Biotinylation in vivo as a sensitive indicator of protein secretion and membrane protein insertion.

Escherichia coli biotin ligase is a cytoplasmic protein which specifically biotinylates the biotin-accepting domains from a variety of organisms. This in vivo biotinylation can be used as a sensitive signal to study protein secretion and membrane protein insertion. When the biotin-accepting domain from the 1.3S subunit of Propionibacterium shermanii transcarboxylase (PSBT) is translationally fused to the periplasmic proteins alkaline phosphatase and maltose-binding protein, there is little or no biotinylation of PSBT in wild-type E. coli. Inhibition of SecA with sodium azide and mutations in SecB, SecD, and SecF, all of which slow down protein secretion, result in biotinylation of PSBT. When PSBT is fused to the E. coli inner membrane protein MalF, it acts as a topological marker: fusions to cytoplasmic domains of MalF are biotinylated, and fusions to periplasmic domains are generally not biotinylated. If SecA is inhibited by sodium azide or if the SecE in the cell is depleted, then the insertion of the MalF second periplasmic domain is slowed down enough that PSBT fusions in this part of the protein become biotinylated. Compared with other protein fusions that have been used to study protein translocation, PSBT fusions have the advantage that they can be used to study the rate of the insertion process.     

The ttsA gene is required for low-calcium-induced type III secretion of Yop proteins and virulence of Yersinia enterocolitica W22703

Pathogenic Yersinia species use a virulence-plasmid encoded type III secretion pathway to escape the innate immune response and to establish infections in lymphoid tissues. At least 22 secretion machinery components are required for type III transport of 14 different Yop proteins, and 10 regulatory factors are responsible for activating this pathway in response to environmental signals. Although the genes for these products are located on the 70-kb virulence plasmid of Yersinia, this extrachromosomal element does not appear to harbor genes that provide for the sensing of environmental signals, such as calcium-, glutamate-, or serum-sensing proteins. To identify such genes, we screened transposon insertion mutants of Y. enterocolitica W22703 for defects in type III secretion and identified ttsA, a chromosomal gene encoding a polytopic membrane protein. ttsA mutant yersiniae synthesize reduced amounts of Yops and display a defect in low-calcium-induced type III secretion of Yop proteins. ttsA mutants are also severely impaired in bacterial motility, a phenotype which is likely due to the reduced expression of flagellar genes. All of these defects were restored by complementation with plasmid-encoded wild-type ttsA. LcrG is a repressor of the Yersinia type III pathway that is activated by an environmental calcium signal. Mutation of the lcrG gene in a ttsA mutant strain restored the type III secretion of Yop proteins, although the double mutant strain secreted Yops in the presence and absence of calcium, similar to the case for mutants that are defective in lcrG gene function alone. To examine the role of ttsA in the establishment of infection, we measured the bacterial dose required to produce an acute lethal disease following intraperitoneal infection of mice. The ttsA insertion caused a greater-than-3-log-unit reduction in virulence compared to that of the parental strain.     

Allele-specific malE mutations that restore interactions between maltose-binding protein and the inner-membrane components of the maltose transport system

Active accumulation of maltose and maltodextrins by Escherichia coli depends on an outer-membrane protein. LamB, a periplasmic maltose-binding protein (MalE, MBP) and three inner-membrane proteins, MalF, MalG and MalK. MalF and MalG are integral transmembrane proteins, while MalK is associated with the inner aspect of the cytoplasmic membrane via an interaction with MalG. Previously we have shown that MBP is essential for movement of maltose across the inner membrane. We have taken advantage of malF and malG mutants in which MBP interacts improperly with the membrane proteins. We describe the properties of malE mutations in which a proper interaction between MBP and defective MalF and MalG proteins has been restored. We found that these malE suppressor mutations are able to restore transport activity in an allele-specific manner. That is, a given malE mutation restores transport activity to different extents in different malF and malG mutants. Since both malF and malG mutations could be suppressed by allele-specific malE suppressors, we propose that, in wild-type bacteria, MBP interacts with sites on both MalF and MalG during active transport. The locations of different malE suppressor mutations indicate specific regions on MBP that are important for interacting with MalF and MalG.     

Biogenesis of MalF and the MalFGK(2) maltose transport complex in Escherichia coli requires YidC

The polytopic inner membrane protein MalF is a constituent of the MalFGK(2) maltose transport complex in Escherichia coli. We have studied the biogenesis of MalF using a combination of in vivo and in vitro approaches. MalF is targeted via the SRP pathway to the Sec/YidC insertion site. Despite close proximity of nascent MalF to YidC during insertion, YidC is not required for the insertion of MalF into the membrane. However, YidC is required for the stability of MalF and the formation of the MalFGK(2) maltose transport complex. Our data indicate that YidC supports the folding of MalF into a stable conformation before it is incorporated into the maltose transport complex.     

Ergosterol content specifies targeting of tail-anchored proteins to mitochondrial outer membranes

Tail-anchored (TA) proteins have a single C-terminal transmembrane domain, making their biogenesis dependent on posttranslational translocation. Despite their importance, no dedicated insertion machinery has been uncovered for mitochondrial outer membrane (MOM) TA proteins. To decipher the molecular mechanisms guiding MOM TA protein insertion, we performed two independent systematic microscopic screens in which we visualized the localization of model MOM TA proteins on the background of mutants in all yeast genes. We could find no mutant in which insertion was completely blocked. However, both screens demonstrated that MOM TA proteins were partially localized to the endoplasmic reticulum (ER) in ∆spf1 cells. Spf1, an ER ATPase with unknown function, is the first protein shown to affect MOM TA protein insertion. We found that ER membranes in ∆spf1 cells become similar in their ergosterol content to mitochondrial membranes. Indeed, when we visualized MOM TA protein distribution in yeast strains with reduced ergosterol content, they phenocopied the loss of Spf1. We therefore suggest that the inherent differences in membrane composition between organelle membranes are sufficient to determine membrane integration specificity in a eukaryotic cell.     

Heat shock induction by a misassembled cytoplasmic membrane protein complex in Escherichia coli

We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli . We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, σ²⁴, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.     

Subunit interactions in ABC transporters: a conserved sequence in hydrophobic membrane proteins of periplasmic permeases defines an important site of interaction with the ATPase subunits.

The cytoplasmic membrane proteins of bacterial binding protein-dependent transporters belong to the superfamily of ABC transporters. The hydrophobic proteins display a conserved, at least 20 amino acid EAA---G---------I-LP region exposed in the cytosol, the EAA region. We mutagenized the EAA regions of MalF and MalG proteins of the Escherichia coli maltose transport system. Substitutions at the same positions in MalF and MalG have different phenotypes, indicating that EAA regions do not act symmetrically. Mutations in malG or malF that slightly affect or do not affect transport, determine a completely defective phenotype when present together. This suggests that EAA regions of MalF and MalG may interact during transport. Maltose-negative mutants fall into two categories with respect to the cellular localization of the MalK ATPase: in the first, MalK is membrane-bound, as in wild-type strains, while in the second, it is cytosolic, as in strains deleted in the malF and malG genes. From maltose-negative mutants of the two categories, we isolated suppressor mutations within malK that restore transport. They map mainly in the putative helical domain of MalK, suggesting that EAA regions may constitute a recognition site for the ABC ATPase helical domain.     

Proper insertion of a complex membrane protein in the absence of its amino-terminal export signal

The MalF protein spans the Escherichia coli cytoplasmic membrane eight times. Deletion of the first transmembrane stretch of MalF, which acts as an export signal, results in a truncated protein that still exhibits high levels of maltose transport activity. These and additional results indicate that the orientation of a membrane protein is not determined by the amino-terminal export signal, topological information is distributed throughout the MalF protein, and insertion of a protein into the cytoplasmic membrane can occur nonsequentially.     

Protein translocation mutants defective in the insertion of integral membrane proteins into the endoplasmic reticulum.

Yeast mutants defective in the translocation of soluble secretory proteins into the lumen of the endoplasmic reticulum (sec61, sec62, sec63) are not impaired in the assembly and glycosylation of the type II membrane protein dipeptidylaminopeptidase B (DPAPB) or of a chimeric membrane protein consisting of the multiple membrane-spanning domain of yeast hydroxymethylglutaryl CoA reductase (HMG1) fused to yeast histidinol dehydrogenase (HIS4C). This chimera is assembled in wild-type or mutant cells such that the His4c protein is oriented to the ER lumen and thus is not available for conversion of cytosolic histidinol to histidine. Cells harboring the chimera have been used to select new translocation defective sec mutants. Temperature-sensitive lethal mutations defining two complementation groups have been isolated: a new allele of sec61 and a single isolate of a new gene sec65. The new isolates are defective in the assembly of DPAPB, as well as the secretory protein alpha-factor precursor. Thus, the chimeric membrane protein allows the selection of more restrictive sec mutations rather than defining genes that are required only for membrane protein assembly. The SEC61 gene was cloned, sequenced, and used to raise polyclonal antiserum that detected the Sec61 protein. The gene encodes a 53-kDa protein with five to eight potential membrane-spanning domains, and Sec61p antiserum detects an integral protein localized to the endoplasmic reticulum membrane. Sec61p appears to play a crucial role in the insertion of secretory and membrane polypeptides into the endoplasmic reticulum.     

Direct detection of bacterial protein secretion using whole colony proteomics

Bacteria use a variety of secretion systems to transport proteins beyond their cell membrane to interact with their environment. For bacterial pathogens, these systems are key virulence determinants that transport bacterial proteins into host cells. Genetic screens to identify bacterial genes required for export have relied on enzymatic or fluorescent reporters fused to known substrates to monitor secretion. However, they cannot be used in analysis of all secretion systems, limiting the implementation across bacteria. Here, we introduce the first application of a modified form of whole colony MALDI-TOF MS to directly detect protein secretion from intact bacterial colonies. We show that this method is able to specifically monitor the ESX-1 system protein secretion system, a major virulence determinant in both mycobacterial and Gram-positive pathogens that is refractory to reporter analysis. We validate the use of this technology as a high throughput screening tool by identifying an ESAT-6 system 1-deficient mutant from a Mycobacterium marinum transposon insertion library. Furthermore, we also demonstrate detection of secreted proteins of the prevalent type III secretion system from the Gram-negative pathogen, Pseudomonas aeruginosa. This method will be broadly applicable to study other bacterial protein export systems and for the identification of compounds that inhibit bacterial protein secretion.     

Mutations in the exocyst component Sec5 disrupt neuronal membrane traffic,but neurotransmitter release persists

The exocyst (Sec6/8) complex is necessary for secretion in yeast and has been postulated to establish polarity by directing vesicle fusion to specific sites along the plasma membrane. The complex may also function in the nervous system, but its precise role is unknown. We have investigated exocyst function in Drosophila with mutations in one member of the complex, sec5. Null alleles die as growth-arrested larvae, whose neuromuscular junctions fail to expand. In culture, neurite outgrowth fails in sec5 mutants once maternal Sec5 is exhausted. Using a trafficking assay, we found impairments in the membrane addition of newly synthesized proteins. In contrast, synaptic vesicle fusion was not impaired. Thus, Sec5 differentiates between two forms of vesicle trafficking: trafficking for cell growth and membrane protein insertion depend on sec5, whereas transmitter secretion does not. In this regard, sec5 differs from the homologs of other yeast exocytosis genes that are required for both neuronal trafficking pathways.     

Structure-function study of MalF protein by random mutagenesis

MalF is one of the two integral inner membrane proteins of the maltose-maltodextrin transport system. To identify functional regions in this protein, we characterized a collection of malF mutants obtained by random mutagenesis. We analyzed their growth on maltose and maltodextrins, the steady-state levels and subcellular localization of the mutant proteins, and the subcellular localization of MalK. Only 2 of the 21 MalF mutant proteins allowed growth on maltose and maltodextrins. Most mutations resulting in immunodetectable proteins mapped to hydrophilic domains, indicating that insertions affecting transmembrane segments gave rise to unstable or lethal proteins. All MalF mutant proteins, even those C-terminally truncated or with large N-terminal deletions, were inserted into the cytoplasmic membrane. Having identified mutations leading to reduced steady-state level, to partial mislocation, and/or to misfolding, we were able to assign to some regions of MalF a role in the assembly of the MalFGK2 complex and/or in the transport mechanism.     

Efficient translocation of positively charged residues of M13 procoat protein across the membrane excludes electrophoresis as the primary force for membrane insertion.

The coat protein of bacteriophage M13 is inserted into the Escherichia coli plasma membrane as a precursor protein, termed procoat, with a typical leader peptide of 23 amino acid residues. Its membrane insertion requires the electrochemical potential but not the cellular components SecA and SecY. Since the electrochemical gradients result in the periplasmic side of the membrane being positively charged, the membrane potential could contribute to the transfer of the negatively charged central region of procoat across the membrane. Here we demonstrate that the central domain following the leader peptide can be translocated across the membrane even when the net charge of the region is changed from -3 to +3. This rules out an electrophoresis-like insertion mechanism for procoat. We also show that the sec independence of procoat insertion is linked to the presence of the second apolar domain. The deletion of most of the second apolar domain from a procoat fusion protein results in sec dependent membrane insertion of the hybrid protein. Moreover, like other proteins that require the sec genes, translocation of this sec dependent procoat protein is inhibited when positively charged residues are introduced after the leader peptide. Loop models involving one or two hydrophobic regions are presented that account for the differences in tolerance of positively charged residues.     

Secretion of a soluble colonization factor by the TCP type 4 pilus biogenesis pathway in Vibrio cholerae

Colonization of the human small intestine by Vibrio cholerae requires the type 4 toxin co-regulated pilus (TCP). Genes encoding the structure and biogenesis functions of TCP are organized within an operon located on the Vibrio Pathogenicity Island (VPI). In an effort to elucidate the functions of proteins involved in TCP biogenesis, in frame deletions of all of the genes within the tcp operon coding for putative pilus biogenesis proteins have been constructed and the resulting mutants characterized with respect to the assembly and function of TCP. As a result of this analysis, we have identified the product of one of these genes, tcpF, as a novel secreted colonization factor. Chromosomal deletion of tcpF yields a mutant that retains in vitro phenotypes associated with the assembly of functional TCP yet is severely attenuated for colonization of the infant mouse intestine. Furthermore, we have determined that the mechanism by which TcpF is translocated across the bacterial outer membrane requires the TCP biogenesis machinery and is independent of the type II extracellular protein secretion (EPS) system. These results suggest a dual role for the TCP biogenesis apparatus in V. cholerae pathogenesis and a novel mechanism of intestinal colonization mediated by a soluble factor.     

The surfactant of Legionella pneumophila Is secreted in a TolC-dependent manner and is antagonistic toward other Legionella species

When Legionella pneumophila grows on agar plates, it secretes a surfactant that promotes flagellum- and pilus-independent "sliding" motility. We isolated three mutants that were defective for surfactant. The first two had mutations in genes predicted to encode cytoplasmic enzymes involved in lipid metabolism. These genes mapped to two adjacent operons that we designated bbcABCDEF and bbcGHIJK. Backcrossing and complementation confirmed the importance of the bbc genes and suggested that the Legionella surfactant is lipid containing. The third mutant had an insertion in tolC. TolC is the outer membrane part of various trimolecular complexes involved in multidrug efflux and type I protein secretion. Complementation of the tolC mutant restored sliding motility. Mutants defective for an inner membrane partner of TolC also lacked a surfactant, confirming that TolC promotes surfactant secretion. L. pneumophila (lspF) mutants lacking type II protein secretion (T2S) are also impaired for a surfactant. When the tolC and lspF mutants were grown next to each other, the lsp mutant secreted surfactant, suggesting that TolC and T2S conjoin to mediate surfactant secretion, with one being the conduit for surfactant export and the other the exporter of a molecule that is required for induction or maturation of surfactant synthesis/secretion. Although the surfactant was not required for the extracellular growth, intracellular infection, and intrapulmonary survival of L. pneumophila, it exhibited antimicrobial activity toward seven other species of Legionella but not toward various non-Legionella species. These data suggest that the surfactant provides L. pneumophila with a selective advantage over other legionellae in the natural environment.     

Isolation of Predation-Deficient Mutants of Bdellovibrio bacteriovorus by Using Transposon Mutagenesis

The availability of the complete genome sequence of Bdellovibrio bacteriovorus provides an opportunity for investigating genes that play a significant role in predation. Using two independently derived facultatively predatory Bdellovibrio strains, we have designed a method to cultivate and screen transposon insertion mutants in 96-well microtiter dishes. Transposon insertion mutants were produced by introducing the plasposon pRL27, which carries a mini-Tn5. Mutants have been screened for predatory activity using 96-well plates. Seventeen independent nonpredatory mutants have been isolated, and DNA flanking the insertion has been sequenced. BLAST analysis revealed that most of these interrupted DNA sequences do not code for known proteins or functions. Two of the inactivated genes were analyzed further: one was found to code for a putative serine protease and the other a probable protein involved in secretion through the outer membrane. The methods described here are the first for the generation and isolation of predation-deficient mutants using random-transposon-insertion mutagenesis. As more mutants are isolated and their gene products analyzed, more light will be shed on how this predator carries out its exclusive life processes and perhaps how these products, or the organism itself, can be used for therapeutic, agricultural, and/or other purposes.     

Autophagosome Requires Specific Early Sec Proteins for Its Formation and NSF/SNARE for Vacuolar Fusion

Double membrane structure, autophagosome, is formed de novo in the process of autophagy in the yeast Saccharomyces cerevisiae, and many Apg proteins participate in this process. To further understand autophagy, we analyzed the involvement of factors engaged in the secretory pathway. First, we showed that Sec18p (N-ethylmaleimide-sensitive fusion protein, NSF) and Vti1p (soluble N-ethylmaleimide-sensitive fusion protein attachment protein, SNARE), and soluble N-ethylmaleimide-sensitive fusion protein receptor are required for fusion of the autophagosome to the vacuole but are not involved in autophagosome formation. Second, Sec12p was shown to be essential for autophagy but not for the cytoplasm to vacuole-targeting (Cvt) (pathway, which shares mostly the same machinery with autophagy. Subcellular fractionation and electron microscopic analyses showed that Cvt vesicles, but not autophagosomes, can be formed in sec12 cells. Three other coatmer protein (COPII) mutants, sec16, sec23, and sec24, were also defective in autophagy. The blockage of autophagy in these mutants was not dependent on transport from endoplasmic reticulum-to-Golgi, because mutations in two other COPII genes, SEC13 and SEC31, did not affect autophagy. These results demonstrate the requirement for subgroup of COPII proteins in autophagy. This evidence demonstrating the involvement of Sec proteins in the mechanism of autophagosome formation is crucial for understanding membrane flow during the process.