Role of the TonB amino terminus in energy transduction between membranes.

Escherichia coli TonB protein is an energy transducer, coupling cytoplasmic membrane energy to active transport of vitamin B12 and iron-siderophores across the outer membrane. TonB is anchored in the cytoplasmic membrane by its hydrophobic amino terminus, with the remainder occupying the periplasmic space. In this report we establish several functions for the hydrophobic amino terminus of TonB. A G-26-->D substitution in the amino terminus prevents export of TonB, suggesting that the amino terminus contains an export signal for proper localization of TonB within the cell envelope. Substitution of the first membrane-spanning domain of the cytoplasmic membrane protein TetA for the TonB amino terminus eliminates TonB activity without altering TonB export, suggesting that the amino terminus contains sequence-specific information. Detectable TonB cross-linking to ExbB is also prevented, suggesting that the two proteins interact primarily through their transmembrane domains. In vivo cleavage of the amino terminus of TonB carrying an engineered leader peptidase cleavage site eliminates (i) TonB activity, (ii) detectable interaction with a membrane fraction having a density intermediate to those of the cytoplasmic and outer membranes, and (iii) cross-linking to ExbB. In contrast, the amino terminus is not required for cross-linking to other proteins with which TonB can form complexes, including FepA. Additionally, although the amino terminus clearly is a membrane anchor, it is not the only means by which TonB associates with the cytoplasmic membrane. TonB lacking its amino-terminal membrane anchor still remains largely associated with the cytoplasmic membrane.     

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.     

The tonB gene of Serratia marcescens: sequence, activity and partial complementation of Escherichia coli tonB mutants

The TonB protein plays a key role in the energy-coupled transport of iron siderophores, of vitamin B12, and of colicins of the B-group across the outer membrane of Escherichia coli. In order to obtain more data about which of its particular amino acid sequences are necessary for TonB function, we have cloned and sequenced the tonB gene of Serratia marcescens. The nucleotide sequence predicts an amino acid sequence of 247 residues (Mr 27,389), which is unusually proline-rich and contains the tandem sequences (Glu-Pro)5 and (Lys-Pro)5. In contrast to the TonB proteins of E. coli and Salmonella typhimurium, translation of the S. marcescens TonB protein starts at the first methionine residue of the open reading frame, which is the only amino acid removed during TonB maturation and export. Only the N-terminal sequence is hydrophobic, suggesting its involvement in anchoring the TonB protein to the cytoplasmic membrane. The S. marcescens tonB gene complemented an E. coli tonB mutant with regard to uptake of iron siderophores, and sensitivity to phages T1 and phi 80, and to colicins B and M. However, an E. coli tonB mutant transformed with the S. marcescens tonB gene remained resistant to colicins Ia and Ib, to colicin B derivatives carrying the amino acid replacements Val/Ala and Val/Gly at position 20 in the TonB box, and they exhibited a tenfold lower activity with colicin D. In addition, the S. marcescens TonB protein did not restore T1 sensitivity of an E. coli exbB tolQ double mutant, as has been found for the overexpressed E. coli TonB protein, indicating a lower activity of the S. marcescens TonB protein. Although the S. marcescens TonB protein was less prone to proteolytic degradation, it was stabilized in E. coli by the ExbBD proteins. In E. coli, TonB activity of S. marcescens depended either on the ExbBD or the TolQR activities.     

A mutation in the amino terminus of a hybrid TrpC-TonB protein relieves overproduction lethality and results in cytoplasmic accumulation.

We have developed a selection for mutations in a trpC-tonB gene fusion that takes advantage of the properties of the plasmid-encoded TrpC-TonB hybrid protein. The TrpC-TonB hybrid protein consists of amino acids 1 through 25 of the normally cytoplasmic protein, TrpC, fused to amino acids 12 through 239 of TonB. It is expressed from the trp promoter and is regulated by the trpR gene and the presence or absence of tryptophan. Under repressing conditions in the presence of tryptophan, the trpC-tonB gene can restore phi 80 sensitivity to a tonB deletion mutant, which indicates that TrpC-TonB can be exported and is functional. High-level expression of TrpC-TonB protein in the absence of tryptophan results in virtually immediate cessation of growth for strains carrying the trpC-tonB plasmid. By selecting for survivors of the induced growth inhibition (overproduction lethality), we have isolated a variety of mutations. Many of the mutations decrease expression of the TrpC-TonB protein, as expected. In addition, three independently isolated mutants expressing normal levels of TrpC-TonB protein result in a Gly----Asp substitution within the hydrophobic amino terminus of TonB. The mutant proteins are designated TrpC-TonBG26D. The mutations are suppressed by prlA alleles, known to suppress export (signal sequence) mutations. TrpC-TonB proteins carrying the Gly----Asp substitution accumulate in the cytoplasm. We conclude that the Gly----Asp substitution is an export mutation. TrpC-TonBG26D protein has been purified and used to raise polyclonal antibodies that specifically recognize both TrpC-TonB protein and wild-type TonB protein.     

Insight from TonB hybrid proteins into the mechanism of iron transport through the outer membrane

We created hybrid proteins to study the functions of TonB. We first fused the portion of Escherichia coli tonB that encodes the C-terminal 69 amino acids (amino acids 170 to 239) of TonB downstream from E. coli malE (MalE-TonB69C). Production of MalE-TonB69C in tonB(+) bacteria inhibited siderophore transport. After overexpression and purification of the fusion protein on an amylose column, we proteolytically released the TonB C terminus and characterized it. Fluorescence spectra positioned its sole tryptophan (W213) in a weakly polar site in the protein interior, shielded from quenchers. Affinity chromatography showed the binding of the TonB C-domain to other proteins: immobilized TonB-dependent (FepA and colicin B) and TonB-independent (FepADelta3-17, OmpA, and lysozyme) proteins adsorbed MalE-TonB69C, revealing a general affinity of the C terminus for other proteins. Additional constructions fused full-length TonB upstream or downstream of green fluorescent protein (GFP). TonB-GFP constructs had partial functionality but no fluorescence; GFP-TonB fusion proteins were functional and fluorescent. The activity of the latter constructs, which localized GFP in the cytoplasm and TonB in the cell envelope, indicate that the TonB N terminus remains in the inner membrane during its biological function. Finally, sequence analyses revealed homology in the TonB C terminus to E. coli YcfS, a proline-rich protein that contains the lysin (LysM) peptidoglycan-binding motif. LysM structural mimicry occurs in two positions of the dimeric TonB C-domain, and experiments confirmed that it physically binds to the murein sacculus. Together, these findings infer that the TonB N terminus remains associated with the inner membrane, while the downstream region bridges the cell envelope from the affinity of the C terminus for peptidoglycan. This architecture suggests a membrane surveillance model of action, in which TonB finds occupied receptor proteins by surveying the underside of peptidoglycan-associated outer membrane proteins.     

The amino-terminal domain of the lamin B receptor is a nuclear envelope targeting signal

The lamin B receptor (LBR) is a polytopic protein of the inner nuclear membrane. It is synthesized without a cleavable amino-terminal signal sequence and composed of a nucleoplasmic amino-terminal domain of 204 amino acids followed by a hydrophobic domain with eight putative transmembrane segments. To identify a nuclear envelope targeting signal, we have examined the cellular localization by immunofluorescence microscopy of chicken LBR, its amino-terminal domain and chimeric proteins transiently expressed in transfected COS-7. Full- length LBR was targeted to the nuclear envelope. The amino-terminal domain, without any transmembrane segments, was transported to the nucleus but excluded from the nucleolus. When the amino-terminal domain of LBR was fused to the amino-terminal side of a transmembrane segment of a type II integral membrane protein of the ER/plasma membrane, the chimeric protein was targeted to the nuclear envelope, likely the inner nuclear membrane. When the amino-terminal domain was deleted from LBR and replaced by alpha-globin, the chimeric protein was retained in the ER. These findings demonstrate that the amino-terminal domain of LBR is targeted to the nucleus after synthesis in the cytoplasm and that this polypeptide can function as a nuclear envelope targeting signal when located at the amino terminus of a type II integral membrane protein synthesized on the ER.     

Specificity of signal peptide recognition in tat-dependent bacterial protein translocation

The bacterial twin arginine translocation (Tat) pathway translocates across the cytoplasmic membrane folded proteins which, in most cases, contain a tightly bound cofactor. Specific amino-terminal signal peptides that exhibit a conserved amino acid consensus motif, S/T-R-R-X-F-L-K, direct these proteins to the Tat translocon. The glucose-fructose oxidoreductase (GFOR) of Zymomonas mobilis is a periplasmic enzyme with tightly bound NADP as a cofactor. It is synthesized as a cytoplasmic precursor with an amino-terminal signal peptide that shows all of the characteristics of a typical twin arginine signal peptide. However, GFOR is not exported to the periplasm when expressed in the heterologous host Escherichia coli, and enzymatically active pre-GFOR is found in the cytoplasm. A precise replacement of the pre-GFOR signal peptide by an authentic E. coli Tat signal peptide, which is derived from pre-trimethylamine N-oxide (TMAO) reductase (TorA), allowed export of GFOR, together with its bound cofactor, to the E. coli periplasm. This export was inhibited by carbonyl cyanide m-chlorophenylhydrazone, but not by sodium azide, and was blocked in E. coli tatC and tatAE mutant strains, showing that membrane translocation of the TorA-GFOR fusion protein occurred via the Tat pathway and not via the Sec pathway. Furthermore, tight cofactor binding (and therefore correct folding) was found to be a prerequisite for proper translocation of the fusion protein. These results strongly suggest that Tat signal peptides are not universally recognized by different Tat translocases, implying that the signal peptides of Tat-dependent precursor proteins are optimally adapted only to their cognate export apparatus. Such a situation is in marked contrast to the situation that is known to exist for Sec-dependent protein translocation.     

Export of hybrid proteins FhuA''-''LacZ and FhuA''-''PhoA to the cell envelope of Escherichia coli K-12.

The fhuA gene of Escherichia coli K-12 encodes an outer membrane protein that acts as the ferrichrome-iron(III) receptor. To determine the export signals and sorting information within FhuA, gene fusions of fhuA'-'lacZ and fhuA'-'phoA were constructed. Although a FhuA'-'LacZ hybrid protein was detected in the Triton X-100-insoluble fraction of the cell envelope, direct immunoelectron microscopic observation showed that this protein remained in the cytoplasm. FhuA'-'PhoA hybrid proteins were all exported across the cytoplasmic membrane. Those hybrids containing up to 88 amino acids of FhuA (FhuA88) fused to PhoA were released along with other periplasmic proteins. Hybrids containing 180 or more amino acids of FhuA (FhuA180) fused to PhoA were associated with the outer membrane. It is proposed that some information inherent in the sequences between FhuA88 and FhuA180 confers stable association with the outer membrane.     

Identification of two Mycobacterium smegmatis lipoproteins exported by a SecA2-dependent pathway

The SecA2 protein is part of a specialized protein export system of mycobacteria. We set out to identify proteins exported to the bacterial cell envelope by the mycobacterial SecA2 system. By comparing the protein profiles of cell wall and membrane fractions from wild-type and DeltasecA2 mutant Mycobacterium smegmatis, we identified the Msmeg1712 and Msmeg1704 proteins as SecA2-dependent cell envelope proteins. These are the first endogenous M. smegmatis proteins identified as dependent on SecA2 for export. Both proteins are homologous to periplasmic sugar-binding proteins of other bacteria, and both contain functional amino-terminal signal sequences with lipobox motifs. These two proteins appeared to be genuine lipoproteins as shown by Triton X-114 fractionation and sensitivity to globomycin, an inhibitor of lipoprotein signal peptidase. The role of SecA2 in the export of these proteins was specific; not all mycobacterial lipoproteins required SecA2 for efficient localization or processing. Finally, Msmeg1704 was recognized by the SecA2 pathway of Mycobacterium tuberculosis, as indicated by the appearance of an export intermediate when the protein was expressed in a DeltasecA2 mutant of M. tuberculosis. Taken together, these results indicate that a select subset of envelope proteins containing amino-terminal signal sequences can be substrates of the mycobacterial SecA2 pathway and that some determinants for SecA2-dependent export are conserved between M. smegmatis and M. tuberculosis.     

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).     

Chloroplast transport of a ribulose bisphosphate carboxylase small subunit-5-enolpyruvyl 3-phosphoshikimate synthase chimeric protein requires part of the mature small subunit in addition to the transit peptide

Ribulose bisphosphate carboxylase small subunit protein is synthesized in the cytoplasm as a precursor and transported into the chloroplast where the amino-terminal portion, the transit peptide, is removed proteolytically. To obtain chloroplast delivery of the 43-kDa 5-enolpyruvyl 3-phosphoshikimate (EPSP) synthase of Salmonella typhimurium, we constructed fusion proteins between the bacterial EPSP synthase and the ribulose bisphosphate carboxylase small subunit. A fusion protein consisting of the transit peptide fused to the EPSP synthase was not transported in vitro or in vivo into chloroplasts. A second fusion protein consisting of the transit peptide and 24 amino acids of the mature small subunit fused to the EPSP synthase was transported both in vitro and in vivo into chloroplasts. It was processed into two polypeptides of 46 and 47 kDa, respectively. This heterogeneity in processing was not caused by the presence of the aroA start codon, since its removal resulted in the same pattern. Substituting 24 different amino acids for the 24 amino acids of the mature small subunit resulted in a fusion protein that was not transported into the chloroplast. It was concluded that a portion of the mature small subunit was needed for efficient chloroplast delivery.     

Information within the mature LamB protein necessary for localization to the outer membrane of E coli K12

It has been proposed that the efficient localization of the outer membrane protein LamB requires a functional signal sequence and at least two additional regions contained within the mature protein. We define these regions more precisely by deletion analysis, and we describe methods for cloning deleterious lacZ fusions onto high-copy-number plasmids and generating in-frame deletions. Analysis of the effects of a series of internal lamB deletions on the export of a LamB-LacZ hybrid protein and of the LamB protein itself indicates that necessary informational signal(s) required for localization lie at the amino-terminal end of the protein. In addition, our analysis indicates that there is a region of information close to or within the fusion joint of the largest lamB-lacZ fusion that increases the efficiency of the export process. A unique deletion that removes a protein segment from amino acid 70 to 200 appears to prevent proteolytic removal of the signal sequence. Nevertheless, the mutant protein is exported to the outer membrane.     

In vivo evidence of TonB shuttling between the cytoplasmic and outer membrane in Escherichia coli

Gram-negative bacteria are able to convert potential energy inherent in the proton gradient of the cytoplasmic membrane into active nutrient transport across the outer membrane. The transduction of energy is mediated by TonB protein. Previous studies suggest a model in which TonB makes sequential and cyclic contact with proteins in each membrane, a process called shuttling. A key feature of shuttling is that the amino-terminal signal anchor must quit its association with the cytoplasmic membrane, and TonB becomes associated solely with the outer membrane. However, the initial studies did not exclude the possibility that TonB was artifactually pulled from the cytoplasmic membrane by the fractionation process. To resolve this ambiguity, we devised a method to test whether the extreme TonB amino-terminus, located in the cytoplasm, ever became accessible to the cys-specific, cytoplasmic membrane-impermeant molecule, Oregon Green(R) 488 maleimide (OGM) in vivo. A full-length TonB and a truncated TonB were modified to carry a sole cysteine at position 3. Both full-length TonB and truncated TonB (consisting of the amino-terminal two-thirds) achieved identical conformations in the cytoplasmic membrane, as determined by their abilities to cross-link to the cytoplasmic membrane protein ExbB and their abilities to respond conformationally to the presence or absence of proton motive force. Full-length TonB could be amino-terminally labelled in vivo, suggesting that it was periplasmically exposed. In contrast, truncated TonB, which did not associate with the outer membrane, was not specifically labelled in vivo. The truncated TonB also acted as a control for leakage of OGM across the cytoplasmic membrane. Further, the extent of labelling for full-length TonB correlated roughly with the proportion of TonB found at the outer membrane. These findings suggest that TonB does indeed disengage from the cytoplasmic membrane during energy transduction and shuttle to the outer membrane.     

A 15-kDa interferon-induced protein is derived by COOH-terminal processing of a 17-kDa precursor

An interferon-induced 15-kDa protein is synthesized from a precursor of higher molecular weight; the precursor contains 165 amino acids (17 kDa), whereas the stable product (15 kDa) contains 156 amino acids. The stable 15-kDa form is derived from the precursor 17-kDa form by the removal of eight amino acids from the COOH terminus and the methionine from the NH2 terminus. The existence of the precursor 17-kDa protein can be demonstrated after brief periods of in vivo labeling with [35S]methionine and by translation of mRNA in vitro.     

In vivo selection and characterization of internal deletions in the lamB::lacZ gene fusion

Strain Pop3299 contains the lamB::lacZ42-12 gene fusion that encodes a hybrid protein that is efficiently exported to the cellular envelope of Escherichia coli. As a result of this efficient export, this strain is killed by the inducer maltose and unable to grow on minimal media supplemented with lactose. In an attempt to isolate mutants in which export of the hybrid protein is altered, we selected Lac+ mutants of strain Pop3299 on lactose tetrazolium media. Unlike mutants previously isolated on lactose minimal media, all the mutants we obtained carried large deletions within the lamB::lacZ gene fusion. Thus, it appears that the type of selection employed affects the type of mutations obtained. We have analyzed the nucleotide sequences of representative mutants, and demonstrate a correlation between the deletion size and the export-related maltose and lactose phenotypes. In addition, we demonstrate that the deletions do not appear to arise from regions of micro-homology.     

Two nuclear export signals specify the cytoplasmic localization of calcineurin B homologous protein 1

We previously showed that calcineurin B homologous protein 1 (CHP1) interacts with nuclear apoptosis-inducing protein kinase DRAK2, and that overexpression of DRAK2 induces the nuclear accumulation of CHP1, although CHP1 usually resides in the cytoplasm [Matsumoto et al. (2001) J. Biochem. 130, 217-225]. Here we show that CHP1 has two functional nuclear export signal (NES) sequences in its carboxyl-terminal region. Treatment of several cell lines with leptomycin B, a specific inhibitor of CRM1-dependent nuclear export, induces the nuclear accumulation of CHP1. Moreover, CHP1-GFP fusion proteins with deletions or point mutations affecting the two putative NES sequences accumulate in the nucleus to a greater extent than wild-type CHP1-GFP. Tagging glutathione S-transferase-GFP fusion protein with each NES sequence caused a shift in their intracellular localization from all over the cells to the cytoplasm. These results suggest that after CHP1 has entered the nucleus, it is exported to the cytoplasm in an NES-dependent manner.     

Export of FepA::PhoA fusion proteins to the outer membrane of Escherichia coli K-12.

A library of fepA::phoA gene fusions was generated in order to study the structure and secretion of the Escherichia coli K-12 ferric enterobactin receptor, FepA. All of the fusion proteins contained various lengths of the amino-terminal portion of FepA fused in frame to the catalytic portion of bacterial alkaline phosphatase. Localization of FepA::PhoA fusion proteins in the cell envelope was dependent on the number of residues of mature FepA present at the amino terminus. Hybrids containing up to one-third of the amino-terminal portion of FepA fractionated with their periplasm, while those containing longer sequences of mature FepA were exported to the outer membrane. Outer membrane-localized fusion proteins expressed FepA sequences on the external face of the outer membrane and alkaline phosphatase moieties in the periplasmic space. From sequence determinations of the fepA::phoA fusion joints, residues within FepA which may be exposed on the periplasmic side of the outer membrane were identified.     

Genomic but not antigenomic hepatitis delta virus RNA is preferentially exported from the nucleus immediately after synthesis and processing

Hepatitis delta virus (HDV) contains a viroid-like circular RNA that replicates via a double rolling circle replication mechanism. It is generally assumed that HDV RNA is synthesized and remains exclusively in the nucleus until being exported to the cytoplasm for virion assembly. Using a [32P]orthophosphate metabolic labeling procedure to study HDV RNA replication (T. B. Macnaughton, S. T. Shi, L. E. Modahl, and M. M. C. Lai. J. Virol. 76:3920-3927, 2002), we unexpectedly found that a significant amount of newly synthesized HDV RNA was detected in the cytoplasm. Surprisingly, Northern blot analysis revealed that the genomic-sense HDV RNA is present almost equally in both the nucleus and cytoplasm, whereas antigenomic HDV RNA was mostly retained in the nucleus, suggesting the specific and highly selective export of genomic HDV RNA. Kinetic studies showed that genomic HDV RNA was exported soon after synthesis. However, only the monomer and, to a lesser extent, the dimer HDV RNAs were exported to the cytoplasm; very little higher-molecular-weight HDV RNA species were detected in the cytoplasm. These results suggest that the cleavage and processing of HDV RNA may facilitate RNA export. The export of genomic HDV RNA was resistant to leptomycin B, indicating that a cell region maintenance 1 (Crm1)-independent pathway was involved. The large form of hepatitis delta antigen (L-HDAg), which is responsible for virus packaging, was not required for RNA export, as a mutant HDV RNA genome unable to synthesize L-HDAg was still exported. The proportions of genomic HDV RNA in the nucleus and cytoplasm remained relatively constant throughout replication, indicating that export of genomic HDV RNA occurred continuously. In contrast, while antigenomic HDV RNA was predominantly in the nucleus, there was a proportionally large fraction of antigenomic HDV RNA in the cytoplasm at early time points of RNA replication. These findings uncover a previously unrecognized presence of HDV RNA in the cytoplasm, which may have implications for viral RNA synthesis and packaging.