Evolutionarily divergent, Na+-regulated H+-transporting membrane-bound pyrophosphatases

2015 ◽  
Vol 467 (2) ◽  
pp. 281-291 ◽  
Author(s):  
Heidi H. Luoto ◽  
Erika Nordbo ◽  
Anssi M. Malinen ◽  
Alexander A. Baykov ◽  
Reijo Lahti
Keyword(s):  
Membrane Vesicles ◽  
Evolutionary Divergence ◽  
Chlorobium Limicola ◽  
Na Transport ◽  
Alkali Cations ◽  
Archaeal Protein ◽  
Bacterial Proteins ◽  
Inverted Membrane Vesicles ◽  
Substrate Conversion ◽  
Membrane Bound

Membrane-bound pyrophosphatase (mPPases) of various types consume pyrophosphate (PPi) to drive active H+ or Na+ transport across membranes. H+-transporting PPases are divided into phylogenetically distinct K+-independent and K+-dependent subfamilies. In the present study, we describe a group of 46 bacterial proteins and one archaeal protein that are only distantly related to known mPPases (23%–34% sequence identity). Despite this evolutionary divergence, these proteins contain the full set of 12 polar residues that interact with PPi, the nucleophilic water and five cofactor Mg2+ ions found in ‘canonical’ mPPases. They also contain a specific lysine residue that confers K+ independence on canonical mPPases. Two of the proteins (from Chlorobium limicola and Cellulomonas fimi) were expressed in Escherichia coli and shown to catalyse Mg2+-dependent PPi hydrolysis coupled with electrogenic H+, but not Na+ transport, in inverted membrane vesicles. Unique features of the new H+-PPases include their inhibition by Na+ and inhibition or activation, depending on PPi concentration, by K+ ions. Kinetic analyses of PPi hydrolysis over wide ranges of cofactor (Mg2+) and substrate (Mg2–PPi) concentrations indicated that the alkali cations displace Mg2+ from the enzyme, thereby arresting substrate conversion. These data define the new proteins as a novel subfamily of H+-transporting mPPases that partly retained the Na+ and K+ regulation patterns of their precursor Na+-transporting mPPases.

scholarly journals Improving cell-free glycoprotein synthesis by characterizing and enriching native membrane vesicles

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Jasmine M. Hershewe ◽  
Katherine F. Warfel ◽  
Shaelyn M. Iyer ◽  
Justin A. Peruzzi ◽  
Claretta J. Sullivan ◽  
...  
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Synthetic Biology ◽  
Membrane Protein ◽  
Membrane Vesicles ◽  
Processing Methods ◽  
Glycoprotein Synthesis ◽  
On Demand ◽  
Free Gene ◽  
Membrane Bound

AbstractCell-free gene expression (CFE) systems from crude cellular extracts have attracted much attention for biomanufacturing and synthetic biology. However, activating membrane-dependent functionality of cell-derived vesicles in bacterial CFE systems has been limited. Here, we address this limitation by characterizing native membrane vesicles in Escherichia coli-based CFE extracts and describing methods to enrich vesicles with heterologous, membrane-bound machinery. As a model, we focus on bacterial glycoengineering. We first use multiple, orthogonal techniques to characterize vesicles and show how extract processing methods can be used to increase concentrations of membrane vesicles in CFE systems. Then, we show that extracts enriched in vesicle number also display enhanced concentrations of heterologous membrane protein cargo. Finally, we apply our methods to enrich membrane-bound oligosaccharyltransferases and lipid-linked oligosaccharides for improving cell-free N-linked and O-linked glycoprotein synthesis. We anticipate that these methods will facilitate on-demand glycoprotein production and enable new CFE systems with membrane-associated activities.

scholarly journals Targeting Mycobacterial F-ATP Synthase C-Terminal α Subunit Interaction Motif on Rotary Subunit γ

Antibiotics ◽  
2021 ◽  
Vol 10 (12) ◽  
pp. 1456
Author(s):  
Amaravadhi Harikishore ◽  
Chui-Fann Wong ◽  
Priya Ragunathan ◽  
Dennis Litty ◽  
Volker Müller ◽  
...  
Keyword(s):  
Atp Synthase ◽  
Atp Hydrolysis ◽  
Atp Synthesis ◽  
Membrane Vesicles ◽  
Α Subunit ◽  
Rotational States ◽  
C Terminus ◽  
Inverted Membrane Vesicles ◽  
Hydrolysis Of ◽  
Micromolar Range

Mycobacteria regulate their energy (ATP) levels to sustain their survival even in stringent living conditions. Recent studies have shown that mycobacteria not only slow down their respiratory rate but also block ATP hydrolysis of the F-ATP synthase (α3:β3:γ:δ:ε:a:b:b’:c9) to maintain ATP homeostasis in situations not amenable for growth. The mycobacteria-specific α C-terminus (α533-545) has unraveled to be the major regulative of latent ATP hydrolysis. Its deletion stimulates ATPase activity while reducing ATP synthesis. In one of the six rotational states of F-ATP synthase, α533-545 has been visualized to dock deep into subunit γ, thereby blocking rotation of γ within the engine. The functional role(s) of this C-terminus in the other rotational states are not clarified yet and are being still pursued in structural studies. Based on the interaction pattern of the docked α533-545 region with subunit γ, we attempted to study the druggability of the α533-545 motif. In this direction, our computational work has led to the development of an eight-featured α533-545 peptide pharmacophore, followed by database screening, molecular docking, and pose selection, resulting in eleven hit molecules. ATP synthesis inhibition assays using recombinant ATP synthase as well as mycobacterial inverted membrane vesicles show that one of the hits, AlMF1, inhibited the mycobacterial F-ATP synthase in a micromolar range. The successful targeting of the α533-545-γ interaction motif demonstrates the potential to develop inhibitors targeting the α site to interrupt rotary coupling with ATP synthesis.

scholarly journals Outer membrane vesicles engineered to express membrane-bound antigen program dendritic cells for cross-presentation to CD8+ T cells

2019 ◽  
Vol 91 ◽  
pp. 248-257 ◽  
Author(s):  
Sjoerd T.T. Schetters ◽  
Wouter S.P. Jong ◽  
Sophie K. Horrevorts ◽  
Laura J.W. Kruijssen ◽  
Steef Engels ◽  
...  
Keyword(s):  
T Cells ◽  
Dendritic Cells ◽  
Outer Membrane ◽  
Cd8 T Cells ◽  
Membrane Vesicles ◽  
Outer Membrane Vesicles ◽  
Cross Presentation ◽  
Membrane Bound

Dissociation of proximal tubular glucose and Na+ reabsorption by amphotericin B

1979 ◽  
Vol 236 (4) ◽  
pp. F392-F397
Author(s):  
P. S. Aronson ◽  
J. P. Hayslett ◽  
M. Kashgarian
Keyword(s):  
Proximal Tubule ◽  
Glucose Uptake ◽  
Amphotericin B ◽  
Membrane Vesicle ◽  
Membrane Vesicles ◽  
Na Transport ◽  
Glucose Reabsorption ◽  
Tubular Lumen ◽  
Proximal Tubular ◽  
Necturus Proximal Tubule

The effect of amphotericin B on glucose and Na+ transport was studied in the Necturus proximal tubule and in microvillus membrane vesicles isolated from the rabbit renal cortex. In the Necturus experiments, the rate constants for disappearance of radiolabeled glucose (kG) and mannitol (kM) from the tubular lumen were determined by stop-flow microperfusion. Saturability and Na+-dependence of glucose reabsorption was confirmed, since kG was reduced by raising intratubular glucose from 1 to 5 mM or by replacing intratubular Na+ with choline. Neither maneuver affected kM. Intratubular amphotericin B (10 microgram/ml), previously shown to stimulate active Na+ reabsorption in the Necturus proximal tubule, inhibited kG with no effect on kM. In the membrane vesicle preparation, amphotericin inhibited the uphill glucose uptake which results from imposing a NaCl gradient from outside to inside, but had no effect on glucose uptake in either the absence of Na+ or in the presence of Na+ when there was no Na+ gradient. Amphotericin B stimulated the uptake of Na+ by the vesicles. The observed dissociation of glucose and Na+ transport by amphotericin B is consistent with the concept that proximal tubular glucose reabsorption is energized by the luminal membrane Na+ gradient and is not directly linked to active Na+ transport per se.

Passive diffusion of nucleosides into Micrococcus sodonensis membrane vesicles

1980 ◽  
Vol 58 (6) ◽  
pp. 457-460 ◽  
Author(s):  
M. A. Pickard
Keyword(s):  
Energy Source ◽  
Membrane Vesicles ◽  
Passive Diffusion ◽  
Membrane Bound ◽  
Nucleoside Uptake ◽  
Membrane Bound Enzymes ◽  
Isolated Membrane Vesicles

Nucleoside entry into isolated membrane vesicles of Micrococcus sodonensis (luteus) was studied using 14C-labelled nucleosides: adenosine, inosine, cytidine, uridine, guanosine, and thymidine. All nucleosides were recovered unmetabolized from the vesicles except adenosine and cytidine which were partly deaminated by membrane-bound enzymes. Vesicle preparations actively transported proline but no energy source was found capable of supporting concentrative nucleoside uptake. The entry of nucleosides into M. sodonensis vesicles was not saturable, nor was there competition between the nucleosides studied for entry. It was concluded that nucleoside entry into M. sodonensis vesicles occurs by passive diffusion.

Sign in / Sign up

Export Citation Format

Share Document