85-like protein P39 influences metabolic homeostasis in mature Arabidopsis thaliana.

The Omp85 proteins form a large membrane protein family in bacteria and eukaryotes. Omp85 proteins are composed of a C-terminal ß-barrel-shaped membrane domain and one or more N-terminal polypeptide transport-associated (POTRA) domains. However, Arabidopsis thaliana contains two genes coding for Omp85 proteins without a POTRA domain. One gene is designated P39, according to the molecular weight of the encoded protein. The protein is targeted to plastids and it was established that p39 has electrophysiological properties similar to other Omp85 family members, particularly to that designated as Toc75V/Oep80. We analysed expression of the gene and characterised two T-DNA insertion mutants, focusing on alterations in photosynthetic activity, plastid ultrastructure, global expression profile and metabolome. We observed pronounced expression of P39, especially in veins. Mutants of P39 show growth aberrations, reduced photosynthetic activity and changes in plastid ultrastructure, particularly in the leaf tip. Further, they display global alteration of gene expression and metabolite content in leaves of mature plants. We conclude that the function of the plastid-localised and vein-specific Omp85 family protein p39 is important, but not essential, for maintenance of metabolic homeostasis of full-grown A. thaliana plants. Further, the function of p39 in veins influences the functionality of other plant tissues. The link connecting p39 function with metabolic regulation in mature A. thaliana is discussed.

Transitions of gene expression induced by short-term blue light.

Blue light modulates many processes in plants and plant cells. It influences global and long-term responses, such as seedling development and phototropism, and induces short-term adaptations like stomatal opening and chloroplast movement. Three genes were identified as important for the latter process, namely PHOT1, PHOT2 and CHUP1. The former two phototropin blue light receptors act in perception of the blue light signal. The protein CHUP1 is localised to the outer envelope membrane of chloroplasts and is involved in chloroplast movement. To explore whether short-term reactions required for chloroplast movement are under transcriptional control, we analysed the transcriptome in wild-type Arabidopsis thaliana, phot1, phot2 and chup1 with different blue light treatments for 5 or 30 min. Blue light-induced changes in transcription depended on illumination time and intensity. Illumination with 100 µmol·m(-2) · s(-1) blue light induced down-regulation of several genes and might point to cascades that could be important for sensing low levels of blue light. Analysis of the transcriptome of the mutants in response to the different light regimes suggests that the transcriptional response to blue light in the wild-type can be attributed to phot1 rather than phot2, suggesting that blue light-induced alteration of expression is a function of phot1. In contrast, the blue light response at the transcriptional level of chup1 plants was unique, and confirmed the higher light sensitivity of this mutant.

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.

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.

Without a little help from 'my' friends: direct insertion of proteins into chloroplast membranes?

The chloroplast membranes are highly regulated and biological active regions of the living plant cell, which carry numerous essential proteinaceous components. For example, in the thylakoid membrane the photosynthesis apparatus, one of the most life-relevant biological machineries, is located. How these membrane proteins are targeted to and inserted into their target membranes was one of the questions we aimed to understand in the last few years. Fifteen years ago little to nothing was known about the targeting and translocation of outer envelope proteins (G.W. Schmidt and L.M. Mishkind, Annu. Rev. Biochem. 55 (1986)). Although several protein assisted pathways for translocation of proteins across the membranes have been characterised, only recent results gave insight into how membrane proteins are inserted into the chloroplast membranes. Here we will focus on the mode of insertion of a class of proteins into the outer envelope and the thylakoid membranes, which share a unique feature: they insert apparently directly into the lipid bilayer, i.e. without the help of a proteinaceous translocation pore.

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.

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.

The central matrix loop drives import of uncoupling protein 1 into mitochondria.

The uncoupling protein (UCP1) is a carrier protein of the inner mitochondrial membrane spanning the bilayer six times. It does not contain a typical amino-terminal targeting signal and the mechanism of targeting and insertion is unknown. Here we focus on the biogenesis of UCP1 by analysing the import signals contained within the three repeated units of the protein. The amino-terminal third of the protein can mediate insertion into the outer membrane and therefore acts as artificial targeting signal when fused to DHFR. However, in the context of full-length UCP, the targeting information contained within the first repeated unit is not sufficient to trigger insertion into the outer membrane. Deletion of either the first or third repeated unit from UCP1 did not reduce import into the inner membrane and bound to the outer membrane receptor protein hTom20 with the characteristics of full-length UCP1. Deletion of the second repeat of UCP1 completely abolished all import into the mitochondria. Consistent with this, the central repeat alone was efficiently imported to the inner membrane and bound hTom20 with the characteristics of UCP1. We conclude that the site for binding hTom20 is within the central repeat and that this domain contains the complete targeting signal for directing UCP1 to the inner membrane.

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.

Cloning and characterization of a 35-kDa mouse mitochondrial outer membrane protein MOM35 with high homology to Tom40.

We have cloned a 35-kDa protein from a mouse cDNA library with a 25% overall amino acid identity to yTom40 and 27% identity to nTom40. This homolog toTom40 was named MOM35. It contains two possible start codons 36 amino acids apart from each other. Both the long and the short version of MOM35 can be imported in vitro into mouse mitochondria. The identified protein is imported into the outer mitochondrial membrane and comprises a trypsin-resistance pattern similar to that of nTom40. Tom40 of N. crassa, S. cerevisiae, and the protein identified herein contains a highly conserved region with possible physiological importance. Subsequent investigation has revealed that this region interacts specifically in vitro with preproteins proposed to be imported by a Tom40-dependent pathway.

Signals and receptors--the translocation machinery on the mitochondrial surface.

Most proteins involved in mitochondrial biogenesis are encoded by the genome of the nucleus. They are synthesized in the cytosol and have to be transported toward and, subsequently, imported into the organelle. This targeting and import process is initiated by the specific mitochondrial targeting signal, which differs pending on the final localization of the protein. The preprotein will be recognized by cytosolic proteins, which function in transport toward the mitochondria and in maintaining the import competent state of the preprotein. The precursor will be transferred onto a multicomponent complex on the outer mitochondrial membrane, formed by receptor proteins and the general insertion pore (GIP). Some proteins are directly sorted into the outer membrane whereas the majority will be transported over the outer membrane through the import channel followed by further distribution of those proteins.

Positively charged residues, the helical conformation and the structural flexibility of the leader sequence of pALDH are important for recognition by hTom20.

Tom20, a mitochondrial outer membrane receptor necessary for protein translocation, was found to interact specifically with mitochondrial preproteins. The interaction of proteins containing an N-terminal matrix targeting signal was enhanced in an hydrophobic environment and the dependence of this interaction on the alpha helical conformation of the presequence was postulated. In order to test this hypothesis and to gain insights about the features of a matrix targeting signal necessary to be recognized by the receptor machinery including Tom20, the interaction of pALDH and signal sequence mutants to Tom20 in the absence and presence of a hydrophobic environment was investigated. Here we present evidence to show that in a hydrophobic environment the interaction between Tom20 and the leader sequence is strongly dependent on the positive charges within the signal sequence as well as on the flexibility of this signal.

Direct membrane insertion of voltage-dependent anion-selective channel protein catalyzed by mitochondrial Tom20.

Insertion of newly synthesized proteins into or across the mitochondrial outer membrane is initiated by import receptors at the surface of the organelle. Typically, this interaction directs the precursor protein into a preprotein translocation pore, comprised of Tom40. Here, we show that a prominent beta-barrel channel protein spanning the outer membrane, human voltage- dependent anion-selective channel (VDAC), bypasses the requirement for the Tom40 translocation pore during biogenesis. Insertion of VDAC into the outer membrane is unaffected by plugging the translocation pore with a partially translocated matrix preprotein, and mitochondria containing a temperature-sensitive mutant of Tom40 insert VDAC at the nonpermissive temperature. Synthetic liposomes harboring the cytosolic domain of the human import receptor Tom20 efficiently insert newly synthesized VDAC, resulting in transbilayer transport of ATP. Therefore, Tom20 transforms newly synthesized cytosolic VDAC into a transmembrane channel that is fully integrated into the lipid bilayer.

Expression, purification, and in vitro characterization of the human outer mitochondrial membrane receptor human translocase of the outer mitochondrial membrane 20.

In order to investigate the biochemical properties of the mitochondrial outer membrane receptor, hTom20, involved in protein recognition, the cytosolic domain of this receptor was overexpressed and purified to homogeneity. A four-step purification including the purification of thrombin is described as well as an analysis of the function of the highly purified hTom20 protein. The receptor was concentrated and the subsequent aggregation behavior was investigated in order to understand the function of the single cysteine in the cytosolic domain as well as the function of the proposed "glutamine face" for the structure of the protein. It was found that specific dimerization of the cytosolic domain of hTom20 is necessary in order to prevent aggregation of the protein. In addition, the cysteine and the glutamine face are important for the stability of the protein. We propose that the function of the cysteine is to promote dimerization as found in the absence of dithiothreitol.

Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL).

The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures. We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase. We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate). The partition coefficients describing the association of unmyristoylated and myristoylated MARCKS-related protein with membranes of different phospholipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-related protein mediate the binding. However, no significant cooperativity is observed between these two domains. Interestingly, MARCKS-related protein binds to TRANSIL membranes more strongly at temperatures below their phase-transition temperature. Taking advantage of this property, MARCKS-related protein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSIL column at 4 degrees C and eluting MRP at room temperature. In conclusion, TRANSIL is a versatile tool to determine the affinity of compounds for phospholipid membranes and to purify membrane-bound proteins. TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes.

Functional and structural properties of the mitochondrial outer membrane receptor Tom20.

Tom20 is an outer mitochondrial membrane protein that functions as a component of the import receptor complex for cytoplasmically synthesized mitochondrial precursor proteins. The human homologue, hTom20, consists of an N-terminal membrane anchor region predicted between aa5-25 and a soluble cytosolic domain from aa30 to 145. To analyze the properties of hTom20, we have expressed several truncations of the cytosolic domain as fusion proteins with glutathione S-transferase. Our studies reveal that the cytosolic region of hTom20 is a monomeric protein in solution containing two domains which are involved in different functions of the receptor. The N-terminal region is involved in membrane binding (aa30-60) and recognition of the cleavable matrix targeting signals (aa50-90). In addition, we have demonstrated that the receptor recognizes the alpha-helical state of the matrix targeting signal. The dissociation constant for this interaction in the presence of a detergent which induces this secondary structure is 0.6 microM, one-fifth the value in the absence of detergent. In aqueous solution, the region between aa30 and 60 is loosely folded and stabilized against proteolytic cleavage by interaction with detergents or a matrix targeting signal. Our work further shows that the remainder of the cytosolic domain of hTom20, aa60-145, is a compactly folded globular domain containing a region (aa90-145) that is critical for the recognition of proteins bearing internal signal sequences such as the uncoupling protein and porin.

Characterization of the N-terminal targeting signal binding domain of the mitochondrial outer membrane receptor, Tom20.

hTom20 is an outer mitochondrial membrane receptor involved in protein translocation. The cytosolic domain (aa30-145) and selected truncated versions of this domain were overexpressed and purified to study the structure-function relationship of this protein. Our studies reveal that the secondary structure of the cytosolic domain is very resistant to unfolding by guanidine-HCl and urea and is stabilized mainly by hydrophobic interactions. However, the tertiary structure of the N-terminal targeting signal binding domain (aa30-90) is more flexible. The first 30 amino acids of the cytosolic domain (aa30-60) are involved in recognizing N-terminal targeting signals and in stabilizing the cytosolic domain on the lipid surface. Moreover, we show that specifically aa30-48 interact with the membrane surface; a construct containing aa48-145 will only bind to the membrane surface in the presence of an N-terminal targeting signal peptide. The C-terminal region of hTom20 (aa141-145) interacts with the N-terminal region of hTom20, helping to stabilize the proper conformation of the N-terminal targeting signal binding domain. Finally, hTom20 interacts with the N-terminal targeting signal of preornithine carbamyl transferase fused to dihydrofolate reductase very weakly (Kd = 8 microM), as would be expected if this interaction was the first in a series orchestrated by the import receptor complex to draw the targeted protein into the mitochondrion.

Interactions of the human mitochondrial protein import receptor, hTom20, with precursor proteins in vitro reveal pleiotropic specificities and different receptor domain requirements.

Tom20 is part of a multiple component, dynamic complex that functions to import specific cytosolic proteins into or through the outer membrane of the mitochondrion. To analyze the contribution of Tom20 to precursor protein recognition, the cytosolic domain of the human mitochondrial import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase and conditions established to measure specific interactions of the receptor component with precursor proteins in vitro. Reconstitution of receptor binding from purified components revealed that a prototypic matrix-destined precursor protein, pODHFR, interacts with Tom20 by a mechanism that is dependent on an active matrix targeting signal but does not require cytosolic components or ATP. Binding was influenced by both salt concentration and detergent. The effect of salt or detergent, however, varied for different precursor proteins. In particular, detergent selectively enhanced binding of pODHFR to receptor, possibly because of induced changes in the structure of the signal sequence. Finally, mutations were introduced into hTom20 which had a dramatic effect on binding of some precursor proteins but not on others. Taken together, the results suggest that hTom20 recognizes and physically interacts with precursor proteins bearing a diverse array of topogenic sequences and that such pleiotropic specificity for these precursor proteins may involve different domains within the receptor molecule.

Human mitochondrial import receptor, Tom20p. Use of glutathione to reveal specific interactions between Tom20-glutathione S-transferase and mitochondrial precursor proteins.

The cytosolic domain of the human mitochondrial protein import receptor, hTom20, has been expressed as a fusion protein with glutathione S-transferase (GST) in bacteria and the purified protein immobilized on Sepharose beads. To discriminate between specific binding of precursor proteins with the receptor and non-specific binding, precursors were recovered as a complex with GST-hTom20 following competitive elution from the beads with reduced glutathione. Here, we describe the specificity of this assay and demonstrate that the cytosolic domain of hTom20 interacts directly with the transcription-translation product of precursor proteins that bear a diverse array of targeting signals. Such proteins include a matrix protein (pODHFR), a polytopic integral protein of the inner membrane (uncoupling protein), a beta-barrel protein of the outer membrane (VDAC/porin) as well as bitopic integral proteins which are inserted into the outer membrane by either an NH2-terminal or COOH-terminal signal anchor sequence (yTom70(1-29)DHFR and Bcl-2, respectively).