Publications (16)83.92 Total impact
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Chapter: Protein Transport Into and Across Haloarchaeal Cytoplasmic Membranes
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ABSTRACT: Several pathways have evolved in the three domains of life to facilitate membrane protein insertion and the transport of proteins across lipid membranes. Haloarchaea employ the universally conserved Sec pathway, which transports unfolded proteins, for the transport of biologically important substrates into and across the membrane. However, they also extensively employ the twin arginine translocation (Tat) system, which transports substrates across the lipid bilayer in a folded conformation. Most haloarchaeal Tat substrates appear to be anchored to cytoplasmic membranes via lipid modifications. In silico analyses suggest that the prominent use of the Tat pathway and the lipid tethering of Tat substrates are traits unique to halophilic archaea. We discuss the selective pressures that may have led to these unique adaptations as well as possible explanations for why they are not observed in halobacteria.06/2011: pages 207-225; -
Article: Identification of surprisingly diverse type IV pili, across a broad range of gram-positive bacteria.
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ABSTRACT: In Gram-negative bacteria, type IV pili (TFP) have long been known to play important roles in such diverse biological phenomena as surface adhesion, motility, and DNA transfer, with significant consequences for pathogenicity. More recently it became apparent that Gram-positive bacteria also express type IV pili; however, little is known about the diversity and abundance of these structures in Gram-positives. Computational tools for automated identification of type IV pilins are not currently available. To assess TFP diversity in Gram-positive bacteria and facilitate pilin identification, we compiled a comprehensive list of putative Gram-positive pilins encoded by operons containing highly conserved pilus biosynthetic genes (pilB, pilC). A surprisingly large number of species were found to contain multiple TFP operons (pil, com and/or tad). The N-terminal sequences of predicted pilins were exploited to develop PilFind, a rule-based algorithm for genome-wide identification of otherwise poorly conserved type IV pilins in any species, regardless of their association with TFP biosynthetic operons (http://signalfind.org). Using PilFind to scan 53 Gram-positive genomes (encoding >187,000 proteins), we identified 286 candidate pilins, including 214 in operons containing TFP biosynthetic genes (TBG+ operons). Although trained on Gram-positive pilins, PilFind identified 55 of 58 manually curated Gram-negative pilins in TBG+ operons, as well as 53 additional pilin candidates in operons lacking biosynthetic genes in ten species (>38,000 proteins), including 27 of 29 experimentally verified pilins. False positive rates appear to be low, as PilFind predicted only four pilin candidates in eleven bacterial species (>13,000 proteins) lacking TFP biosynthetic genes. We have shown that Gram-positive bacteria contain a highly diverse set of type IV pili. PilFind can be an invaluable tool to study bacterial cellular processes known to involve type IV pilus-like structures. Its use in combination with other currently available computational tools should improve the accuracy of predicting the subcellular localization of bacterial proteins.PLoS ONE 01/2011; 6(12):e28919. · 4.09 Impact Factor -
Article: Haloferax volcanii flagella are required for motility but are not involved in PibD-dependent surface adhesion.
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ABSTRACT: Although the genome of Haloferax volcanii contains genes (flgA1-flgA2) that encode flagellins and others that encode proteins involved in flagellar assembly, previous reports have concluded that H. volcanii is nonmotile. Contrary to these reports, we have now identified conditions under which H. volcanii is motile. Moreover, we have determined that an H. volcanii deletion mutant lacking flagellin genes is not motile. However, unlike flagella characterized in other prokaryotes, including other archaea, the H. volcanii flagella do not appear to play a significant role in surface adhesion. While flagella often play similar functional roles in bacteria and archaea, the processes involved in the biosynthesis of archaeal flagella do not resemble those involved in assembling bacterial flagella but, instead, are similar to those involved in producing bacterial type IV pili. Consistent with this observation, we have determined that, in addition to disrupting preflagellin processing, deleting pibD, which encodes the preflagellin peptidase, prevents the maturation of other H. volcanii type IV pilin-like proteins. Moreover, in addition to abolishing swimming motility, and unlike the flgA1-flgA2 deletion, deleting pibD eliminates the ability of H. volcanii to adhere to a glass surface, indicating that a nonflagellar type IV pilus-like structure plays a critical role in H. volcanii surface adhesion.Journal of bacteriology 04/2010; 192(12):3093-102. · 3.94 Impact Factor -
Article: Mutational and bioinformatic analysis of haloarchaeal lipobox-containing proteins.
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ABSTRACT: A conserved lipid-modified cysteine found in a protein motif commonly referred to as a lipobox mediates the membrane anchoring of a subset of proteins transported across the bacterial cytoplasmic membrane via the Sec pathway. Sequenced haloarchaeal genomes encode many putative lipoproteins and recent studies have confirmed the importance of the conserved lipobox cysteine for signal peptide processing of three lipobox-containing proteins in the model archaeon Haloferax volcanii. We have extended these in vivo analyses to additional Hfx. volcanii substrates, supporting our previous in silico predictions and confirming the diversity of predicted Hfx. volcanii lipoproteins. Moreover, using extensive comparative secretome analyses, we identified genes encodining putative lipoproteins across a wide range of archaeal species. While our in silico analyses, supported by in vivo data, indicate that most haloarchaeal lipoproteins are Tat substrates, these analyses also predict that many crenarchaeal species lack lipoproteins altogether and that other archaea, such as nonhalophilic euryarchaeal species, transport lipoproteins via the Sec pathway. To facilitate the identification of genes that encode potential haloarchaeal Tat-lipoproteins, we have developed TatLipo, a bioinformatic tool designed to detect lipoboxes in haloarchaeal Tat signal peptides. Our results provide a strong foundation for future studies aimed at identifying components of the archaeal lipoprotein biogenesis pathway.Archaea 01/2010; 2010. -
Article: Haloferax volcanii twin-arginine translocation substates include secreted soluble, C-terminally anchored and lipoproteins.
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ABSTRACT: Recent in silico and in vivo studies have suggested that the majority of proteins destined for secretion in the haloarchaea are trafficked through the twin-arginine translocation (Tat) pathway. The presence of lipobox motifs in most haloarchaeal Tat signal sequences is intriguing as: (i) bioinformatic searches of archaeal genomes have not identified lipoprotein biogenesis enzymes and (ii) there are no known Tat substrates containing both a twin-arginine and a bona fide lipobox. We have examined six computationally designated Tat substrates in the haloarchaeon Haloferax volcanii to verify previous computational predictions and to initiate studies of lipoprotein biogenesis via the Tat pathway. Our results confirmed that the six candidate proteins were not only Tat substrates, but also belonged to diverse classes of secretory proteins. Analysis of predicted lipoprotein Tat substrates revealed that they are anchored to the archaeal membrane in a cysteine-dependent manner. Interestingly, despite the absence of an archaeal lipoprotein signal peptidase II (SPase II) homologue, the SPase II inhibitor globomycin impeded cell growth and specifically prevented maturation of lipoproteins. Together, this work not only represents the first experimental demonstration of a lipoprotein Tat substrate, but also indicates the presence of an unidentified lipoprotein biogenesis pathway in archaea.Molecular Microbiology 01/2008; 66(6):1597-606. · 5.01 Impact Factor -
Article: Identification of diverse archaeal proteins with class III signal peptides cleaved by distinct archaeal prepilin peptidases.
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ABSTRACT: Most secreted archaeal proteins are targeted to the membrane via a tripartite signal composed of a charged N terminus and a hydrophobic domain, followed by a signal peptidase-processing site. Signal peptides of archaeal flagellins, similar to class III signal peptides of bacterial type IV pilins, are distinct in that their processing sites precede the hydrophobic domain, which is crucial for assembly of these extracytoplasmic structures. To identify the complement of archaeal proteins with class III signal sequences, a PERL program (FlaFind) was written. A diverse set of proteins was identified, and many of these FlaFind positives were encoded by genes that were cotranscribed with homologs of pilus assembly genes. Moreover, structural conservation of primary sequences between many FlaFind positives and subunits of bacterial pilus-like structures, which have been shown to be critical for pilin assembly, have been observed. A subset of pilin-like FlaFind positives contained a conserved domain of unknown function (DUF361) within the signal peptide. Many of the genes encoding these proteins were in operons that contained a gene encoding a novel euryarchaeal prepilin-peptidase, EppA, homolog. Heterologous analysis revealed that Methanococcus maripaludis DUF361-containing proteins were specifically processed by the EppA homolog of this archaeon. Conversely, M. maripaludis preflagellins were cleaved only by the archaeal preflagellin peptidase FlaK. Together, the results reveal a diverse set of archaeal proteins with class III signal peptides that might be subunits of as-yet-undescribed cell surface structures, such as archaeal pili.Journal of Bacteriology 03/2007; 189(3):772-8. · 3.83 Impact Factor -
Article: The twin-arginine translocation pathway is a major route of protein export in Streptomyces coelicolor.
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ABSTRACT: The twin-arginine translocation (Tat) pathway is a protein transport system for the export of folded proteins. Substrate proteins are targeted to the Tat translocase by N-terminal signal peptides harboring a distinctive R-R-x-Phi-Phi "twin-arginine" amino acid motif. Using a combination of proteomic techniques, the protein contents from the cell wall of the model Gram-positive bacterium Streptomyces coelicolor were identified and compared with that of mutant strains defective in Tat transport. The proteomic experiments pointed to 43 potentially Tat-dependent extracellular proteins. Of these, 25 were verified as bearing bona fide Tat-targeting signal peptides after independent screening with a facile, rapid, and sensitive reporter assay. The identified Tat substrates, among others, include polymer-degrading enzymes, phosphatases, and binding proteins as well as enzymes involved in secondary metabolism. Moreover, in addition to predicted extracellular substrates, putative lipoproteins were shown to be Tat-dependent. This work provides strong experimental evidence that the Tat system is used as a major general export pathway in Streptomyces.Proceedings of the National Academy of Sciences 12/2006; 103(47):17927-32. · 9.68 Impact Factor -
Article: Archaeal and bacterial SecD and SecF homologs exhibit striking structural and functional conservation.
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ABSTRACT: The majority of secretory proteins are translocated into and across hydrophobic membranes via the universally conserved Sec pore. Accessory proteins, including the SecDF-YajC Escherichia coli membrane complex, are required for efficient protein secretion. E. coli SecDF-YajC has been proposed to be involved in the membrane cycling of SecA, the cytoplasmic bacterial translocation ATPase, and in the stabilizing of SecG, a subunit of the Sec pore. While there are no identified archaeal homologs of either SecA or SecG, many archaea possess homologs of SecD and SecF. Here, we present the first study that addresses the function of archaeal SecD and SecF homologs. We show that the SecD and SecF components in the model archaeon Haloferax volcanii form a cytoplasmic membrane complex in the native host. Furthermore, as in E. coli, an H. volcanii deltasecFD mutant strain exhibits both severe cold sensitivity and a Sec-specific protein translocation defect. Taken together, these results demonstrate significant functional conservation among the prokaryotic SecD and SecF homologs despite the distinct composition of their translocation machineries.Journal of Bacteriology 03/2006; 188(4):1251-9. · 3.83 Impact Factor -
Article: Protein transport in Archaea: Sec and twin arginine translocation pathways.
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ABSTRACT: The transport of proteins into and across hydrophobic membranes is an essential cellular process. The majority of proteins that are translocated in an unfolded conformation traverse the membrane by way of the universally conserved Sec pathway, whereas the twin arginine translocation pathway is responsible for the transport of folded proteins across the membrane. Structural, biochemical and genetic analyses of these processes in Archaea have revealed unique archaeal features, and have also provided a better understanding of these pathways in organisms of all domains. Further study of these pathways in Archaea might elucidate fundamental principles involved in each type of transport and could help determine their relative costs and benefits as well as evolutionary adaptations in protein secretion strategies.Current Opinion in Microbiology 01/2006; 8(6):713-9. · 7.93 Impact Factor -
Article: Genetic and biochemical analysis of the twin-arginine translocation pathway in halophilic archaea.
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ABSTRACT: The twin-arginine translocation (Tat) pathway is present in a wide variety of prokaryotes and is capable of exporting partially or fully folded proteins from the cytoplasm. Although diverse classes of proteins are transported via the Tat pathway, in most organisms it facilitates the secretion of a relatively small number of substrates compared to the Sec pathway. However, computational evidence suggests that haloarchaea route nearly all secreted proteins to the Tat pathway. We have expanded previous computational analyses of the haloarchaeal Tat pathway and initiated in vivo characterization of the Tat machinery in a model haloarchaeon, Haloferax volcanii. Consistent with the predicted usage of the this pathway in the haloarchaea, we determined that three of the four identified tat genes in Haloferax volcanii are essential for viability when grown aerobically in complex medium. This represents the first report of an organism that requires the Tat pathway for viability when grown under such conditions. Deletion of the nonessential gene had no effect on the secretion of a verified substrate of the Tat pathway. The two TatA paralogs TatAo and TatAt were detected in both the membrane and cytoplasm and could be copurified from the latter fraction. Using size exclusion chromatography to further characterize cytoplasmic and membrane TatA proteins, we find these proteins present in high-molecular-weight complexes in both cellular fractions.Journal of Bacteriology 01/2006; 187(23):8104-13. · 3.83 Impact Factor -
Article: The Haloferax volcanii FtsY homolog is critical for haloarchaeal growth but does not require the A domain.
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ABSTRACT: The targeting of many Sec substrates to the membrane-associated translocation pore requires the cytoplasmic signal recognition particle (SRP). In Eukarya and Bacteria it has been shown that membrane docking of the SRP-substrate complex occurs via the universally conserved SRP receptor (Sralpha/beta and FtsY, respectively). While much has been learned about the archaeal SRP in recent years, few studies have examined archaeal Sralpha/FtsY homologs. In the present study the FtsY homolog of Haloferax volcanii was characterized in its native host. Disruption of the sole chromosomal copy of ftsY in H. volcanii was possible only under conditions where either the full-length haloarchaeal FtsY or an amino-terminally truncated version of this protein lacking the A domain, was expressed in trans. Subcellular fractionation analysis of H. volcanii ftsY deletion strains expressing either one of the complementing proteins revealed that in addition to a cytoplasmic pool, both proteins cofractionate with the haloarchaeal cytoplasmic membrane. Moreover, membrane localization of the universally conserved SRP subunit SRP54, the key binding partner of FtsY, was detected in both H. volcanii strains. These analyses suggest that the H. volcanii FtsY homolog plays a crucial role but does not require its A domain for haloarchaeal growth.Journal of Bacteriology 07/2005; 187(12):4015-22. · 3.83 Impact Factor -
Article: Diversity and evolution of protein translocation.
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ABSTRACT: Cells need to translocate proteins into and across hydrophobic membranes in order to interact with the extracellular environment. Although a subset of proteins are thought to spontaneously insert into lipid bilayers, translocation of most transported proteins requires additional cellular components. Such components catalyze efficient lateral transport into or across cellular membranes in prokaryotes and eukaryotes. These include, among others, the conserved YidC/Oxa1/Alb3 proteins as well as components of the Sec and the Tat pathways. Our current knowledge of the function and distribution of these components and their corresponding pathways in organisms of the three domains of life is reviewed. On the basis of this information, the evolution of protein translocation is discussed.Annual Review of Microbiology 02/2005; 59:91-111. · 14.35 Impact Factor -
Article: Translocation of proteins across archaeal cytoplasmic membranes.
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ABSTRACT: All cells need to transport proteins across hydrophobic membranes. Several mechanisms have evolved to facilitate this transport, including: (i) the universally-conserved Sec system, which transports proteins in an unfolded conformation and is thought to be the major translocation pathway in most organisms and (ii) the Tat system, which transports proteins that have already obtained some degree of tertiary structure. Here, we present the current understanding of these processes in the domain Archaea, and how they compare to the corresponding pathways in bacteria and eukaryotes.FEMS Microbiology Reviews 03/2004; 28(1):3-24. · 10.96 Impact Factor -
Article: Prokaryotic utilization of the twin-arginine translocation pathway: a genomic survey.
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ABSTRACT: The twin-arginine translocation (Tat) pathway, which has been identified in plant chloroplasts and prokaryotes, allows for the secretion of folded proteins. However, the extent to which this pathway is used among the prokaryotes is not known. By using a genomic approach, a comprehensive list of putative Tat substrates for 84 diverse prokaryotes was established. Strikingly, the results indicate that the Tat pathway is utilized to highly varying extents. Furthermore, while many prokaryotes use this pathway predominantly for the secretion of redox proteins, analyses of the predicted substrates suggest that certain bacteria and archaea secrete mainly nonredox proteins via the Tat pathway. While no correlation was observed between the number of Tat machinery components encoded by an organism and the number of predicted Tat substrates, it was noted that the composition of this machinery was specific to phylogenetic taxa.Journal of Bacteriology 03/2003; 185(4):1478-83. · 3.83 Impact Factor -
Article: Adaptation of protein secretion to extremely high-salt conditions by extensive use of the twin-arginine translocation pathway.
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ABSTRACT: Halophilic archaea thrive in environments with salt concentrations approaching saturation. However, little is known about the way in which these organisms stabilize their secreted proteins in such 'hostile' conditions. Here, we present data suggesting that the utilization of protein translocation pathways for protein secretion by the Halobacteriaceae differs significantly from that of non-haloarchaea, and most probably represents an adaptation to the high-salt environment. Although most proteins are secreted via the general secretion (Sec) machinery, the twin-arginine translocation (Tat) pathway is mainly used for the secretion of redox proteins and is distinct from the Sec pathway, in that it allows cytoplasmic folding of secreted proteins. tatfind (developed in this study) was used for systematic whole-genome analysis of Halobacterium sp. NRC-1 and several other prokaryotes to identify putative Tat substrates. Our analyses revealed that the vast majority of haloarchaeal secreted proteins were predicted substrates of the Tat pathway. Strikingly, most of these putative Tat substrates were non-redox proteins, the homologues of which in non-haloarchaea were identified as putative Sec substrates. We confirmed experimentally that the secretion of one such putative Tat substrate depended on the twin-arginine motif in its signal sequence. This extensive utilization of the Tat pathway in haloarchaea suggests an evolutionary adaptation to high-salt conditions by allowing cytoplasmic folding of secreted proteins before their secretion.Molecular Microbiology 09/2002; 45(4):943-50. · 5.01 Impact Factor -
Article: In vivo analysis of an essential archaeal signal recognition particle in its native host.
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ABSTRACT: The evolutionarily conserved signal recognition particle (SRP) plays an integral role in Sec-mediated cotranslational protein translocation and membrane protein insertion, as it has been shown to target nascent secretory and membrane proteins to the bacterial and eukaryotic translocation pores. However, little is known about its function in archaea, since characterization of the SRP in this domain of life has thus far been limited to in vitro reconstitution studies of heterologously expressed archaeal SRP components identified by sequence comparisons. In the present study, the genes encoding the SRP54, SRP19, and 7S RNA homologs (hv54h, hv19h, and hv7Sh, respectively) of the genetically and biochemically tractable archaeon Haloferax volcanii were cloned, providing the tools to analyze the SRP in its native host. As part of this analysis, an hv54h knockout strain was created. In vivo characterization of this strain revealed that the archaeal SRP is required for viability, suggesting that cotranslational protein translocation is an essential process in archaea. Furthermore, a method for the purification of this SRP employing nickel chromatography was developed in H. volcanii, allowing the successful copurification of (i) Hv7Sh with a histidine-tagged Hv54h, as well as (ii) Hv54h and Hv7Sh with a histidine-tagged Hv19h. These results provide the first in vivo evidence that these components interact in archaea. Such copurification studies will provide insight into the significance of the similarities and differences of the protein-targeting systems of the three domains of life, thereby increasing knowledge about the recognition of translocated proteins in general.Journal of Bacteriology 07/2002; 184(12):3260-7. · 3.83 Impact Factor
Top co-authors
Top Journals
Institutions
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2002–2011
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University of Pennsylvania
- Department of Biology
Philadelphia, PA, USA
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2007
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University of Groningen
- Groningen Biomolecular Sciences and Biotechnology Institute (GBB)
Groningen, Province of Groningen, Netherlands
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