YscP and YscU Regulate Substrate Specificity of the<i>Yersinia</i>Type III Secretion System

Edqvist, Petra J.; Olsson, Jan; Lavander, Moa; Sundberg, Lena; Forsberg, Åke; Wolf‐Watz, Hans; Lloyd, Scott A.

doi:10.1128/jb.185.7.2259-2266.2003

Cited by 112 publications

(152 citation statements)

References 51 publications

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“…Type III virulence homologues of flhB apparently play a similar role in switching export specificity from needle-type substrates to later substrates. In Yersinia pseudotuberculosis, mutations in the cytoplasmic domain of the inner membrane protein YscU (FlhB homologue) suppress the yscP (fliK homologue) polyneedle phenotype (39). In addition, it has been shown that specific cleavage occurs at the equivalent site (Asn-263/Pro-264) of YscU, and mutations at this site have complex effects on late substrate secretion and growth (40).…”

Section: Discussionmentioning

confidence: 99%

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

Ferris

Furukawa²,

Minamino

et al. 2005

Journal of Biological Chemistry

130

138

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The bacterial flagellum is a predominantly cell-external supermacromolecular construction whose structural components are exported by a flagellum-specific export apparatus. One of the export apparatus proteins, FlhB, regulates the substrate specificity of the entire apparatus; i.e. it has a role in the ordered export of the two main groups of flagellar structural proteins such that the cell-proximal components (rod-/hook-type proteins) are exported before the cell-distal components (filament-type proteins). The controlled switch between these two export states is believed to be mediated by conformational changes in the structure of the C-terminal cytoplasmic domain of FlhB (FlhB C ), which is consistently and specifically cleaved into two subdomains (FlhB CN and FlhB CC ) that remain tightly associated with each other. The cleavage event has been shown to be physiologically significant for the switch. In this study, the mechanism of FlhB cleavage has been more directly analyzed. We demonstrate that cleavage occurs in a heterologous host, Saccharomyces cerevisiae, deficient in vacuolar proteinases A and B. In addition, we find that cleavage of a slow-cleaving variant, FlhB C (P270A), is stimulated in vitro at alkaline pH. We also show by analytical gel-filtration chromatography and analytical ultracentrifugation experiments that both FlhB C and FlhB C (P270A) are monomeric in solution, and therefore self-proteolysis is unlikely. Finally, we provide evidence via peptide analysis and FlhB cleavage variants that the tertiary structure of FlhB plays a significant role in cleavage. Based on these results, we propose that FlhB cleavage is an autocatalytic process.A large percentage of the bacterial flagellar structure lies outside of the cell envelope, thus requiring that the vast majority of the subunits that compose the flagellum be exported from the cytosol across both the inner and outer membranes. Salmonella employs a type III export pathway to accomplish this (1, 2). It is a Sec-independent pathway that utilizes a flagellum-specific export apparatus to transport flagellar components across the inner membrane. These exported proteins then travel the length of the developing flagellum within an interior channel prior to their incorporation at the structure's cell-distal end (3-6) (the developing structure therefore facilitates export across the outer membrane). At least nine flagellar proteins are involved in the flagellumspecific export apparatus (7). Six are integral membrane components (FlhA, FlhB, FliO, FliP, FliQ, and FliR) postulated to be located within the basal body MS ring (8, 9), and three are soluble components: an ATPase (FliI) that drives export (10), a regulator of the ATPase (FliH) (11-13), and a general chaperone (FliJ) (14, 15).One of the integral membrane proteins, FlhB, has been found to play a central role in export substrate-specificity switching; i.e. regulation of the order in which flagellar subunits are exported, such that proteins incorporated into the cell-proximal rod and hook structure...

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Section: Discussionmentioning

confidence: 99%

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

Ferris

Furukawa²,

Minamino

et al. 2005

Journal of Biological Chemistry

130

138

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“…[8][9][10] Therefore, it has been proposed that autocleavage of the YscU cytoplasmic domain results in a conformational change that triggers the recognition and export of translocators at the proper time during the assembly of the type III secretion apparatus. 9,[11][12][13] We report here the crystal structures of two noncleaved forms of YscU, YscU N263A and YscU N263A/ P264A , and an atomic resolution structure of the wildtype cleaved form, YscU cleaved , to elucidate structural changes that occur upon cleavage. These crystallographic studies reveal the conformational changes induced upon auto-cleavage of YscU and provide structural insights into the cleavage mechanism, at very high resolution, that confirm and extend the information gleaned from structures of YscU homologs from Shigella flexneri (Spa40), enteropathogenic Escherichia coli (EscU) and Salmonella typhimurium (SpaS).…”

Section: Introductionmentioning

confidence: 99%

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

et al. 2009

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Crystal structures of cleaved and uncleaved forms of the YscU cytoplasmic domain, an essential component of the type III secretion system (T3SS) in Yersinia pestis, have been solved by single-wavelength anomolous dispersion and refined with X-ray diffraction data extending up to atomic resolution (1.13 Å ). These crystallographic studies provide structural insights into the conformational changes induced upon auto-cleavage of the cytoplasmic domain of YscU. The structures indicate that the cleaved fragments remain bound to each other. The conserved NPTH sequence that contains the site of the N263-P264 peptide bond cleavage is found on a b-turn which, upon cleavage, undergoes a major reorientation of the loop away from the catalytic N263, resulting in altered electrostatic surface features at the site of cleavage. Additionally, a significant conformational change was observed in the N-terminal linker regions of the cleaved and noncleaved forms of YscU which may correspond to the molecular switch that influences substrate specificity. The YscU structures determined here also are in good agreement with the auto-cleavage mechanism described for the flagellar homolog FlhB and E. coli EscU.

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“…This hypothesis is corroborated by the finding that single point mutations in the C-terminal regions of FlhB, YscU, and homologs can restore the wild-type phenotype in T3S4 mutants of S. Typhimurium, Y. pseudotuberculosis, EPEC, and X. campestris pv. vesicatoria (148,298,330,602,626) (Table 7). It is assumed that the introduction of point mutations into the C-terminal domains of YscU/FlhB family members leads to a conformational change that is permissive for the substrate specificity switch.…”

Section: T3s4 Proteins and Their Interplay With Yscu/flhb Family Membersmentioning

confidence: 99%

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

Büttner

2012

Microbiol Mol Biol Rev

363

482

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SUMMARY Flagellar and translocation-associated type III secretion (T3S) systems are present in most Gram-negative plant- and animal-pathogenic bacteria and are often essential for bacterial motility or pathogenicity. The architectures of the complex membrane-spanning secretion apparatuses of both systems are similar, but they are associated with different extracellular appendages, including the flagellar hook and filament or the needle/pilus structures of translocation-associated T3S systems. The needle/pilus is connected to a bacterial translocon that is inserted into the host plasma membrane and mediates the transkingdom transport of bacterial effector proteins into eukaryotic cells. During the last 3 to 5 years, significant progress has been made in the characterization of membrane-associated core components and extracellular structures of T3S systems. Furthermore, transcriptional and posttranscriptional regulators that control T3S gene expression and substrate specificity have been described. Given the architecture of the T3S system, it is assumed that extracellular components of the secretion apparatus are secreted prior to effector proteins, suggesting that there is a hierarchy in T3S. The aim of this review is to summarize our current knowledge of T3S system components and associated control proteins from both plant- and animal-pathogenic bacteria.

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YscP and YscU Regulate Substrate Specificity of theYersiniaType III Secretion System

Abstract: Pathogenic Yersinia species use a type III secretion system to inhibit phagocytosis by eukaryotic cells. At 37°C, the secretion system is assembled, forming a needle-like structure on the bacterial cell surface.

Cited by 112 publications

References 51 publications

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

FlhB Regulates Ordered Export of Flagellar Components via Autocleavage Mechanism

Atomic resolution structure of the cytoplasmic domain of Yersinia pestis YscU, a regulatory switch involved in type III secretion

Protein Export According to Schedule: Architecture, Assembly, and Regulation of Type III Secretion Systems from Plant- and Animal-Pathogenic Bacteria

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