scholarly journals Characterization of Transmembrane Segments 3, 4, and 5 of MalF by Mutational Analysis

2001 ◽  
Vol 183 (1) ◽  
pp. 375-381 ◽  
Author(s):  
Angelika Steinke ◽  
Sandra Grau ◽  
Amy Davidson ◽  
Eckhard Hofmann ◽  
Michael Ehrmann
Keyword(s):  
Structural Model ◽  
Cytoplasmic Membrane ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Transport Channel ◽  
Transmembrane Segments ◽  
Maltose Transporter ◽  
Membrane Spanning ◽  
Membrane Components

ABSTRACT MalF and MalG are the cytoplasmic membrane components of the binding protein-dependent ATP binding cassette maltose transporter inEscherichia coli. They are thought to form the transport channel and are thus of critical importance for the mechanism of transport. To study the contributions of individual transmembrane segments of MalF, we isolated 27 point mutations in membrane-spanning segments 3, 4, and 5. These data complement a previous study, which described the mutagenesis of membrane-spanning segments 6, 7, and 8. While most of the isolated mutations appear to cause assembly defects, L323Q in helix 5 could interfere more directly with substrate specificity. The phenotypes and locations of the mutations are consistent with a previously postulated structural model of MalF.

scholarly journals Interaction and localization studies of enteropathogenic Escherichia coli type IV bundle-forming pilus outer membrane components

Microbiology ◽  
2006 ◽  
Vol 152 (8) ◽  
pp. 2405-2420 ◽  
Author(s):  
Anu Daniel ◽  
Aparna Singh ◽  
Lynette J. Crowther ◽  
Paula J. Fernandes ◽  
Wiebke Schreiber ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Outer Membrane ◽  
Cytoplasmic Membrane ◽  
Complete Characterization ◽  
Type Iv Pili ◽  
Amino Terminus ◽  
Type Iv ◽  
Membrane Components ◽  
Nucleotide Binding Proteins

Typical enteropathogenic Escherichia coli strains express an established virulence factor belonging to the type IV pili family, called the bundle-forming pilus (BFP). BFP are present on the cell surface as bundled filamentous appendages, and are assembled and retracted by proteins encoded by the bfp operon. These proteins assemble to form a molecular machine. The BFP machine may be conceptually divided into three components: the cytoplasmic membrane (CM) subassembly, which is composed of CM proteins and cytoplasmic nucleotide-binding proteins; the outer membrane (OM) subassembly and the pilus itself. The authors have previously characterized the CM subassembly and the pilus. In this study, a more complete characterization of the OM subassembly was carried out using a combination of biochemical, biophysical and genetic approaches. It is reported that targeting of BfpG to the OM was influenced by the secretin BfpB. BfpG and BfpU interacted with the amino terminus of BfpB. BfpU had a complex cellular distribution pattern and, along with BfpB and BfpG, was part of the OM subassembly.

scholarly journals The FtsH Protein Accumulates at the Septum ofBacillus subtilis during Cell Division and Sporulation

2000 ◽  
Vol 182 (13) ◽  
pp. 3870-3873 ◽  
Author(s):  
Wolfgang Wehrl ◽  
Michael Niederweis ◽  
Wolfgang Schumann
Keyword(s):  
Bacillus Subtilis ◽  
Cell Division ◽  
Cytoplasmic Membrane ◽  
Point Mutations ◽  
Filamentous Growth ◽  
Coding Region ◽  
Enhanced Fluorescence ◽  
Ofbacillus Subtilis ◽  
Transmembrane Segments ◽  
Dividing Cells

ABSTRACT The ftsH gene encodes an ATP- and Zn2+-dependent metalloprotease which is anchored to the cytoplasmic membrane via two transmembrane segments in such a way that the very short amino- and the long carboxy termini are exposed to the cytoplasm. Deletion of the ftsH gene in Bacillus subtilis results in a pleiotropic phenotype such as filamentous growth. This observation prompted us to ask whether ftsH is involved in cell division. A translational fusion was constructed between the complete coding region of ftsH andgfp + the latter carrying five point mutations to obtain enhanced fluorescence. We detected that the FtsH protein accumulates in the midcell septum of dividing cells, and during sporulation first in the asymmetrically located septa of sporulating cells and later in the membrane which engulfs the forespore. These observations revealed a new function of FtsH.

scholarly journals Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis

2020 ◽  
Vol 16 (12) ◽  
pp. e1009148
Author(s):  
Hualiang Pi ◽  
Michelle L. Chu ◽  
Samuel J. Ivan ◽  
Casey J. Latario ◽  
Allen M. Toth ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Bacillus Anthracis ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Cell Envelope ◽  
Point Mutations ◽  
Rate Limiting Step ◽  
Envelope Stress ◽  
Mechanistic Understanding

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Mutational analysis of the SNF3 glucose transporter of Saccharomyces cerevisiae

1990 ◽  
Vol 10 (3) ◽  
pp. 1105-1115
Author(s):  
L Marshall-Carlson ◽  
J L Celenza ◽  
B C Laurent ◽  
M Carlson
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Glucose Transporter ◽  
Mutational Analysis ◽  
Point Mutations ◽  
Alpha Helix ◽  
Glucose Transporters ◽  
Hydrophobic Region ◽  
C Terminus ◽  
Transport Defect ◽  
Membrane Spanning

The SNF3 gene of Saccharomyces cerevisiae encodes a high-affinity glucose transporter that is homologous to mammalian glucose transporters. Point mutations affecting the function of the transporter were recovered from the genomes of four snf3 mutants and characterized. Two of the mutations introduced a charged amino acid into the first and second predicted membrane-spanning regions, respectively. The analogs of a bifunctional SNF3-lacZ fusion containing these two mutations were constructed, and the mutant fusion proteins were not localized to the plasma membrane, as judged by immunofluorescence microscopy. The third mutation produced a valine-to-isoleucine substitution in hydrophobic region 8, and the corresponding mutant fusion protein was correctly localized. The finding that this conservative change causes a transport defect is consistent with the possibility that this transmembrane region, which could exist as an amphipathic alpha-helix, forms part of the glucose channel through the membrane. The fourth snf3 allele harbored an ochre mutation midway through the coding sequence. We have also constructed mutations in the cloned SNF3 gene. A major difference between the yeast SNF3 protein and mammalian glucose transporters is the presence in the SNF3 protein of an additional 303 amino acids at the C terminus. Analysis of a series of C-terminal deletions and fusions to lacZ showed that this C-terminal region is important, but not essential, for transport function. We also report the genetic mapping of the SNF3 locus on the left arm of chromosome IV.

scholarly journals Characterization of the scrambling domain of the TMEM16 family

2017 ◽  
Vol 114 (24) ◽  
pp. 6274-6279 ◽  
Author(s):  
Sayuri Gyobu ◽  
Kenji Ishihara ◽  
Jun Suzuki ◽  
Katsumori Segawa ◽  
Shigekazu Nagata
Keyword(s):  
Plasma Membranes ◽  
Point Mutations ◽  
Transmembrane Segment ◽  
Stepping Stones ◽  
Transmembrane Segments ◽  
Outer Leaflet ◽  
Intracellular Ca ◽  
A Cell ◽  
Phospholipid Scrambling

The TMEM16 protein family has 10 members, each of which carries 10 transmembrane segments. TMEM16A and 16B are Ca2+-activated Cl− channels. Several other members, including TMEM16F, promote phospholipid scrambling between the inner and outer leaflets of a cell membrane in response to intracellular Ca2+. However, the mechanism by which TMEM16 proteins translocate phospholipids in plasma membranes remains elusive. Here we show that Ca2+-activated, TMEM16F-supported phospholipid scrambling proceeds at 4 °C. Similar to TMEM16F and 16E, seven TMEM16 family members were found to carry a domain (SCRD; scrambling domain) spanning the fourth and fifth transmembrane segments that conferred scrambling ability to TMEM16A. By introducing point mutations into TMEM16F, we found that a lysine in the fourth transmembrane segment of the SCRD as well as an arginine in the third and a glutamic acid in the sixth transmembrane segment were important for exposing phosphatidylserine from the inner to the outer leaflet. However, their role in internalizing phospholipids was limited. Our results suggest that TMEM16 provides a cleft containing hydrophilic “stepping stones” for the outward translocation of phospholipids.

Abstract 307: Structural Characterization of Calcineurin A-Calsarcin 1 Assembly

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Carlos R Koscky Paier ◽  
Alisson C Cardoso ◽  
Tatiani L Brenneli ◽  
Rodrigo V Honorato ◽  
Fábio C Gozzo ◽  
...  
Keyword(s):  
Structural Characterization ◽  
Molecular Mechanisms ◽  
Structural Model ◽  
Mutational Analysis ◽  
Structural Similarity ◽  
Dynamics Simulation ◽  
Allosteric Site ◽  
In Silico Docking ◽  
Close Proximity

Signaling by the calcium-dependent phosphatase calcineurin (Cn) plays key roles in regulating cardiac development, hypertrophy, and pathological remodeling. Cn binds to and is negatively regulated by calsarcins (CS), a family of muscle-specific proteins. However, the molecular mechanisms involved in the inhibition of Cn by CS remain unclear. Understanding the architecture and structure of Cn-CS complex is critical to unravel the regulation of Cn by CS. Here we combined biochemical assays, chemical cross-linking coupled to mass spectrometry experiments (MS/MS), mutational analysis and a modeling strategy for structural characterization of CnA-CS1 assembly. The MS/MS data obtained from the cross-linked peptides of both proteins were used to guide an in silico docking of their polypeptide models. The protein complex models with the smallest estimated binding energy were clustered according to structural similarity and submitted to molecular dynamics simulation. The interacting surface of CnA was mapped in a pocket between the 1st and 3rd α-helixes and surrounding loops, while the corresponding surface of CS1 was mapped to the carboxyterminal loops within the Leu179-Phe185, Phe195-Ser199 and Thr250-Leu264 regions. Notably, the region of CnA that interacts with CS1 was found to be located in close proximity, but not coincident, to the β-sheet 14, the main binding site for the PxIxIT sequence of NFAT. Experiments performed with several CnA (FLAG-CnA) and CS1 (myc-CS1) mutants were used to validate the structural model of the CnA-CS1 assembly. The Lys40 (CnA) and Glu254 (CS1) residues were identified as critical for the complex stability. The model that emerges from this study supports the notion that CS1 interacts with an allosteric site to inhibit the activity of CnA. Alternatively, the close proximity of the CS1 to NFAT interacting site supports an interference of CS1 on the ability of CnA to bind and activate NFAT.

scholarly journals The mechanoelectrical response of the cytoplasmic membrane of Vibrio cholerae

2013 ◽  
Vol 142 (1) ◽  
pp. 75-85 ◽  
Author(s):  
Ian Rowe ◽  
Merina Elahi ◽  
Anwar Huq ◽  
Sergei Sukharev
Keyword(s):  
Vibrio Cholerae ◽  
Cytoplasmic Membrane ◽  
Mechanosensitive Channel ◽  
Inward Rectification ◽  
Electrogenic Transport ◽  
E Coli ◽  
Esherichia Coli ◽  
Membrane Components

Persistence of Vibrio cholerae in waters of fluctuating salinity relies on the capacity of this facultative enteric pathogen to adapt to varying osmotic conditions. In an event of osmotic downshift, osmolytes accumulated inside the bacterium can be quickly released through tension-activated channels. With the newly established procedure of giant spheroplast preparation from V. cholerae, we performed the first patch-clamp characterization of its cytoplasmic membrane and compared tension-activated currents with those in Esherichia coli. Saturating pressure ramps revealed two waves of activation belonging to the ∼1-nS mechanosensitive channel of small conductance (MscS)-like channels and ∼3-nS mechanosensitive channel of large conductance (MscL)-like channels, with a pressure midpoint ratio p0.5MscS/p0.5MscL of 0.48. We found that MscL-like channels in V. cholerae present at a density three times higher than in E. coli, and yet, these vibrios were less tolerant to large osmotic downshocks. The Vibrio MscS-like channels exhibit characteristic inward rectification and subconductive states at depolarizing voltages; they also adapt and inactivate at subsaturating tensions and recover within 2 s upon tension release, just like E. coli MscS. Trehalose, a compatible internal osmolyte accumulated under hypertonic conditions, significantly shifts activation curves of both MscL- and MscS-like channels toward higher tensions, yet does not freely partition into the channel pore. Direct electrophysiology of V. cholerae offers new avenues for the in situ analysis of membrane components critical for osmotic survival and electrogenic transport in this pathogen.

The topogenic fate of the polytopic transmembrane proteins, synaptophysin and connexin, is determined by their membrane-spanning domains

1995 ◽  
Vol 108 (3) ◽  
pp. 883-894 ◽  
Author(s):  
R.E. Leube
Keyword(s):  
Endoplasmic Reticulum ◽  
Gap Junctions ◽  
De Novo ◽  
Point Mutations ◽  
Transmembrane Proteins ◽  
Transmembrane Domains ◽  
Parent Molecule ◽  
Transmembrane Segments ◽  
Genes Expression ◽  
Membrane Spanning

The synaptophysins and connexins are polytopic transmembrane proteins of similar secondary structure that accumulate as multiple homo-oligomers in specialized membrane regions, the presynaptic transmitter vesicles or gap junctions. Transfection and expression of the respective genes in cultured epithelial cells results in the de novo formation of either small cytoplasmic, synaptophysin-rich vesicles, or functional gap junctions consisting of clustered connexin molecules. To examine the molecular requirements for the specific enrichment and topogenesis of both types of molecule, chimeric cDNAs were constructed composed of different parts of the rat synaptophysin and rat liver connexin32 genes. Expression of the encoded chimeric polypeptides in hepatocellular carcinoma-derived cells showed that only chimeras with all four transmembrane domains from either parent molecule were delivered to their specific destination. In contrast, chimeras with transmembrane domains from both connexin32 and synaptophysin were always retained in the endoplasmic reticulum. The topogenic nature of the transmembrane domains was further demonstrated by deletion mutagenesis, indicating that removal of cytoplasmic end domains or intravesicular loops does not abolish targeting. On the other hand, excision of individual transmembrane domains or introduction of point mutations in transmembrane segments resulted in retention in the endoplasmic reticulum.

scholarly journals Directed evolution reveals the mechanism of HitRS signal transduction in Bacillus anthracis

2020 ◽  
Author(s):  
Hualiang Pi ◽  
Michelle L. Chu ◽  
Samuel J. Ivan ◽  
Casey J. Latario ◽  
Allen M. Toth ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Bacillus Anthracis ◽  
Mutational Analysis ◽  
Hydrophobic Interactions ◽  
Cell Envelope ◽  
Point Mutations ◽  
Rate Limiting Step ◽  
Envelope Stress ◽  
Mechanistic Understanding

AbstractBacterial two component systems (TCSs) have been studied for decades; however, most work has focused on individual domains or proteins. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that has been implicated in detecting cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. This study establishes the use of unbiased genetic selections to study TCS signaling, provides a comprehensive mechanistic understanding of an entire TCS, and lays the foundation for development of novel antimicrobial therapeutics against this important infectious threat.

scholarly journals Structural basis for functional interactions in dimers of SLC26 transporters

2019 ◽  
Author(s):  
Yung-Ning Chang ◽  
Eva A. Jaumann ◽  
Katrin Reichel ◽  
Julia Hartmann ◽  
Dominik Oliver ◽  
...  
Keyword(s):  
Structural Model ◽  
Lipid Membrane ◽  
Md Simulations ◽  
Structural Basis ◽  
Distance Measurements ◽  
Transmembrane Segments ◽  
The Family ◽  
Stas Domain ◽  
Functional Relevance

AbstractThe SLC26 family of transporters maintains anion equilibria in all kingdoms of life. The family shares a 7 + 7 transmembrane segments inverted repeat architecture with the SLC4 and SLC23 families, but holds a regulatory STAS domain in addition. While the only experimental SLC26 structure is monomeric, SLC26 proteins form structural and functional dimers in the lipid membrane. Here we resolve the structure of an SLC26 dimer embedded in a lipid membrane and characterize its functional relevance by combining PELDOR distance measurements and biochemical studies with MD simulations and spin-label ensemble refinement. Our structural model reveals a unique interface different from the SLC4 and SLC23 families. The functionally relevant STAS domain exerts a stabilizing effect on regions central in this dimer. Characterization of heterodimers indicates that protomers in the dimer functionally interact. The combined structural and functional data define the framework for a mechanistic understanding of functional cooperativity in SLC26 dimers.

Sign in / Sign up

Export Citation Format

Share Document