scholarly journals The KtrA and KtrE Subunits Are Required for Na+-Dependent K+ Uptake by KtrB across the Plasma Membrane in Synechocystis sp. Strain PCC 6803

2010 ◽  
Vol 192 (19) ◽  
pp. 5063-5070 ◽  
Author(s):  
Lalu Zulkifli ◽  
Masaro Akai ◽  
Asuka Yoshikawa ◽  
Mie Shimojima ◽  
Hiroyuki Ohta ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Homology Model ◽  
Atomic Scale ◽  
Site Directed Mutagenesis ◽  
Dependent Manner ◽  
Transport Activity ◽  
Charged Residues ◽  
Uptake Activity ◽  
Extracellular Side

ABSTRACT The Na+-dependent K+ uptake KtrABE system is essential for the adaptation of Synechocystis to salinity stress and high osmolality. While KtrB forms the K+-translocating pore, the role of the subunits KtrA and KtrE for Ktr function remains elusive. Here, we characterized the role of KtrA and KtrE in Ktr-mediated K+ uptake and in modulating Na+ dependency. Expression of KtrB alone in a K+ uptake-deficient Escherichia coli strain conferred low K+ uptake activity that was not stimulated by Na+. Coexpression of both KtrA and KtrE with KtrB increased the K+ transport activity in a Na+-dependent manner. KtrA and KtrE were found to be localized to the plasma membrane in Synechocystis. Site-directed mutagenesis was used to analyze the role of single charged residues in KtrB for Ktr function. Replacing negatively charged residues facing the extracellular space with residues of the opposite charge increased the apparent Km for K+ in all cases. However, none of the mutations eliminated the Na+ dependency of Ktr-mediated K+ transport. Mutations of residues on the cytoplasmic side had larger effects on K+ uptake activity than those of residues on the extracellular side. Further analysis revealed that replacement of R262, which is well conserved among Ktr/Trk/HKT transporters in the third extracellular loop, by Glu abolished transport activity. The atomic-scale homology model indicated that R262 might interact with E247 and D261. Based on these data, interaction of KtrA and KtrE with KtrB increased the K+ uptake rate and conferred Na+ dependency.

scholarly journals Potential transceptor AtNRT1.13 modulates shoot architecture and flowering time in a nitrate-dependent manner

2021 ◽  
Author(s):  
Hui-Yu Chen ◽  
Shan-Hua Lin ◽  
Ling-Hsin Cheng ◽  
Jeng-Jong Wu ◽  
Yi-Chen Lin ◽  
...  
Keyword(s):  
Flowering Time ◽  
Nitrate Transport ◽  
Dependent Manner ◽  
Transport Activity ◽  
Node Number ◽  
Shoot Architecture ◽  
Glucuronidase Reporter ◽  
Uptake Activity ◽  
Delayed Flowering ◽  
Severe Growth

Abstract Compared with root development regulated by external nutrients, less is known about how internal nutrients are monitored to control plasticity of shoot development. In this study, we characterize an Arabidopsis thaliana transceptor, NRT1.13 (NPF4.4), of the NRT1/PTR/NPF family. Different from most NRT1 transporters, NRT1.13 does not have the conserved proline residue between transmembrane domains 10 and 11; an essential residue for nitrate transport activity in CHL1/NRT1.1/NPF6.3. As expected, when expressed in oocytes, NRT1.13 showed no nitrate transport activity. However, when Ser 487 at the corresponding position was converted back to proline, NRT1.13 S487P regained nitrate uptake activity, suggesting that wild-type NRT1.13 cannot transport nitrate but can bind it. Subcellular localization and β-glucuronidase reporter analyses indicated that NRT1.13 is a plasma membrane protein expressed at the parenchyma cells next to xylem in the petioles and the stem nodes. When plants were grown with a normal concentration of nitrate, nrt1.13 showed no severe growth phenotype. However, when grown under low-nitrate conditions, nrt1.13 showed delayed flowering, increased node number, retarded branch outgrowth, and reduced lateral nitrate allocation to nodes. Our results suggest that NRT1.13 is required for low-nitrate acclimation and that internal nitrate is monitored near the xylem by NRT1.13 to regulate shoot architecture and flowering time.

scholarly journals Functional Role of Charged Residues in the Transmembrane Segments of the Yeast Plasma Membrane H+-ATPase

2000 ◽  
Vol 275 (21) ◽  
pp. 15709-15716 ◽  
Author(s):  
Valery V. Petrov ◽  
Kristine P. Padmanabha ◽  
Robert K. Nakamoto ◽  
Kenneth E. Allen ◽  
Carolyn W. Slayman
Keyword(s):  
Plasma Membrane ◽  
Functional Role ◽  
Yeast Plasma Membrane ◽  
Transmembrane Segments ◽  
Charged Residues

scholarly journals The action of α-adrenergic agonists on plasma-membrane calcium fluxes in perfused rat liver

1984 ◽  
Vol 220 (1) ◽  
pp. 43-50 ◽  
Author(s):  
P H Reinhart ◽  
W M Taylor ◽  
F L Bygrave
Keyword(s):  
Plasma Membrane ◽  
Rat Liver ◽  
Potential Role ◽  
Dependent Manner ◽  
Perfused Rat Liver ◽  
Adrenergic Agonists ◽  
Mediated Effects ◽  
Alpha Agonist ◽  
Alpha Adrenergic Agonist

The effect of alpha-adrenergic agonists on Ca2+ fluxes was examined in the perfused rat liver by using a combination of Ca2+-electrode and 45Ca2+-uptake techniques. We showed that net Ca2+ fluxes can be described by the activities of separate Ca2+-uptake and Ca2+-efflux components, and that alpha-adrenergic agonists modulate the activity of both components in a time-dependent manner. Under resting conditions, Ca2+-uptake and -efflux activities are balanced, resulting in Ca2+ cycling across the plasma membrane. The alpha-adrenergic agonists vasopressin and angiotensin, but not glucagon, stimulate the rate of both Ca2+ efflux and Ca2+ uptake. During the first 2-3 min of alpha-agonist administration the effect on the efflux component is the greater, the net effect being efflux of Ca2+ from the cell. After 3-4 min of phenylephrine treatment, net Ca2+ movements are essentially complete, however, the rate of Ca2+ cycling is significantly increased. After removal of the alpha-agonist a large stimulation of the rate of Ca2+ uptake leads to the net accumulation of Ca2+ by the cell. The potential role of these Ca2+ flux changes in the expression of alpha-adrenergic-agonist-mediated effects is discussed.

scholarly journals Cysteine residues in the Na+/dicarboxylate co-transporter, NaDC-1

1999 ◽  
Vol 344 (1) ◽  
pp. 205-209 ◽  
Author(s):  
Ana M. PAJOR ◽  
Sally J. KRAJEWSKI ◽  
Nina SUN ◽  
Rama GANGULA
Keyword(s):  
Protein Trafficking ◽  
Direct Relationship ◽  
Transmembrane Domain ◽  
Cysteine Residue ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Directed Mutagenesis ◽  
Transport Activity ◽  
Specific Reagent

The role of cysteine residues in the Na+/dicarboxylate co-transporter (NaDC-1) was tested using site-directed mutagenesis. The transport activity of NaDC-1 was not affected by mutagenesis of any of the 11 cysteine residues, indicating that no individual cysteine residue is necessary for function. NaDC-1 is sensitive to inhibition by the impermeant cysteine-specific reagent, p-chloromercuribenzenesulphonate (pCMBS). The pCMBS-sensitive residues in NaDC-1 are Cys-227, found in transmembrane domain 5, and Cys-476, located in transmembrane domain 9. Although cysteine residues are not required for function in NaDC-1, their presence appears to be important for protein stability or trafficking to the plasma membrane. There was a direct relationship between the number of cysteine residues, regardless of location, and the transport activity and expression of NaDC-1. The results indicate that mutagenesis of multiple cysteine residues in NaDC-1 may alter the shape or configuration of the protein, leading to alterations in protein trafficking or stability.

scholarly journals Regulation of the electrogenic H+ channel in the plasma membrane of neutrophils: possible role of phospholipase A2, internal and external protons

1993 ◽  
Vol 292 (2) ◽  
pp. 445-450 ◽  
Author(s):  
A Kapus ◽  
K Suszták ◽  
E Ligeti
Keyword(s):  
Plasma Membrane ◽  
Phospholipase A2 ◽  
Neutrophil Granulocytes ◽  
Dependent Manner ◽  
Concentration Dependent Manner ◽  
Conducting Pathway ◽  
External Ph ◽  
Force Relationship ◽  
Stimulation Of

Possible factors regulating the opening of and the rate of H+ flux through a recently described, Cd(2+)-sensitive, phorbol ester- and arachidonic acid (AA)-activatable H(+)-conducting pathway in the plasma membrane of neutrophil granulocytes were investigated. (1) The phospholipase A2 blocker p-bromophenacyl bromide (BPB) inhibited the phorbol 12-myristate 13-acetate (PMA)-induced activation of this channel in a concentration-dependent manner (IC50, 4 microM). (2) Neither BPB nor the protein kinase C (PKC) inhibitor staurosporine influenced the AA-elicited stimulation of this route. (3) Intracellular acidification (cytoplasmic pH below 6.9) itself is capable of activating an electrogenic, Cd(2+)-sensitive H+ efflux indicating that protons can open up this route in the absence of any other stimulator. (4) PMA significantly decreases the intracellular H+ concentration ([H+]i) threshold for the opening of the channel, thus providing a conductive state at resting pH values, and elevates the rate of H+ efflux at any [H+]i. (5) Changes in external pH also modify the operation of the channel: above an extracellular pH (pH(o)) value of 7.4, the H(+)-flux/driving force relationship is approx. 5-fold greater than below this value. Our results suggest a multifactorial regulation of the electrogenic H+ channel: most probably PKC activates the channel indirectly, via stimulation of phospholipase A2 that subsequently liberates AA. In addition to this, the channel conductance seems to be promoted by internal H+ and inhibited by external H+.

scholarly journals Sec61p Contributes to Signal Sequence Orientation According to the Positive-Inside Rule

2004 ◽  
Vol 15 (3) ◽  
pp. 1470-1478 ◽  
Author(s):  
Veit Goder ◽  
Tina Junne ◽  
Martin Spiess
Keyword(s):  
Protein Targeting ◽  
Signal Sequence ◽  
Site Directed Mutagenesis ◽  
Diagnostic Model ◽  
Hydrophobic Core ◽  
Flanking Sequence ◽  
Charged Residues ◽  
Signal Anchor ◽  
A Charge

Protein targeting to the endoplasmic reticulum is mediated by signal or signal-anchor sequences. They also play an important role in protein topogenesis, because their orientation in the translocon determines whether their N- or C-terminal sequence is translocated. Signal orientation is primarily determined by charged residues flanking the hydrophobic core, whereby the more positive end is predominantly positioned to the cytoplasmic side of the membrane, a phenomenon known as the “positive-inside rule.” We tested the role of conserved charged residues of Sec61p, the major component of the translocon in Saccharomyces cerevisiae, in orienting signals according to their flanking charges by site-directed mutagenesis by using diagnostic model proteins. Mutation of R67, R74, or E382 in Sec61p reduced C-terminal translocation of a signal-anchor protein with a positive N-terminal flanking sequence and increased it for signal-anchor proteins with positive C-terminal sequences. These mutations produced a stronger effect on substrates with greater charge difference across the hydrophobic core of the signal. For some of the substrates, a charge mutation in Sec61p had a similar effect as one in the substrate polypeptides. Although these three residues do not account for the entire charge effect in signal orientation, the results show that Sec61p contributes to the positive-inside rule.

scholarly journals Palmitoylation-dependent Estrogen Receptor α Membrane Localization: Regulation by 17β-Estradiol

2005 ◽  
Vol 16 (1) ◽  
pp. 231-237 ◽  
Author(s):  
Filippo Acconcia ◽  
Paolo Ascenzi ◽  
Alessio Bocedi ◽  
Enzo Spisni ◽  
Vittorio Tomasi ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Estrogen Receptor ◽  
Plasma Membrane ◽  
Estrogen Receptor Α ◽  
Membrane Localization ◽  
Target Cells ◽  
Dependent Manner ◽  
Caveolin 1 ◽  
17Β Estradiol

A fraction of the nuclear estrogen receptor α (ERα) is localized to the plasma membrane region of 17β-estradiol (E2) target cells. We previously reported that ERα is a palmitoylated protein. To gain insight into the molecular mechanism of ERα residence at the plasma membrane, we tested both the role of palmitoylation and the impact of E2 stimulation on ERα membrane localization. The cancer cell lines expressing transfected or endogenous human ERα (HeLa and HepG2, respectively) or the ERα nonpalmitoylable Cys447Ala mutant transfected in HeLa cells were used as experimental models. We found that palmitoylation of ERα enacts ERα association with the plasma membrane, interaction with the membrane protein caveolin-1, and nongenomic activities, including activation of signaling pathways and cell proliferation (i.e., ERK and AKT activation, cyclin D1 promoter activity, DNA synthesis). Moreover, E2 reduces both ERα palmitoylation and its interaction with caveolin-1, in a time- and dose-dependent manner. These data point to the physiological role of ERα palmitoylation in the receptor localization to the cell membrane and in the regulation of the E2-induced cell proliferation.

Ezrin has properties to self-associate at the plasma membrane

1994 ◽  
Vol 107 (9) ◽  
pp. 2509-2521 ◽  
Author(s):  
C. Andreoli ◽  
M. Martin ◽  
R. Le Borgne ◽  
H. Reggio ◽  
P. Mangeat
Keyword(s):  
Plasma Membrane ◽  
Recombinant Baculovirus ◽  
Dependent Manner ◽  
Physiological Properties ◽  
Self Association ◽  
A Site ◽  
Apical Membranes ◽  
Gastric Parietal Cells ◽  
Overlay Assay

Ezrin, a member of a family of proteins involved in the interaction of the microfilament cytoskeleton with the plasma membrane, plays a role in membrane translocation in gastric parietal cells (Hanzel, D., Reggio, H., Bretscher, A., Forte, J. G. and Mangeat, P. (1991). EMBO J. 10, 2363–2373). Human ezrin was expressed in and purified from Escherichia coli. It possesses all the major biophysical, immunological and physiological properties of natural ezrin. Upon microinjection in live gastric HGT-1 cells, ezrin was incorporated into the dorsal microvilli, a site where the endogeneous protein is localized. By coimmunoprecipitation and ezrin-affinity assays, two HGT-1 cell proteins of 77 and 72 kDa behaved as ezrin-binding proteins. In enriched gastric apical membranes, 125I-ezrin labelled proteins of 80, 77 and 72 kDa by overlay assay. The 80 kDa protein was identified as ezrin and the 77 and 72 kDa proteins as gastric forms of proteins structurally related to ezrin, such as radixin and moesin. In insect cells infected with a recombinant baculovirus, one-third of over-expressed ezrin accumulated at the plasma membrane. Ezrin bound a 77 kDa endogenous peripheral membrane protein, behaving as an insect counterpart of the mammalian ezrin family. In addition to the respective role of the amino- and carboxyl-terminal domains of ezrin in linking the membrane and the cytoskeleton (Algrain, M., Turunen, O., Vaheri, A., Louvard, D. and Arpin, M. (1993). J. Cell Biol. 120, 129–139), both domains interacted synergistically in a salt-dependent manner to trigger self-association of ezrin. Ezrin's self-association properties could represent another way of regulating the number of ezrin molecules bound at specific membrane sites.

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