Abstract: Failure to detoxify the intermediary metabolite glyoxylate in human hepatocytes underlies the metabolic pathology of two potentially lethal hereditary calcium oxalate kidney stone diseases, PH (primary hyperoxaluria) types 1 and 2. In order to define more clearly the roles of enzymes involved in the metabolism of glyoxylate, we have established singly, doubly and triply transformed CHO (Chinese-hamster ovary) cell lines, expressing all combinations of normal human AGT (alanine:glyoxylate aminotransferase; the enzyme deficient in PH1), GR/HPR (glyoxylate/hydroxypyruvate reductase; the enzyme deficient in PH2), and GO (glycolate oxidase). We have embarked on the preliminary metabolic analysis of these transformants by studying the indirect toxicity of glycolate as a simple measure of the net intracellular production of glyoxylate. Our results show that glycolate is toxic only to those cells expressing GO and that this toxicity is diminished when AGT and/or GR/HPR are expressed in addition to GO. This finding indicates that we have been able to reconstruct the glycolate-->glyoxylate, glyoxylate-->glycine, and glyoxylate-->glycolate metabolic pathways, catalysed by GO, AGT, and GR/HPR respectively, in cells that do not normally express them. These results are compatible with the findings in PH1 and PH2, in which AGT and GR/HPR deficiencies lead to increased oxalate synthesis, due to the failure to detoxify its immediate precursor glyoxylate. These CHO cell transformants have a potential use as a cell-based bioassay for screening small molecules that stabilize AGT or GR/HPR and might have use in the treatment of PH1 or PH2.
Abstract: Superficially similar cleavable targeting signals specify whether lumenal proteins are transported across the thylakoid membrane by a Sec- or ΔpH-dependent pathway. A twin-arginine motif is essential but not sufficient to direct ΔpH-dependent targeting; here we show that a second determinant is located in the hydrophobic region. A highly hydrophobic amino acid is found either two or three residues C-terminal to the twin-arginine in all known transfer peptides for the ΔpH-dependent system, and substitution of this residue in the 23-kDa (23K) peptide markedly inhibits translocation. Further, whereas the insertion of twin-arginine in a Sec-dependent precursor does not permit efficient ΔpH-dependent targeting, the simultaneous presence of a leucine at the +3 position (relative to the RR) enables the peptide to function as efficiently as an authentic transfer peptide. RRNVL, RRAAL and RRALA within a Sec targeting signal all support efficient ΔpH-dependent targeting, RRNVA is less effective and RRNAA/RRNAG are totally ineffective. We conclude that the core signal for this pathway is a twin-arginine together with an adjacent hydrophobic determinant. Copyright (C) 1998 Federation of European Biochemical Societies.
Abstract: Thylakoid transfer signals carry information specifying translocation by either a Sec- or delta pH-dependent protein translocator in the chloroplast thylakoid membrane, yet all resemble classical signal peptides in overall structural terms. Comparison of known transfer signals reveals two differences: (a) signals for the delta pH-driven system invariably contain a critical twin-arginine (Arg-Arg) motif prior to the hydrophobic (H) domain, whereas known Sec-dependent signals contain lysine, and (b) the H-domains of Sec-dependent signals are generally longer. Previous work has shown that a twin-Arg motif before the H-domain is critical for targeting by the delta pH-dependent pathway; in this report we show that the charge characteristics of this region are not important for sorting by the Sec pathway. Twin-Lys, twin-Arg or single Arg are all acceptable to the Sec system, although single Lys/Arg is preferred. The single Lys in pre-plastocyanin can even be replaced by an uncharged residue without apparent effect. We have also generated a pre-plastocyanin mutant containing an H-domain which, in terms of hydropathy profile, is identical to that of a delta pH-dependent protein. This mutant is also transported efficiently by the Sec system, demonstrating that hydrophobicity per se is not a key sorting determinant. However, the characteristics of the H-domain may be important in avoiding a different form of mis-targeting: to the endoplasmic reticulum. Thylakoid signal peptides have undergone substantial structural changes during the evolution of the chloroplast from endosymbiotic cyanobacterium: plastid-encoded and cyanobacterial signals contain H-domains that are highly hydrophobic and enriched in Leu and aromatic residues, whereas nuclear-encoded counterparts are Ala-rich and far less hydrophobic. We speculate that this trend may reflect a need to avoid mistargeting through recognition by cytosolic signal recognition particle, which preferentially interacts with more hydrophobic signal peptides.
Abstract: Cleavable N-terminal targeting signals direct the translocation of lumenal proteins across the chloroplast thylakoid membrane by either a Sec-type or delta pH-driven protein translocase. The targeting signals specify choice of translocation pathway, yet all resemble typical bacterial 'signal' peptides in possessing a charged N-terminus (N-domain), hydrophobic core region (H-domain) and more polar C-terminal region (C-domain). We have previously shown that a twin-arginine motif in the N-domain is essential for targeting by the delta pH-dependent pathway, but it has remained unclear why targeting signals for this system (transfer peptides) are not recognized by the Sec apparatus. We show here that the conserved charge distribution around the H-domain in the 23K transfer peptide (twin-Arg in the N-domain, Lys in the C-domain) constitutes a 'Sec-avoidance' signal. The C-domain Lys, while not important for delta pH-dependent targeting, is the only barrier to Sec-dependent translocation; its removal generates an apparently perfect signal peptide. Conversely, insertion of twin-Arg into the N-domain of a Sec substrate has little effect, as has insertion of a C-domain Lys, but the combined substitutions almost totally block transport. We also show that the 23K mature protein is incapable of being targeted by the Sec pathway, and it is proposed that the role of the Sec-avoidance motif in the transfer peptide is to prevent futile interactions with the Sec apparatus.
Abstract: 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.
Abstract: The spinach triose phosphate/phosphate translocator and the 37-kDa protein are both integral components of the chloroplast inner envelope membrane. They are synthesized in the cytosol with N-terminal extensions, the transit peptides, that are different in structural terms from those of imported stromal or thylakoid proteins. In order to determine if these N-terminal extensions are essential for the correct localization to the envelope membrane, they were linked to the mature parts of thylakoid membrane proteins, the light-harvesting chlorophyll a/b binding protein and the CF0II-subunit of the thylakoid ATP synthase, respectively. In addition, the transit peptide of the CF0II-subunit that contains signals for the transport across both the envelope and the thylakoid membrane was fused to the mature parts of both envelope membrane proteins. The chimeric proteins were imported into isolated spinach chloroplasts, and the intraorganellar routing of the proteins was analyzed. The results obtained show that the N-terminal extensions of both envelope membrane proteins possess a stroma-targeting function only and that the information for the integration into the envelope membrane is contained in the mature parts of the proteins. At least part of the integration signal is provided by hydrophobic domains in the mature sequences since the removal of such a hydrophobic segment from the 37-kDa protein leads to missorting of the protein to the stroma and the thylakoid membrane.
Abstract: The primary sequences of the chloroplast triose phosphate/phosphate translocator precursor proteins from C4-plants (maize mesophyll cells and Flaveria trinervia) and from the C3-type Flaveria pringlei were determined. The mature parts of these translocators possess 83-94% identical amino acid residues. The C4-translocator protein can be correctly targeted to C3-type chloroplasts and inserted into the envelope membrane. Expression of the mature parts of these chloroplast translocators (cTPT) in transformed yeast cells and subsequent reconstitution of the functional proteins reveals the difference between the recombinant translocator proteins from the two cell types with respect to the transport of phosphoenolpyruvate. Comparison of the cTPT sequences from F. pringlei and F. trinervia in combination with computer-aided molecular modelling of the substrate translocation pore leads to the suggestion, that only minor exchanges of amino acid residues between the C3- and C4-translocator proteins are sufficient to extend their substrate specificities to recognize also phosphoenolpyruvate.
Abstract: Porins are voltage-gated diffusion pores found in all eukaryotic kingdoms. Here we describe, for the first time, the identification and characterization of two cDNAs encoding porins from plants. Peptide sequences obtained from a 30-kDa protein of envelope membranes from pea root plastids allowed the isolation of two cDNA clones from pea and maize. On the protein level, both proteins are homologous by 58%. Sequence comparison against the Swiss-Prot sequence data base revealed a homology of about 25% to mitochondrial porins from fungi and human. Computer-aided predictions of the secondary structure of the plant porins revealed the presence of 16 antiparallel beta-strands that are also found in mitochondrial porins. Porins from non-green plastids and from the outer mitochondrial membrane were reconstituted into planar lipid bilayers. The proteins showed high pore-forming activities and similar single-channel conductances. In vitro translated porin was preferentially imported only into non-green plastids but not into chloroplasts. To our knowledge, this is the first example of selective import of a plastid protein into different types of plastids. This finding is in line with the observation that an immunoreactive 30-kDa band was only found in non-green plastids and mitochondria but not in chloroplasts. We conclude that mitochondria and non-green plastids possess homologous porin proteins, whereas chloroplasts are characterized by a different type of porin.
Abstract: The amino-terminal transit sequences of two preproteins destined for the chloroplast inner envelope membrane show similarities to mitochondrial presequences in the prevalence of positive charges and the potential formation of an amphipathic alpha-helix. We studied if these preproteins could be imported into mitochondria and found a low, yet significant import into isolated plant mitochondria. The plant mitochondria were previously shown not to import precursors of chloroplast stromal or thylakoidal proteins. To analyze the specificity of import into mitochondria we used the established import systems of fungal mitochondria. The envelope preproteins were efficiently imported into Saccharomyces cerevisiae or Neurospora crassa mitochondria. Their import showed the characteristics of specific mitochondrial protein uptake, including a requirement for the main receptor MOM19 (mitochondrial outer membrane protein of 19 kDa) and a membrane potential across the inner membrane, and depended on the presence of the chloroplast transit sequence. We conclude that some chloroplast transit sequences contain sufficient information for specific interaction with mitochondrial import receptors (at least from fungal sources).
Abstract: The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane. Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template. This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence. The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da. It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues. Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca. 25 kDa) is due to its abnormal amino acid composition. Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane. Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.
