scholarly journals Mitochondrial outer and inner membrane fusion requires a modified carrier protein

2009 ◽  
Vol 184 (4) ◽  
pp. 569-581 ◽  
Author(s):  
Suzanne Hoppins ◽  
Jennifer Horner ◽  
Cheng Song ◽  
J. Michael McCaffery ◽  
Jodi Nunnari
Keyword(s):  
Functional Analysis ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Outer Membrane Protein ◽  
Carrier Protein ◽  
Inner Membrane ◽  
The Third ◽  
Membrane Tethering ◽  
Related Proteins

In yeast, three proteins are essential for mitochondrial fusion. Fzo1 and Mgm1 are conserved guanosine triphosphatases that reside in the outer and inner membranes, respectively. At each membrane, these conserved proteins are required for the distinct steps of membrane tethering and lipid mixing. The third essential component is Ugo1, an outer membrane protein in the mitochondrial transport protein family. We show that Ugo1 is a modified member of this family, containing three transmembrane domains and existing as a dimer, a structure that is critical for the fusion function of Ugo1. Our functional analysis of Ugo1 indicates that it is required distinctly for both outer and inner membrane fusion after membrane tethering, indicating that it operates at the lipid-mixing step of fusion. This role is distinct from the fusion dynamin-related proteins and thus demonstrates that at each membrane, a single fusion protein is not sufficient to drive the lipid-mixing step, but instead, this step requires a more complex assembly of proteins.

scholarly journals Assembly and Channel Opening of Outer Membrane Protein in Tripartite Drug Efflux Pumps of Gram-negative Bacteria

2012 ◽  
Vol 287 (15) ◽  
pp. 11740-11750 ◽  
Author(s):  
Yongbin Xu ◽  
Arne Moeller ◽  
So-Young Jun ◽  
Minho Le ◽  
Bo-Young Yoon ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Membrane Fusion Protein ◽  
Docking Model ◽  
Channel Opening

Gram-negative bacteria are capable of expelling diverse xenobiotic substances from within the cell by use of three-component efflux pumps in which the energy-activated inner membrane transporter is connected to the outer membrane channel protein via the membrane fusion protein. In this work, we describe the crystal structure of the membrane fusion protein MexA from the Pseudomonas aeruginosa MexAB-OprM pump in the hexameric ring arrangement. Electron microscopy study on the chimeric complex of MexA and the outer membrane protein OprM reveals that MexA makes a tip-to-tip interaction with OprM, which suggests a docking model for MexA and OprM. This docking model agrees well with genetic results and depicts detailed interactions. Opening of the OprM channel is accompanied by the simultaneous exposure of a protein structure resembling a six-bladed cogwheel, which intermeshes with the complementary cogwheel structure in the MexA hexamer. Taken together, we suggest an assembly and channel opening model for the MexAB-OprM pump. This study provides a better understanding of multidrug resistance in Gram-negative bacteria.

scholarly journals Coexpression of MmpS5 and MmpL5 Contributes to Both Efflux Transporter MmpL5 Trimerization and Drug Resistance in Mycobacterium tuberculosis

mSphere ◽  
2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Kentaro Yamamoto ◽  
Noboru Nakata ◽  
Tetsu Mukai ◽  
Ikuro Kawagishi ◽  
Manabu Ato
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Membrane Protein ◽  
Fusion Protein ◽  
Membrane Fusion ◽  
Single Molecule ◽  
Efflux Transporter ◽  
Inner Membrane ◽  
Content Type ◽  
Membrane Fusion Protein ◽  
Resistance Nodulation Division

ABSTRACT The increasing occurrence of multidrug-resistant Mycobacterium tuberculosis (Mtb) is a serious threat to global public health. Among the many mechanisms of drug resistance, only resistance-nodulation-division (RND)-type multidrug efflux systems can simultaneously render bacteria tolerant to numerous toxic compounds, including antibiotics. The elevated expression of RND-type xenobiotic efflux transporter complexes, which consist of an inner membrane transporter, membrane fusion protein, and outer membrane channel, plays a major role in multidrug resistance. Among the 14 mycobacterial membrane protein large (MmpL) proteins identified as inner membrane transporters of Mtb, MmpL5 is known to participate in the acquisition of resistance to bedaquiline and clofazimine. MmpL5 exports these drugs by forming a complex with the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5). However, the role of MmpS5 in the efflux of antituberculous drugs by MmpL5 remains unclear. In this study, we focused on the in vivo dynamics of MmpL5 using green fluorescent protein (GFP). Single-molecule observations of MmpL5 showed substantial lateral displacements of MmpL5-GFP without the expression of MmpS5. Nondiffusing MmpL5-GFP foci typically showed three-step photobleaching, suggesting that MmpL5 formed a homotrimeric functional complex on the inner membrane in the presence of MmpS5. These results suggest that the expression of MmpS5 facilitates the assembly of monomeric MmpL5 into a homotrimer that is anchored to the inner membrane to transport various antimycobacterial drugs. IMPORTANCE It has been reported that mycobacterial membrane protein large 5 (MmpL5), a resistance-nodulation-division (RND)-type inner membrane transporter in Mycobacterium tuberculosis (Mtb), is involved in the transport of antimycobacterial drugs. However, the functional roles of the membrane fusion protein mycobacterial membrane protein small 5 (MmpS5), organized as an operon with MmpL5, are unclear. Via the single-molecule imaging of MmpL5, we uncovered the maintenance of the functional trimeric complex structure of MmpL5 in the presence of MmpS5. These findings demonstrate that the assembly mechanisms of mycobacterial RND efflux systems are the dynamically regulated process through interactions among the components. This represents the first report of the single-molecule observation of Mtb efflux transporters, which may enhance our understanding of innate antibiotic resistance.

scholarly journals Opening of the Outer Membrane Protein Channel in Tripartite Efflux Pumps Is Induced by Interaction with the Membrane Fusion Partner

2010 ◽  
Vol 286 (7) ◽  
pp. 5484-5493 ◽  
Author(s):  
Thamarai K. Janganan ◽  
Li Zhang ◽  
Vassiliy N. Bavro ◽  
Dijana Matak-Vinkovic ◽  
Nelson P. Barrera ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Membrane Fusion ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Efflux Pumps ◽  
Fusion Partner ◽  
Protein Channel

scholarly journals Epitope Mapping of the Outer Membrane Protein P5-Homologous Fimbrin Adhesin of Nontypeable Haemophilus influenzae

2000 ◽  
Vol 68 (4) ◽  
pp. 2119-2128 ◽  
Author(s):  
Laura A. Novotny ◽  
Joseph A. Jurcisek ◽  
Michael E. Pichichero ◽  
Lauren O. Bakaletz
Keyword(s):  
Membrane Protein ◽  
Haemophilus Influenzae ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Interaction Analysis ◽  
Immune Recognition ◽  
Mature Protein ◽  
The Third ◽  
Binding Domains ◽  
Adherence Inhibition

ABSTRACT To identify potential immunodominant and/or adhesin binding domains of the outer membrane protein P5-homologous fimbrin adhesin of nontypeable Haemophilus influenzae (NTHI), three sets of synthetic peptides were synthesized and assayed in an adherence inhibition assay, by Western blotting, and in a biomolecular interaction analysis (BIA) system. The first series of 34 8- to 10-mer peptides represented the entire mature protein sequentially. The second set of four peptides (each 19 to 28 residues) represented the four predicted major surface-exposed regions (or loops) of this adhesin. The third series of seven peptides (each 27 to 34 residues) were specifically designed to map the third surface-exposed region. Data obtained by BIA indicated limited reactivity of a panel of high-titered immune chinchilla sera to the 8- to 10-mer peptides representing the mature protein, likely because these linear peptides did not represent continuous epitopes. However, several of these short peptides did inhibit adherence of multiple NTHI strains to a human respiratory epithelial cell. Overall, greatest relative reactivity in both BIA and adherence inhibition assays was demonstrated against, or shown by, peptides mapping to the third and fourth predicted surface-exposed regions of this adhesin, thereby indicating the presence of immunodominant and adhesin binding domains at these sites. Middle ear fluids sequentially recovered from a chinchilla with an ongoing NTHI-induced otitis media (OM) as well as sera from children with OM due to NTHI also reacted exclusively with peptides representing the third and fourth surface-exposed regions of the P5-fimbrin adhesin, indicating a similarity in immune recognition of this bacterial protein by these two hosts. Collectively, these data together with the previously demonstrated protective efficacy of immunogens derived from this adhesin in chinchilla models support the continued development of P5-fimbrin based vaccine components.

scholarly journals Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197Conjugate Vaccine

2011 ◽  
Vol 18 (5) ◽  
pp. 724-729 ◽  
Author(s):  
Zengzu Lai ◽  
John R. Schreiber
Keyword(s):  
T Cell ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Conjugate Vaccine ◽  
Outer Membrane Protein ◽  
Protein Complex ◽  
Carrier Protein ◽  
Conjugate Vaccines ◽  
Membrane Protein Complex ◽  
Content Type

ABSTRACTBacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. TheHaemophilus influenzaetype b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM197conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM197conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

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