Preproteins of chloroplast envelope inner membrane contain targeting information for receptor-dependent import into fungal mitochondria

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).     

Old and new pathways of protein export in chloroplasts and bacteria

Targeting of chloroplast proteins to the thylakoid membrane is analogous to bacterial secretion, and much of what we know has been learned from secretory mechanisms in Escherichia coli. However, chloroplasts also use a delta pH-dependent pathway to target thylakoid proteins, at least some of which are folded before transport. Previously, this pathway seemed to have no cognate in bacteria, but recent results have shown that the HCF106 gene in maize encodes a component of this pathway and has bacterial homologues. This delta pH-dependent pathway might be an ancient conserved mechanism for protein translocation that evolved before the endosymbiotic origin of plastids and mitochondria.     

Lipoprotein nature of Bacillus licheniformis membrane penicillinase

Membrane penicillinase (penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) from Bacillus licheniformis bears a striking resemblance to the major outer membrane lipoprotein of Escherichia coli. It can be specifically labeled in vivo with [3H]glycerol, [35S]cysteine, or [3H]palmitate but not by [32P]orthophosphate. The labeled residues are located at or near the NH2 terminus of the membrane penicillinase because they can be completely removed by trypsin which cleaves a hydrophobic peptide(s) from the NH2 terminus, thereby rendering the enzyme hydrophilic. The membrane penicillinase produced by the 749/C gene carried in E. coli on phage lambda is similar to the enzyme formed in strain 749/C itself. The peptide antibiotic globomycin, which prevents processing of the E. coli prolipoprotein, severely inhibited the attachment of [3H]palmitate or [3H]glycerol to the 749/C enzyme (either in B. licheniformis 749/C or in E. coli), blocked the accumulation of penicillinase in the plasma membrane, and enhanced the formation of exoenzyme. Under the same conditions, globomycin does not prevent the attachment of palmitate or glycerol to the E. coli prolipoprotein but inhibits processing of the modified precursor to the mature lipoprotein. These results are in contrast with the lack of effect of globomycin on the RTEM-beta-lactamase of E. coli which has no detectable hydrophobic membrane form and was not labeled with palmitate or glycerol.     

Sub-mitochondrial localization of the catalytic subunit of pyruvate dehydrogenase phosphatase

Using a specific antibody against the PDP catalytic subunit, PDPc, precise localization of this subunit in mitochondria was performed. Sub-fractionation of purified mitochondria by controlled swelling processes led to the isolation of outer membranes, matrix space and inner membrane vesicles which were purified on a sucrose density gradient. In this study, we demonstrated that PDPc was not recovered as a soluble protein in the matrix space but was associated with the inner membrane. Moreover, Triton X-114 phase partitioning performed on inner membranes showed that PDPc behaved both as a hydrophilic and as a hydrophobic protein, thus suggesting two different forms of this enzyme.     

Heat-stress response of maize mitochondria

We have identified maize (Zea mays L. inbred B73) mitochondrial homologs of the Escherichia coli molecular chaperones DnaK (HSP70) and GroEL (cpn60) using two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblots. During heat stress (42 degrees C for 4 h), levels of HSP70 and cpn60 proteins did not change significantly. In contrast, levels of two 22-kD proteins increased dramatically (HSP22). Monoclonal antibodies were developed to maize HSP70, cpn60, and HSP22. The monoclonal antibodies were characterized with regard to their cross-reactivity to chloroplastic, cytosolic, and mitochondrial fractions, and to different plant species. Expression of mitochondrial HSP22 was evaluated with regard to induction temperature, time required for induction, and time required for degradation upon relief of stress. Maximal HSP22 expression occurred in etiolated seedling mitochondria after 5 h of a +13 degrees C heat stress. Upon relief of heat stress, the HSP22 proteins disappeared with a half-life of about 4 h and were undetectable after 21 h of recovery. Under continuous heat-stress conditions, the level of HSP22 remained high. A cDNA for maize mitochondrial HSP22 was cloned and extended to full length with sequences from an expressed sequence tag database. Sequence analysis indicated that HSP22 is a member of the plant small heat-shock protein superfamily.     

Bacterial hemolysins and leukotoxins affect target cells by forming large exogenous pores into their plasma membrane: Escherichia coli hemolysin A as a case example

Many bacteria include among their virulence factors exoproteins which exert leukocidal and cytolytic functions and have the ability to form pores in model membranes. We show that, at least in the case of the RTX hemolysin produced by Escherichia coli (HlyA), formation of pores in planar lipid membranes is parallelled by opening of strikingly similar channels in the plasma membrane of exposed macrophages. Formation of such lesions in leukocytes can give rise to a variety of effects leading altogether to a diminished immune response towards the invasive bacteria.     

Bioreduction of Tempone and spin-labeled gentamicin by gram-negative bacteria: kinetics and effect of ultrasound

The primary objective of this study is the investigation of bioreduction kinetics of hydrophilic spin probes, 2,2,6,6, -tetramethyl-4-oxo-piperidinyl-1-oxyl (Tempone), and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tensions, with emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into Pseudomonas aeruginosa and Escherichia coli cells. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. In cell suspensions, both Tempone and spin-labeled gentamicin were localized predominantly in the aqueous phase surrounding the cells. However, a gradual reduction of the probes in contact with the cells indicated that the probes penetrated through the outer membrane and periplasmic space into the cytoplasmic membrane, where the electron transport chains and other metabolic activities of gram-negative bacteria are localized. The kinetics of probe reduction depended on oxygen tension and presence of electron transport chain blockers. It was found that probe penetration rate through the outer cell membrane affected the rate of probe reduction; damaging the permeability barrier by cell incubation with EDTA or by powerful insonation above the cavitation threshold increased the rate of probe reduction. In contrast, insonation below the cavitation threshold did not affect the rate of probe reduction. These findings imply that the recently observed synergistic effect between hydrophilic antibiotics and low frequency ultrasound in killing gram-negative bacteria did not result from the enhanced antibiotic penetration through bacterial cell walls.     

The protein import receptor MOM19 of yeast mitochondria

We have identified the protein import receptor MOM19 of Saccharomyces cerevisiae mitochondria. MOM19 is exposed on the outer membrane surface and present in the mitochondrial receptor complex. Antibodies raised against MOM19 strongly inhibited the import of preproteins into isolated yeast mitochondria. Fab fragments prepared from the antibodies showed the same inhibitory effect. By using mutant mitochondria, which lacked the second import receptor MOM72, we found that the import of preproteins via MOM19 did not require the presence of MOM72. We conclude that MOM19 is required for preprotein translocation across the yeast mitochondrial outer membrane and is able to function independently of the receptor MOM72.     

Host H-2 haplotype modulates the induction of host-versus-graft disease after the induction of neonatal tolerance to H-2 alloantigens

Infection of Nicotiana benthamiana cells with cymbidium ringspot (CymRSV) and carnation Italian ringspot (CIRV) viruses results in the formation of conspicuous membranous bodies [multivesicular bodies (MVBs)], which develop from modified peroxisomes or mitochondria, respectively. The organelle targeting signal is located in the proteins of 33 kDa (CymRSV) or 36 kDa (CIRV) encoded by ORF 1, which contain an N-terminal hydrophilic portion followed by two predicted hydrophobic transmembrane segments. Biochemical analysis showed that the 33- and 36-kDa proteins are integral membrane proteins. By exchanging small portions of the ORF 1 sequence between the infectious full-length clones of the two viruses, hybrid constructs were obtained of which the in vitro synthesized RNA was inoculated to N. benthamiana plants and protoplasts. The structure of infectious clones suggested that both the N-terminal hydrophilic region and the transmembrane segments of the ORF 1-encoded proteins specify which organelle is involved in the synthesis of MVBs. Mutational analysis of the CIRV 36-kDa protein also suggested the presence of an internal mitochondrial targeting sequence similar to that found in several normal host proteins that are synthesized in the cytoplasm and transported to mitochondria. The CymRSV 33-kDa protein did not contain the obvious consensus signals thought to be characteristic of proteins targeted to peroxisomes, and an mitochondrial targeting sequence motif was not evident.     

Solid-state REDOR NMR distance measurements at the ligand site of a bacterial chemotaxis membrane receptor

The Escherichia coli serine receptor senses serine levels in the environment and transmits this information across the bacterial inner membrane to modulate a protein phosphorylation cascade which controls swimming behavior. Solid-state nuclear magnetic resonance (NMR) has been used to characterize specific structural features of the ligand binding site interactions in the intact, membrane-bound Ser receptor. Rotational-echo double-resonance (REDOR) experiments on [15N]Ser bound to a [1-13C]Phe-receptor preparation are used to measure distances between the ligand amino group and the carbonyls of two phenylalanine residues in the ligand binding pocket. The results indicate two 4.0 +/- 0.2 A distances, in excellent agreement with the X-ray crystal structure of a soluble fragment of the homologous aspartate receptor [Milburn et al. (1991) Science 254, 1342-1347]. These results confirm the similarity of the binding sites of the Asp and Ser receptors, and demonstrate the feasibility of using solid-state NMR measurements to obtain specific structural information on the 120 kDa intact receptor for probing transmembrane signaling mechanisms.     

FtsY, the prokaryotic signal recognition particle receptor homologue, is essential for biogenesis of membrane proteins

In mammalian cells, many secretory proteins are targeted to the endoplasmic reticulum co-translationally, by the signal recognition particle (SRP) and its receptor. In Escherichia coli, the targeting of secretory proteins to the inner membrane can be accomplished post-translationally. Unexpectedly, despite this variance, E. coli contains essential genes encoding Ffh and FtsY with a significant similarity to proteins of the eukaryotic SRP machinery. In this study, we investigated the possibility that the prokaryotic SRP-like machinery is involved in biogenesis of membrane proteins in E. coli. The data presented here demonstrate that the SRP-receptor homologue, FtsY, is indeed essential for expression of integral membrane proteins in E. coli, indicating that, in the case of this group of proteins, FtsY and the mammalian SRP receptor have similar functions.     

The translocation-associated tox1 locus of Cochliobolus heterostrophus is two genetic elements on two different chromosomes

Previously, Tox1 was defined as a single genetic element controlling the difference between races of Cochliobolus heterostrophus: race T is highly virulent on T-cytoplasm corn and produces the polyketide T-toxin; race O is weakly virulent and does not produce T-toxin. Here we report that Tox1 is two loci, Tox1A and Tox1B, on two different chromosomes. Evidence for two loci derives from: (1) the appearance of 25% Tox+ progeny in crosses between induced Tox1(-) mutants, one defective at Tox1A, the other at Tox1B; (2) the ability of Tox1A- + Tox1B- heterokaryons to complement for T-toxin production; and (3) electrophoretic karyotypes proving that Tox1(-) mutations are physically located on two different chromosomes. Data showing Tox1 as a single genetic element are reconciled with those proving it is two loci by the fact that Tox1 is inseparably linked to the breakpoints of a reciprocal translocation; the translocation results in a four-armed linkage group. In crosses where the translocation is heterozygous (i.e., race T by race O), all markers linked to the four-armed intersection appear linked to each other; in crosses between induced Tox1(-) mutants, complications due to the translocation are eliminated and the two loci segregate independently.     

Localization of histidine residues responsible for heme axial ligation in cytochrome b556 of complex II (succinate:ubiquinone oxidoreductase) in Escherichia coli

Complex II (succinate:ubiquinone oxidoreductase) from Escherichia coli contains four different subunits. Two of the subunits (SDHC and SDHD) are hydrophobic and anchor the two more hydrophilic (flavin and iron-sulfur) subunits (SDHA and SDHB) to the cytoplasmic membrane. Previous studies have shown that the complex of SDHC/SDHD is required to maintain the heme B component of the enzyme and that the heme B is ligated to the protein by two histidine ligands. In the current work, the histidines within SDHC and SDHD have been systematically mutated. SDHC-His91 and SDHD-His14 were eliminated as potential ligands by these studies. SDHC-His84 and SDHD-His71 have been identified as the most likely heme axial ligands in the E. coli enzyme, suggesting that the heme bridges these two subunits in the membrane. Furthermore, the results show that the four-subunit Complex II assembles and retains function despite the absence of the heme B prosthetic group in the membrane. The results do not rule out completely SDHC-His30 as a candidate for heme ligation, but do show that mutation at this position prevents assembly of Complex II in the membrane.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II and analyses of the deduced protein sequence

The complete nucleotide sequence of the gene encoding the Corynebacterium glutamicum mannose enzyme II (EIIMan) was determined. The gene consisted of 2052 base pairs encoding a protein of 683 amino acid residues; the molecular mass of the protein subunit was calculated to be 72570 Da. The N-terminal hydrophilic domain of EIIMan showed 39.7% homology with a C-terminal hydrophilic domain of Escherichia coli glucose-specific enzyme II (EIIGlc). Similar homology was shown between the C-terminal sequence of EIIMan and the E. coli glucose-specific enzyme III (EIIIGlc), or the EIII-like domain of Streptococcus mutans sucrose-specific enzyme II. Sequence comparison with other EIIs showed that EIIMan contained residues His-602 and Cys-28 which were homologous to the potential phosphorylation sites of EIIIGlc, or EIII-like domains, and hydrophilic domains (IIB) of several EIIs, respectively.     

Guinea worm disease--a chance for successful eradication in the Volta Region, Ghana

Parsley cells recognize the fungal phytopathogen Phytophthora sojae through a plasma membrane receptor. A 13 amino acid oligopeptide fragment (Pep-13) of a 42 kDa fungal cell wall glycoprotein was shown to bind to the receptor and stimulate a complex defense response in cultured parsley cells. The Pep-13 binding site solubilized from parsley microsomal membranes by non-ionic detergents exhibited the same ligand affinity and ligand specificity as the membrane-bound receptor. Chemical crosslinking and photoaffinity labeling assays with [125I]Pep-13 revealed that a monomeric 100 kDa integral plasma membrane protein is sufficient for ligand binding and may thus constitute the ligand binding domain of the receptor. Ligand affinity chromatography of solubilized microsomal membrane protein on immobilized Pep-13 yielded a 5000-fold enrichment of specific receptor activity.     

A MADS-box homologue in Ustilago maydis regulates the expression of pheromone-inducible genes but is nonessential

Mating and pathogenic development in the smut fungus Ustilago maydis are controlled by a pheromone/receptor system and two homeodomain proteins, bEp and bWp, which form heterodimers in nonallelic combinations. We describe the isolation of a gene, umc1, encoding a MADS-box protein, which displays significant similarity to the Saccharomyces cerevisiae MCM1 gene. umc1 complemented the viability defect of yeast mcm1 mutants. In U. maydis, umc1 deletion mutants were viable and pathogenic development was unaffected. Nevertheless, the basal expression levels of several pheromone-inducible genes were significantly reduced leading to an attenuated mating reaction. In contrast to S. cerevisiae, where Mcm1p plays a crucial role in the cell-type specific expression of a- and alpha-specific genes, the U. maydis umc1 gene appears to have only a modulatory effect on the expression of mating type-specific genes.     

Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases

The gene rafY from the plasmid pRSD2, which enables Escherichia coli to grow on raffinose, was transferred into expression plasmid pUSL77. The protein was expressed in the porin-deficient Escherichia coli strain KS26 and was isolated and purified to homogeneity. The pure protein was reconstituted into lipid bilayer membranes. It formed an ion-permeable channel with a single-channel conductance of 2.9 nS of the open state in 1 M KCl, which is approximately twice of that of the general diffusion pores OmpF and OmpC of E. coli outer membrane. At lower pH the channel exhibited rapid flickering between three substates of the open channel. The RafY channel appears to be wide and water filled and has a small selectivity for cations over anions. Although RafY is part of an uptake and fermentation system for raffinose it does not contain a binding site for carbohydrates. Our results suggest that RafY is a general diffusion pore with a diameter, larger than that of the general diffusion porins OmpF and OmpC, that allows the diffusion of high-molecular-mass carbohydrates through the outer membrane.     

Transfer of rps19 to the nucleus involves the gain of an RNP-binding motif which may functionally replace RPS13 in Arabidopsis mitochondria

The discovery of disrupted rps19 genes in Arabidopsis mitochondria prompted speculation about the transfer to the nuclear compartment. We here describe the functional gene transfer of rps19 into the nucleus of Arabidopsis. Molecular cloning and sequence analysis of rps19 show that the nuclear gene encodes a long N-terminal extension. Import studies of the precursor protein indicate that only a small part of this extension is cleaved off during import. The larger part of the extension, which shows high similarity to conserved RNA-binding domains of the RNP-CS type, became part of the S19 protein. In the Escherichia coli ribosome S19 forms an RNA-binding complex as heterodimer with S13. By using immuno-analysis and import studies we show that a eubacterial-like S13 protein is absent from Arabidopsis mitochondria, and is not substituted by either a chloroplastic or a cytosolic homologue of this ribosomal protein. We therefore propose that either a highly diverged or missing RPS13 has been functionally replaced by an RNP domain that most likely derived from a glycine-rich RNA-binding protein. These results represent the first case of a functional replacement of a ribosomal protein by a common RNA-binding domain and offer a new view on the flexibility of biological systems in using well-adapted functional domains for different jobs.