scholarly journals Membrane Association and Multimerization of Secreton Component PulC

1999 ◽  
Vol 181 (13) ◽  
pp. 4004-4011 ◽  
Author(s):  
Odile M. Possot ◽  
Manon Gérard-Vincent ◽  
Anthony P. Pugsley
Keyword(s):  
Outer Membrane ◽  
Klebsiella Oxytoca ◽  
Subcellular Fractionation ◽  
Whole Cells ◽  
Outer Membranes ◽  
Specific Serum ◽  
Signal Anchor ◽  
Anchor Sequence ◽  
Exoprotein Secretion

ABSTRACT The PulC component of the Klebsiella oxytocapullulanase secretion machinery (the secreton) was found by subcellular fractionation to be associated with both the cytoplasmic (inner) and outer membranes. Association with the outer membrane was independent of other secreton components, including the outer membrane protein PulD (secretin). The association of PulC with the inner membrane is mediated by the signal anchor sequence located close to its N terminus. These results suggest that PulC forms a bridge between the two membranes that is disrupted when bacteria are broken open for fractionation. Neither the signal anchor sequence nor the cytoplasmic N-terminal region that precedes it was found to be required for PulC function, indicating that PulC does not undergo sequence-specific interactions with other cytoplasmic membrane proteins. Cross-linking of whole cells resulted in the formation of a ca. 110-kDa band that reacted with PulC-specific serum and whose detection depended on the presence of PulD. However, antibodies against PulD failed to react with this band, suggesting that it could be a homo-PulC trimer whose formation requires PulD. The data are discussed in terms of the possible role of PulC in energy transduction for exoprotein secretion.

scholarly journals Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence.

1993 ◽  
Vol 268 (34) ◽  
pp. 25265-25268 ◽  
Author(s):  
M Nguyen ◽  
D G Millar ◽  
V W Yong ◽  
S J Korsmeyer ◽  
G C Shore
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Signal Anchor ◽  
Anchor Sequence

scholarly journals XpsG, the major pseudopilin in Xanthomonas campestris pv. campestris, forms a pilus-like structure between cytoplasmic and outer membranes

2002 ◽  
Vol 365 (1) ◽  
pp. 205-211 ◽  
Author(s):  
Nien-Tai HU ◽  
Wei-Ming LEU ◽  
Meng-Shiunn LEE ◽  
Avon CHEN ◽  
Shu-Chung CHEN ◽  
...  
Keyword(s):  
Xanthomonas Campestris ◽  
Klebsiella Oxytoca ◽  
Subcellular Fractionation ◽  
Soluble Form ◽  
Wild Type ◽  
Outer Membranes ◽  
Extracellular Milieu ◽  
Large Complex ◽  
Xanthomonas Campestris Pv Campestris

GspG, -H, -I, -J and -K proteins are members of the pseudopilin family. They are the components required for the type II secretion pathway, which translocates proteins across the outer membrane of Gram-negative bacteria to the extracellular milieu. They were predicted to form a pilus-like structure, and this has been shown for PulG of Klebsiella oxytoca by using electron microscopy. In the present study, we performed biochemical analyses of the XpsG protein of Xanthomonas campestris pv. campestris and observed that it is a pillar-like structure spanning the cytoplasmic and outer membranes. Subcellular fractionation revealed a soluble form (SF) of XpsG, in addition to the membrane form. Chromatographic analysis of SF XpsG in the absence of a detergent indicated that it is part of a large complex (>440kDa). In vitro studies indicated that XpsG is prone to aggregate in the absence of a detergent. We isolated and characterized a non-functional mutant defective in forming the large complex. It did not interfere with the function of wild-type XpsG and was not detectable in the SF. Moreover, unlike wild-type XpsG, which was distributed in both the cytoplasmic and outer membranes, it appeared only in the cytoplasmic membrane. When wild-type XpsG was co-expressed with His6-tagged XpsH but not with untagged XpsH, SF XpsG bound to nickel and co-eluted with XpsH. This result suggests the presence of other pseudopilin components in the XpsG-containing large-sized molecules.

An Artificial Mitochondrial Tail Signal/Anchor Sequence Confirms a Requirement for Moderate Hydrophobicity for Targeting

2007 ◽  
Vol 27 (6) ◽  
pp. 385-401 ◽  
Author(s):  
Binks W. Wattenberg ◽  
Denise Clark ◽  
Stephanie Brock
Keyword(s):  
Amino Acids ◽  
Endoplasmic Reticulum ◽  
Outer Membrane ◽  
Helical Structure ◽  
Mitochondrial Outer Membrane ◽  
Carboxy Terminus ◽  
Charged Amino Acids ◽  
Hydrophobic Amino Acids ◽  
Signal Anchor ◽  
Anchor Sequence

Tail-anchored proteins are a group of membrane proteins oriented with their amino terminus in the cytoplasm and their carboxy terminus embedded in intracellular membranes. This group includes the apoptosis-mediating proteins of the Bcl-2 family as well as the vesicle targeting proteins of the SNARE group, among others. A stretch of hydrophobic amino acids at the extreme carboxy terminus of these proteins serves both as a membrane anchor and as a targeting signal. Tail-anchored proteins are differentially targeted to either the endoplasmic reticulum or the mitochondrial outer membrane and the mechanism which accomplishes this selective targeting is poorly understood. Here we define important characteristics of the signal/anchor region which directs proteins to the mitochondrial outer membrane. We have created an artificial sequence consisting of a stretch of 16 leucines bounded by positively charged amino acids. Using this template we demonstrate that moderate hydrophobicity distinguishes the mitochondrial tail-anchor sequence from that of the endoplasmic reticulum tail-anchor sequence. A change as small as introduction of a single polar residue into a sequence that otherwise targets to the endoplasmic reticulum can substantially switch targeting to the mitochondrial outer membrane. Further we show that a mitochondrially targeted tail-anchor has a higher propensity for the formation of alpha-helical structure than a sequence directing tail-anchored proteins to the endoplasmic reticulum.

scholarly journals The Peripheral Membrane Subunits of the SAM Complex Function Codependently in Mitochondrial Outer Membrane Biogenesis

2008 ◽  
Vol 19 (1) ◽  
pp. 126-136 ◽  
Author(s):  
Nickie C. Chan ◽  
Trevor Lithgow
Keyword(s):  
Outer Membrane ◽  
Mitochondrial Protein ◽  
Complex Function ◽  
Mitochondrial Outer Membrane ◽  
Outer Membranes ◽  
Protein Substrates ◽  
Complex Functions ◽  
Outer Membrane Biogenesis ◽  
Substrate Proteins

The sorting and assembly machinery (SAM) complex functions in the assembly of β-barrel proteins into the mitochondrial outer membrane. It is related to the Omp85/YaeT machinery in bacterial outer membranes, but the eukaryotic SAM complex is distinguished by two peripheral subunits, Sam37 and Sam35, that sit on the cytosolic face of the complex. The function of these subunits in β-barrel protein assembly is currently unclear. By screening a library of sam35 mutants, we show that 13 distinct alleles were each specifically suppressed by overexpression of SAM37. Two of these mutants, sam35-409 and sam35-424, show distinct phenotypes that enable us to distinguish the function of Sam35 from that of Sam37. Sam35 is required for the SAM complex to bind outer membrane substrate proteins: destabilization of Sam35 inhibits substrate binding by Sam50. Sam37 acts later than Sam35, apparently to assist release of substrates from the SAM complex. Very different environments surround bacteria and mitochondria, and we discuss the role of Sam35 and Sam37 in terms of the problems peculiar to mitochondrial protein substrates.

scholarly journals A signal-anchor sequence selective for the mitochondrial outer membrane.

1992 ◽  
Vol 119 (6) ◽  
pp. 1451-1457 ◽  
Author(s):  
H M McBride ◽  
D G Millar ◽  
J M Li ◽  
G C Shore
Keyword(s):  
Amino Acids ◽  
Outer Membrane ◽  
Mitochondrial Outer Membrane ◽  
Hybrid Protein ◽  
Transmembrane Segment ◽  
Structural Domains ◽  
Signal Anchor ◽  
Anchor Sequence ◽  
Rate Limiting

pOMD29 is a hybrid protein containing the NH2-terminal topogenic sequence of a bitopic, integral protein of the outer mitochondrial membrane in yeast, OMM70, fused to dihydrofolate reductase. The topogenic sequence consists of two structural domains: an NH2-terminal basic region (amino acids 1-10) and an apolar region which is the predicted transmembrane segment (amino acids 11-29). The transmembrane segment alone was capable of targeting and inserting the hybrid protein into the outer membrane of intact mitochondria from rat heart in vitro. The presence of amino acids 1-10 enhanced the rate of import, and this increased rate depended, in part, on the basic amino acids located at positions 2, 7, and 9. Deletion of a large portion of the transmembrane segment (amino acids 16-29) resulted in a protein that exhibited negligible import in vitro. Insertion of pOMD29 into the outer membrane was not competed by import of excess precursor protein destined for the mitochondrial matrix, indicating that the two proteins may have different rate-limiting steps during import. We propose that the structural domains within amino acids 1-29 of pOMD29 cooperate to form a signal-anchor sequence, the characteristics of which suggest a model for proper sorting to the mitochondrial outer membrane.

On the Role of Transferrin in 67Ga Uptake by Tumor Cells

1988 ◽  
Vol 27 (04) ◽  
pp. 151-153
Author(s):  
P. Thouvenot ◽  
F. Brunotte ◽  
J. Robert ◽  
L. J. Anghileri
Keyword(s):  
Specific Binding ◽  
Subcellular Fractionation ◽  
Animal Blood ◽  
Tumor Bearing ◽  
Cell Structures ◽  
The Difference ◽  
Sarcoma Cells ◽  
In Vitro Uptake

In vitro uptake of 67Ga-citrate and 59Fe-citrate by DS sarcoma cells in the presence of tumor-bearing animal blood plasma showed a dramatic inhibition of both 67Ga and 59Fe uptakes: about ii/io of 67Ga and 1/5o of the 59Fe are taken up by the cells. Subcellular fractionation appears to indicate no specific binding to cell structures, and the difference of binding seems to be related to the transferrin chelation and transmembrane transport differences

A ROLE OF PROTEINS OF THE OUTER MEMBRANE OF Shewanella oneidensis MR-1 BACTERIA IN FORMATION AND STABILIZATION OF SILVER SULFIDE NANOPARTICLES

2015 ◽  
pp. 41-48 ◽  
Author(s):  
T. A. Voeikova ◽  
A. S. Shebanova ◽  
Yu. D. Ivanov ◽  
A. L. Kaysheva ◽  
L. M. Novikova ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Shewanella Oneidensis ◽  
Silver Sulfide ◽  
Silver Sulfide Nanoparticles

scholarly journals Dissecting the contribution of O-Antigen and proteins to the immunogenicity of Shigella sonnei generalized modules for membrane antigens (GMMA)

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Francesca Mancini ◽  
Gianmarco Gasperini ◽  
Omar Rossi ◽  
Maria Grazia Aruta ◽  
Maria Michelina Raso ◽  
...  
Keyword(s):  
Protective Immunity ◽  
Critical Role ◽  
Shigella Sonnei ◽  
Added Value ◽  
Protein Antigens ◽  
Outer Membranes ◽  
O Antigen ◽  
Membrane Antigens ◽  
Specific Igg

AbstractGMMA are exosomes released from engineered Gram-negative bacteria resembling the composition of outer membranes. We applied the GMMA technology for the development of an O-Antigen (OAg) based vaccine against Shigella sonnei, the most epidemiologically relevant cause of shigellosis. S. sonnei OAg has been identified as a key antigen for protective immunity, and GMMA are able to induce anti-OAg-specific IgG response in animal models and healthy adults. The contribution of protein-specific antibodies induced upon vaccination with GMMA has never been fully elucidated. Anti-protein antibodies are induced in mice upon immunization with either OAg-negative and OAg-positive GMMA. Here we demonstrated that OAg chains shield the bacteria from anti-protein antibody binding and therefore anti-OAg antibodies were the main drivers of bactericidal activity against OAg-positive bacteria. Interestingly, antibodies that are not targeting the OAg are functional against OAg-negative bacteria. The immunodominant protein antigens were identified by proteomic analysis. Our study confirms a critical role of the OAg on the immune response induced by S. sonnei GMMA. However, little is known about OAg length and density regulation during infection and, therefore, protein exposure. Hence, the presence of protein antigens on S. sonnei GMMA represents an added value for GMMA vaccines compared to other OAg-based formulations.

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