Interaction of PomB with the Third Transmembrane Segment of PomA in the Na+-Driven Polar Flagellum of Vibrio alginolyticus

ABSTRACT The marine bacterium Vibrio alginolyticus has four motor components, PomA, PomB, MotX, and MotY, responsible for its Na+-driven flagellar rotation. PomA and PomB are integral inner membrane proteins having four and one transmembrane segments (TMs), respectively, which are thought to form an ion channel complex. First, site-directed Cys mutagenesis was systematically performed from Asp-24 to Glu-41 of PomB, and the resulting mutant proteins were examined for susceptibility to a sulfhydryl reagent. Secondly, the Cys substitutions at the periplasmic boundaries of the PomB TM (Ser-38) and PomA TMs (Gly-23, Ser-34, Asp-170, and Ala-178) were combined. Cross-linked products were detected for the combination of PomB-S38C and PomA-D170C mutant proteins. The Cys substitutions in the periplasmic boundaries of PomA TM3 (from Met-169 to Asp-171) and the PomB TM (from Leu-37 to Ser-40) were combined to construct a series of double mutants. Most double mutations reduced the motility, whereas each single Cys substitution slightly affected it. Although the motility of the strain carrying PomA-D170C and PomB-S38C was significantly inhibited, it was recovered by reducing reagent. The strain with this combination showed a lower affinity for Na+ than the wild-type combination. PomA-D148C and PomB-P16C, which are located at the cytoplasmic boundaries of PomA TM3 and the PomB TM, also formed the cross-linked product. From these lines of evidence, we infer that TM3 of PomA and the TM of PomB are in close proximity over their entire length and that cooperation between these two TMs is required for coupling of Na+ conduction to flagellar rotation.

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Proline residues in transmembrane segment IV are critical for activity, expression and targeting of the Na+/H+ exchanger isoform 1

Biochemical Journal ◽

10.1042/bj20030884 ◽

2004 ◽

Vol 379 (1) ◽

pp. 31-38 ◽

Cited By ~ 63

Author(s):

Emily R. SLEPKOV ◽

Signy CHOW ◽

M. Joanne LEMIEUX ◽

Larry FLIEGEL

Keyword(s):

Plasma Membrane ◽

Membrane Protein ◽

Mammalian Cells ◽

Integral Membrane Protein ◽

Amide Hydrogen ◽

Wild Type ◽

Transmembrane Segment ◽

Mutant Proteins ◽

Transmembrane Segments ◽

Induced Changes

NHE1 (Na+/H+ exchanger isoform 1) is a ubiquitously expressed integral membrane protein that regulates intracellular pH in mammalian cells. Proline residues within transmembrane segments have unusual properties, acting as helix breakers and increasing flexibility of membrane segments, since they lack an amide hydrogen. We examined the importance of three conserved proline residues in TM IV (transmembrane segment IV) of NHE1. Pro167 and Pro168 were mutated to Gly, Ala or Cys, and Pro178 was mutated to Ala. Pro168 and Pro178 mutant proteins were expressed at levels similar to wild-type NHE1 and were targeted to the plasma membrane. However, the mutants P167G (Pro167→Gly), P167A and P167C were expressed at lower levels compared with wild-type NHE1, and a significant portion of P167G and P167C were retained intracellularly, possibly indicating induced changes in the structure of TM IV. P167G, P167C, P168A and P168C mutations abolished NHE activity, and P167A and P168G mutations caused markedly decreased activity. In contrast, the activity of the P178A mutant was not significantly different from that of wild-type NHE1. The results indicate that both Pro167 and Pro168 in TM IV of NHE1 are required for normal NHE activity. In addition, mutation of Pro167 affects the expression and membrane targeting of the exchanger. Thus both Pro167 and Pro168 are strictly required for NHE function and may play critical roles in the structure of TM IV of the NHE.

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Key Role of Cysteine Residues in Catalysis and Subcellular Localization of Sulfur Oxygenase-Reductase of Acidianus tengchongensis

Applied and Environmental Microbiology ◽

10.1128/aem.71.2.621-628.2005 ◽

2005 ◽

Vol 71 (2) ◽

pp. 621-628 ◽

Cited By ~ 37

Author(s):

Zhi-Wei Chen ◽

Cheng-Ying Jiang ◽

Qunxin She ◽

Shuang-Jiang Liu ◽

Pei-Jin Zhou

Keyword(s):

Cytoplasmic Membrane ◽

Sulfur Oxidation ◽

Site Directed Mutagenesis ◽

Cysteine Residues ◽

Wild Type ◽

Immune Electron Microscopy ◽

Mutant Proteins ◽

Close Proximity ◽

Catalytic Sites

ABSTRACT Analysis of known sulfur oxygenase-reductases (SORs) and the SOR-like sequences identified from public databases indicated that they all possess three cysteine residues within two conserved motifs (V-G-P-K-V-C31 and C101-X-X-C104; numbering according to the Acidianus tengchongensis numbering system). The thio-modifying reagent N-ethylmaleimide and Zn2+ strongly inhibited the activities of the SORs of A. tengchongensis, suggesting that cysteine residues are important. Site-directed mutagenesis was used to construct four mutant SORs with cysteines replaced by serine or alanine. The purified mutant proteins were investigated in parallel with the wild-type SOR. Replacement of any cysteine reduced SOR activity by 98.4 to 100%, indicating that all the cysteine residues are crucial to SOR activities. Circular-dichroism and fluorescence spectrum analyses revealed that the wild-type and mutant SORs have similar structures and that none of them form any disulfide bond. Thus, it is proposed that three cysteine residues, C31 and C101-X-X-C104, in the conserved domains constitute the putative binding and catalytic sites of SOR. Furthermore, enzymatic activity assays of the subcellular fractions and immune electron microscopy indicated that SOR is not only present in the cytoplasm but also associated with the cytoplasmic membrane of A. tengchongensis. The membrane-associated SOR activity was colocalized with the activities of sulfite:acceptor oxidoreductase and thiosulfate:acceptor oxidoreductase. We tentatively propose that these enzymes are located in close proximity on the membrane to catalyze sulfur oxidation in A. tengchongensis.

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Role of the N- and C-terminal regions of FliF, the MS ring component in Vibrio flagellar basal body

10.1101/2021.01.04.425360 ◽

2021 ◽

Author(s):

Seiji Kojima ◽

Hiroki Kajino ◽

Keiichi Hirano ◽

Yuna Inoue ◽

Hiroyuki Terashima ◽

...

Keyword(s):

Basal Body ◽

Ring Formation ◽

Terminal Region ◽

Wild Type ◽

Transmembrane Segment ◽

Bacterial Flagellum ◽

Transmembrane Segments ◽

Ring Assembly ◽

C Terminus ◽

N Terminus

AbstractThe MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues at the N-terminus (ΔN30 and ΔN50), and 83 (ΔC83) or 110 residues (ΔC110) at the C-terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When co-expressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF, in the MS ring fraction, suggesting that the N-terminus interacts with FlhF. MS ring formation is probably inefficient without an additional factor or FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as with wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles; the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly.ImportanceThe bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N and C terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N-terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C-terminus is essential for motor formation or function.

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CRISPR Mutants of Three Y Chromosome Genes Suggest Gradual Evolution of Fertility Functions in Drosophila melanogaster

10.1101/2020.01.30.926543 ◽

2020 ◽

Author(s):

Yassi Hafezi ◽

Samantha R. Sruba ◽

Steven R. Tarrash ◽

Mariana F. Wolfner ◽

Andrew G. Clark

Keyword(s):

Drosophila Melanogaster ◽

Y Chromosome ◽

Nonsynonymous Substitution ◽

Wild Type ◽

Protein Coding ◽

Repeat Elements ◽

The Third ◽

Gradual Evolution ◽

Complex Functions ◽

Lines Of Evidence

ABSTRACTGene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating “junk”. Yet multiple lines of evidence indicate these regions may be an important source of variation that could drive adaptation and species divergence, particularly through regulation of fertility. The ∼40 Mb Y chromosome of Drosophila melanogaster contains only 16 known protein-coding genes and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. We devised a genetic strategy for recovering and retaining stocks with sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three Y-linked genes. Two genes, PRY and FDY, had no previously identified functions. We found that PRY mutant males are sub-fertile, but FDY mutant males had no detectable fertility defects. FDY, the newest known gene on the Y chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene, CCY, had been predicted but never formally shown to be required for male fertility. CRISPR-targeting and RNAi of CCY caused male sterility. Surprisingly, however, our CCY mutants were sterile even in the presence of an extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to dominant sterility. Our approach provides an important step toward understanding the complex functions of the Y chromosome and parsing which functions are accomplished by genes versus repeat elements.

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Dissecting Fertility Functions of Drosophila Y Chromosome Genes with CRISPR

Genetics ◽

10.1534/genetics.120.302672 ◽

2020 ◽

Vol 214 (4) ◽

pp. 977-990 ◽

Cited By ~ 2

Author(s):

Yassi Hafezi ◽

Samantha R. Sruba ◽

Steven R. Tarrash ◽

Mariana F. Wolfner ◽

Andrew G. Clark

Keyword(s):

Male Fertility ◽

Nonsynonymous Substitution ◽

Wild Type ◽

Protein Coding ◽

Repeat Elements ◽

Protein Coding Genes ◽

The Third ◽

Link Type ◽

Complex Functions ◽

Lines Of Evidence

Gene-poor, repeat-rich regions of the genome are poorly understood and have been understudied due to technical challenges and the misconception that they are degenerating “junk.” Yet multiple lines of evidence indicate these regions may be an important source of variation that could drive adaptation and species divergence, particularly through regulation of fertility. The ∼40 Mb Y chromosome of Drosophila melanogaster contains only 16 known protein-coding genes, and is highly repetitive and entirely heterochromatic. Most of the genes originated from duplication of autosomal genes and have reduced nonsynonymous substitution rates, suggesting functional constraint. We devised a genetic strategy for recovering and retaining stocks with sterile Y-linked mutations and combined it with CRISPR to create mutants with deletions that disrupt three Y-linked genes. Two genes, PRY and FDY, had no previously identified functions. We found that PRY mutant males are subfertile, but FDY mutant males had no detectable fertility defects. FDY, the newest known gene on the Y chromosome, may have fertility effects that are conditional or too subtle to detect. The third gene, CCY, had been predicted but never formally shown to be required for male fertility. CRISPR targeting and RNA interference of CCY caused male sterility. Surprisingly, however, our CCY mutants were sterile even in the presence of an extra wild-type Y chromosome, suggesting that perturbation of the Y chromosome can lead to dominant sterility. Our approach provides an important step toward understanding the complex functions of the Y chromosome and parsing which functions are accomplished by genes vs. repeat elements.

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Determinants of cardiac Na+/Ca2+exchanger temperature dependence: NH2-terminal transmembrane segments

AJP Cell Physiology ◽

10.1152/ajpcell.00558.2001 ◽

2002 ◽

Vol 283 (2) ◽

pp. C512-C520 ◽

Cited By ~ 8

Author(s):

Christian Marshall ◽

Chadwick Elias ◽

Xiao-Hua Xue ◽

Hoa Dinh Le ◽

Alexander Omelchenko ◽

...

Keyword(s):

Temperature Dependence ◽

Temperature Sensitivity ◽

Xenopus Oocytes ◽

Energy Of Activation ◽

Wild Type ◽

Transmembrane Segment ◽

Inhibitory Peptide ◽

Transmembrane Segments ◽

Lower Temperature ◽

Patch Technique

The cardiac Na+/Ca2+ exchanger (NCX) in trout exhibits profoundly lower temperature sensitivity in comparison to the mammalian NCX. In this study, we attempt to characterize the regions of the NCX molecule that are responsible for its temperature sensitivity. Chimeric NCX molecules were constructed using wild-type trout and canine NCX cDNA and expressed in Xenopus oocytes. NCX-mediated currents were measured at 7, 14, and 30°C using the giant excised-patch technique. By using this approach, the differential temperature dependence of NCX was found to reside within the NH2-terminal region of the molecule. Specifically, we found that ∼75% of the Na+/Ca2+ exchange differential energy of activation is attributable to sequence differences in the region that include the first four transmembrane segments, and the remainder is attributable to transmembrane segment five and the exchanger inhibitory peptide site.

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Role of the N- and C-Terminal Regions of FliF, the MS Ring Component in the Vibrio Flagellar Basal Body

Journal of Bacteriology ◽

10.1128/jb.00009-21 ◽

2021 ◽

Vol 203 (9) ◽

Author(s):

Seiji Kojima ◽

Hiroki Kajino ◽

Keiichi Hirano ◽

Yuna Inoue ◽

Hiroyuki Terashima ◽

...

Keyword(s):

Basal Body ◽

Ring Formation ◽

Terminal Region ◽

Wild Type ◽

Transmembrane Segment ◽

Content Type ◽

Bacterial Flagellum ◽

Transmembrane Segments ◽

C Terminus ◽

N Terminus

ABSTRACT The MS ring is a part of the flagellar basal body and formed by 34 subunits of FliF, which consists of a large periplasmic region and two transmembrane segments connected to the N- and C-terminal regions facing the cytoplasm. A cytoplasmic protein, FlhF, which determines the position and number of the basal body, supports MS ring formation in the membrane in Vibrio species. In this study, we constructed FliF deletion mutants that lack 30 or 50 residues from the N terminus (ΔN30 and ΔN50) and 83 (ΔC83) or 110 residues (ΔC110) at the C terminus. The N-terminal deletions were functional and conferred motility of Vibrio cells, whereas the C-terminal deletions were nonfunctional. The mutants were expressed in Escherichia coli to determine whether an MS ring could still be assembled. When coexpressing ΔN30FliF or ΔN50FliF with FlhF, fewer MS rings were observed than with the expression of wild-type FliF in the MS ring fraction, suggesting that the N terminus interacts with FlhF. MS ring formation is probably inefficient without FlhF. The deletion of the C-terminal cytoplasmic region did not affect the ability of FliF to form an MS ring because a similar number of MS rings were observed for ΔC83FliF as for wild-type FliF, although further deletion of the second transmembrane segment (ΔC110FliF) abolished it. These results suggest that the terminal regions of FliF have distinct roles, the N-terminal region for efficient MS ring formation and the C-terminal region for MS ring function. The second transmembrane segment is indispensable for MS ring assembly. IMPORTANCE The bacterial flagellum is a supramolecular architecture involved in cell motility. At the base of the flagella, a rotary motor that begins to construct an MS ring in the cytoplasmic membrane comprises 34 transmembrane proteins (FliF). Here, we investigated the roles of the N- and C-terminal regions of FliF, which are MS rings. Unexpectedly, the cytoplasmic regions of FliF are not indispensable for the formation of the MS ring, but the N terminus appears to assist in ring formation through recruitment of FlhF, which is essential for flagellar formation. The C terminus is essential for motor formation or function.

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Cysteine-Scanning Mutagenesis of the Periplasmic Loop Regions of PomA, a Putative Channel Component of the Sodium-Driven Flagellar Motor in Vibrio alginolyticus

Journal of Bacteriology ◽

10.1128/jb.182.4.1001-1007.2000 ◽

2000 ◽

Vol 182 (4) ◽

pp. 1001-1007 ◽

Cited By ~ 12

Author(s):

Yukako Asai ◽

Tomokazu Shoji ◽

Ikuro Kawagishi ◽

Michio Homma

Keyword(s):

Structure And Function ◽

Vibrio Alginolyticus ◽

Membrane Topology ◽

Flagellar Motor ◽

Transmembrane Segment ◽

Transmembrane Segments ◽

Cysteine Scanning ◽

Loop Regions ◽

Cysteine Scanning Mutagenesis ◽

And Function

ABSTRACT The sodium-driven motor consists of the products of at least four genes, pomA, pomB, motX, andmotY, in Vibrio alginolyticus. PomA and PomB, which are homologous to the MotA and MotB components of proton-driven motors, have four transmembrane segments and one transmembrane segment, respectively, and are thought to form an ion channel. In PomA, two periplasmic loops were predicted at positions 21 to 36 between membrane segments 1 and 2 (loop1-2) and at positions 167 to 180 between membrane segments 3 and 4 (loop3-4). To characterize the two periplasmic loop regions, which may have a role as an ion entrance for the channel, we carried out cysteine-scanning mutagenesis. The T186 residue in the fourth transmembrane segment and the D71, D148, and D202 residues in the predicted cytoplasmic portion of PomA were also replaced with Cys. Only two mutations, M179C and T186C, conferred a nonmotile phenotype. Many mutations in the periplasmic loops and all of the cytoplasmic mutations did not abolish motility, though the five successive substitutions from M169C to K173C of loop3-4 impaired motility. In some mutants that retained substantial motility, motility was inhibited by the thiol-modifying reagents dithionitrobenzoic acid and N-ethylmaleimide. The profiles of inhibition by the reagents were consistent with the membrane topology predicted from the hydrophobicity profiles. Furthermore, from the profiles of labeling by biotin maleimide, we predicted more directly the membrane topology of loop3-4. None of the loop1-2 residues were labeled, suggesting that the environments around the two loops are very different. A few of the mutations were characterized further. The structure and function of the loop regions are discussed.

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Twelve-Transmembrane-Segment (TMS) Version (ΔTMS VII-VIII) of the 14-TMS Tet(L) Antibiotic Resistance Protein Retains Monovalent Cation Transport Modes but Lacks Tetracycline Efflux Capacity

Journal of Bacteriology ◽

10.1128/jb.183.8.2667-2671.2001 ◽

2001 ◽

Vol 183 (8) ◽

pp. 2667-2671 ◽

Cited By ~ 21

Author(s):

Jie Jin ◽

Arthur A. Guffanti ◽

Catherine Beck ◽

Terry A. Krulwich

Keyword(s):

Escherichia Coli ◽

Antibiotic Resistance ◽

Monovalent Cation ◽

Cation Transport ◽

Wild Type ◽

Transmembrane Segment ◽

Transmembrane Segments ◽

Resistance Protein ◽

Transport Modes ◽

Better Than

ABSTRACT A “Tet(L)-12” version of Tet(L), a tetracycline efflux protein with 14 transmembrane segments (TMS), was constructed by deletion of two central TMS. Tet(L)-12 catalyzed Na+/H+antiport and antiport with K+ as a coupling ion as well as or better than wild-type Tet(L) but exhibited no tetracycline-Me2+/H+ antiport inEscherichia coli vesicles.

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A Role for the S0 Transmembrane Segment in Voltage-dependent Gating of BK Channels

The Journal of General Physiology ◽

10.1085/jgp.200609662 ◽

2007 ◽

Vol 129 (3) ◽

pp. 209-220 ◽

Cited By ~ 33

Author(s):

Olga M. Koval ◽

Yun Fan ◽

Brad S. Rothberg

Keyword(s):

Channel Gating ◽

Large Change ◽

Bk Channels ◽

Bk Channel ◽

Voltage Sensor ◽

K Channel ◽

Wild Type ◽

Transmembrane Segment ◽

Amino Terminal ◽

Transmembrane Segments

BK (Maxi-K) channel activity is allosterically regulated by a Ca2+ sensor, formed primarily by the channel's large cytoplasmic carboxyl tail segment, and a voltage sensor, formed by its transmembrane helices. As with other voltage-gated K channels, voltage sensing in the BK channel is accomplished through interactions of the S1–S4 transmembrane segments with the electric field. However, the BK channel is unique in that it contains an additional amino-terminal transmembrane segment, S0, which is important in the functional interaction between BK channel α and β subunits. In this study, we used perturbation mutagenesis to analyze the role of S0 in channel gating. Single residues in the S0 region of the BK channel were substituted with tryptophan to give a large change in side chain volume; native tryptophans in S0 were substituted with alanine. The effects of the mutations on voltage- and Ca2+-dependent gating were quantified using patch-clamp electrophysiology. Three of the S0 mutants (F25W, L26W, and S29W) showed especially large shifts in their conductance–voltage (G-V) relations along the voltage axis compared to wild type. The G-V shifts for these mutants persisted at nominally 0 Ca2+, suggesting that these effects cannot arise simply from altered Ca2+ sensitivity. The basal open probabilities for these mutants at hyperpolarized voltages (where voltage sensor activation is minimal) were similar to wild type, suggesting that these mutations may primarily perturb voltage sensor function. Further analysis using the dual allosteric model for BK channel gating showed that the major effects of the F25W, L26W, and S29W mutations could be accounted for primarily by decreasing the equilibrium constant for voltage sensor movement. We conclude that S0 may make functional contact with other transmembrane regions of the BK channel to modulate the equilibrium between resting and active states of the channel's voltage sensor.

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