scholarly journals Specific cardiolipin-SecY interactions are required for proton-motive-force stimulation of protein secretion

2017 ◽  
Author(s):  
Robin A. Corey ◽  
Euan Pyle ◽  
William J. Allen ◽  
Marina Casiraghi ◽  
Bruno Miroux ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Protein Secretion ◽  
Binding Sites ◽  
Protein Transport ◽  
Proton Motive Force ◽  
Lipid Binding ◽  
Proton Exchange ◽  
Motive Force ◽  
Coarse Grained ◽  
Stimulation Of

AbstractThe transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of pre-proteins across the plasma membrane, powered by ATP hydrolysis and the trans-membrane proton-motive-force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly by cardiolipin, a specialised phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific cardiolipin binding sites on the Thermotoga maritima SecA-SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry and biochemical analysis of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional cardiolipin binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for cardiolipin in the conferral of PMF-stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery and thereby stimulate protein transport, by an as yet unknown mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, towards investigation of both the nature and functional implications of protein-lipid interactions.Significance StatementMany proteins are located in lipid membranes surrounding cells and cellular organelles. The membrane can impart important structural and functional effects on the protein, making understanding of this interaction critical. Here, we apply computational simulation to the identification of conserved lipid binding sites on an important highly conserved bacterial membrane protein, the Sec translocase (SecA-SecYEG), which uses ATP and the proton motive force (PMF) to secrete proteins across the bacterial plasma membrane. We experimentally validate and reveal the conserved nature of these binding sites, and use functional analyses to investigate the biological significance of this interaction. We demonstrate that these interactions are specific, transient, and critical for both ATP- and PMF- driven protein secretion.

scholarly journals Specific cardiolipin–SecY interactions are required for proton-motive force stimulation of protein secretion

2018 ◽  
Vol 115 (31) ◽  
pp. 7967-7972 ◽  
Author(s):  
Robin A. Corey ◽  
Euan Pyle ◽  
William J. Allen ◽  
Daniel W. Watkins ◽  
Marina Casiraghi ◽  
...  
Keyword(s):  
Binding Sites ◽  
Protein Transport ◽  
Membrane Lipids ◽  
Proton Motive Force ◽  
Proton Exchange ◽  
Motive Force ◽  
Coarse Grained ◽  
In Vitro Mutagenesis ◽  
Stimulation Of

The transport of proteins across or into membranes is a vital biological process, achieved in every cell by the conserved Sec machinery. In bacteria, SecYEG combines with the SecA motor protein for secretion of preproteins across the plasma membrane, powered by ATP hydrolysis and the transmembrane proton-motive force (PMF). The activities of SecYEG and SecA are modulated by membrane lipids, particularly cardiolipin (CL), a specialized phospholipid known to associate with a range of energy-transducing machines. Here, we identify two specific CL binding sites on the Thermotoga maritima SecA–SecYEG complex, through application of coarse-grained molecular dynamics simulations. We validate the computational data and demonstrate the conserved nature of the binding sites using in vitro mutagenesis, native mass spectrometry, biochemical analysis, and fluorescence spectroscopy of Escherichia coli SecYEG. The results show that the two sites account for the preponderance of functional CL binding to SecYEG, and mediate its roles in ATPase and protein transport activity. In addition, we demonstrate an important role for CL in the conferral of PMF stimulation of protein transport. The apparent transient nature of the CL interaction might facilitate proton exchange with the Sec machinery, and thereby stimulate protein transport, by a hitherto unexplored mechanism. This study demonstrates the power of coupling the high predictive ability of coarse-grained simulation with experimental analyses, toward investigation of both the nature and functional implications of protein–lipid interactions.

scholarly journals Structure, function, and regulation of myosin 1C.

2005 ◽  
Vol 52 (2) ◽  
pp. 373-380 ◽  
Author(s):  
Barbara Barylko ◽  
Gwanghyun Jung ◽  
Joseph P Albanesi
Keyword(s):  
Plasma Membrane ◽  
Binding Sites ◽  
Subcellular Location ◽  
Lipid Binding ◽  
Tail Domain ◽  
Anionic Lipid ◽  
Myosin I ◽  
Docking Proteins ◽  
Neck Domain ◽  
Anionic Lipids

Myosin 1C, the first mammalian single-headed myosin to be purified, cloned, and sequenced, has been implicated in the translocation of plasma membrane channels and transporters. Like other forms of myosin I (of which eight exist in humans) myosin 1C consists of motor, neck, and tail domains. The neck domain binds calmodulins more tightly in the absence than in the presence of Ca(2+). Release of calmodulins exposes binding sites for anionic lipids, particularly phosphoinositides. The tail domain, which has an isoelectic point of 10.5, interacts with anionic lipid headgroups. When both neck and tail lipid binding sites are engaged, the myosin associates essentially irreversibly with membranes. Despite this tight membrane binding, it is widely believed that myosin 1C docking proteins are necessary for targeting the enzyme to specific subcellular location. The search for these putative myosin 1C receptors is an active area of research.

Ethanol Dissipates the Proton-motive Force across the Plasma Membrane of Saccharomyces cerevisiae

Microbiology ◽  
1986 ◽  
Vol 132 (2) ◽  
pp. 369-377 ◽  
Author(s):  
C. P. Cartwright ◽  
J.-R. juroszek ◽  
M. J. Beavan ◽  
F. M. S. Ruby ◽  
S. M. F. De Morais ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Plasma Membrane ◽  
Proton Motive Force ◽  
Motive Force

scholarly journals Mechanism of glucose and maltose transport in plasma-membrane vesicles from the yeast Candida utilis

1997 ◽  
Vol 321 (2) ◽  
pp. 487-495 ◽  
Author(s):  
Peter J. A. van den BROEK ◽  
Angeline E. van GOMPEL ◽  
Marijke A. H. LUTTIK ◽  
Jack T. PRONK ◽  
Carla C. M. van LEEUWEN
Keyword(s):  
Plasma Membrane ◽  
Candida Utilis ◽  
Plasma Membranes ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Transport Systems ◽  
Sugar Accumulation ◽  
Plasma Membrane Vesicles ◽  
Maltose Transport

Transport of glucose and maltose was studied in plasma-membrane vesicles from Candida utilis. The yeast was grown on a mixture of glucose and maltose in aerobic carbon-limited continuous cultures which enabled transport to be studied for both sugars with the same vesicles. Vesicles were prepared by fusion of isolated plasma membranes with proteoliposomes containing bovine heart cytochrome coxidase as a proton-motive-force-generating system. Addition of reduced cytochrome cgenerated a proton-motive force, consisting of a membrane potential, negative inside, and a pH gradient, alkaline inside. Energization led to accumulation of glucose and maltose in these vesicles, reaching accumulation ratios of about 40Ő50. Accumulation also occurred in the presence of valinomycin or nigericin, but was prevented by a combination of the two ionophores or by uncoupler, showing that glucose and maltose transport are dependent on the proton-motive force. Comparison of sugar accumulation with quantitative data on the proton-motive force indicated a 1:1 H+/sugar stoichiometry for both transport systems. Efflux of accumulated glucose was observed on dissipation of the proton-motive force. Exchange and counterflow experiments confirmed the reversible character of the H+Őglucose symporter. In contrast, uncoupler or a mixture of valinomycin plus nigericin induced only a slow efflux of accumulated maltose. Moreover under counterflow conditions, the expected transient accumulation was small. Thus the H+Őmaltose symporter has some characteristics of a carrier that is not readily reversible. It is concluded that in C. utilisthe transport systems for glucose and maltose are both driven by the proton-motive force, but the mechanisms are different.

scholarly journals K+/H+ antiport in the tobacco hornworm midgut: the K(+)-transporting component of the K+ pump.

1994 ◽  
Vol 196 (1) ◽  
pp. 361-373 ◽  
Author(s):  
A Lepier ◽  
M Azuma ◽  
W R Harvey ◽  
H Wieczorek
Keyword(s):  
Plasma Membrane ◽  
Plasma Membranes ◽  
Tobacco Hornworm ◽  
Proton Motive Force ◽  
Motive Force ◽  
Apical Plasma Membrane ◽  
Lectin Staining ◽  
Sds Page ◽  
K Transport ◽  
Midgut Lumen

The midgut of the tobacco hornworm secretes K+ across the apical plasma membrane of its goblet cells. This secondary K+ transport results from K+/H+ antiport energized by the proton-motive force generated by a primary, H(+)-transporting plasma membrane V-ATPase. Thus, the lepidopteran midgut constitutes a well-established example of the emerging concept that the proton-motive force is an alternative to the classical sodium-motive force for the energization of animal plasma membranes. K+/H+ antiport in the tobacco hornworm midgut is electrophoretic, exchanging 2H+ for 1K+. Under physiological conditions, it is energized by the voltage component of the proton-motive force. The strong coupling of electrophoretic K+/2H+ antiport with the electrogenic V-ATPase provides, in principle, the minimal device for the alkalization of the midgut lumen to pH values higher than 11. K+/H+ antiport is insensitive to bafilomycin A1, but is inhibited by amiloride or Concanavalin A. Lectin staining of blots after SDS-PAGE revealed several glycosylated polypeptides in the goblet cell apical membrane which are not part of the V-ATPase and thus are candidates for the antiporter protein. Current efforts are focused on the isolation of the K+/H+ antiporter.

scholarly journals Type 4 Pilus Biogenesis and Type II-Mediated Protein Secretion by Vibrio cholerae Occur Independently of the TonB-Facilitated Proton Motive Force

2002 ◽  
Vol 184 (8) ◽  
pp. 2305-2309 ◽  
Author(s):  
Niranjan Bose ◽  
Shelley M. Payne ◽  
Ronald K. Taylor
Keyword(s):  
Vibrio Cholerae ◽  
Protein Secretion ◽  
Proton Motive Force ◽  
Extracellular Protein ◽  
Motive Force ◽  
Type Ii ◽  
Pilus Biogenesis ◽  
Toxin Coregulated Pilus

ABSTRACT In Vibrio cholerae, elaboration of toxin-coregulated pilus and protein secretion by the extracellular protein secretion apparatus occurred in the absence of both TonB systems. In contrast, the cognate putative ATPases were required for each process and could not substitute for each other.

scholarly journals Structure of the hexameric fungal plasma membrane proton pump in its auto-inhibited state

2021 ◽  
Author(s):  
Sabine Heit ◽  
Maxwell M.G. Geurts ◽  
Bonnie J. Murphy ◽  
Robin A. Corey ◽  
Deryck J. Mills ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Molecular Basis ◽  
Proton Motive Force ◽  
Coarse Grained ◽  
Physiological Relevance ◽  
Glucose Signalling ◽  
Inhibitory Domain ◽  
Dynamics Simulations ◽  
P Type ◽  
Plasma Membrane Proton Pump

AbstractThe fungal plasma membrane H+-ATPase Pma1 is a vital enzyme, generating a proton-motive force that drives the import of essential nutrients. Auto-inhibited Pma1 hexamers in starving fungi are activated by glucose signalling resulting in phosphorylation of the auto-inhibitory domain. As related P-type ATPases are not known to oligomerise, the physiological relevance of Pma1 hexamers remains unknown. We have determined the structure of hexameric Pma1 from Neurospora crassa by cryo-EM at 3.3 Å resolution, elucidating the molecular basis for hexamer formation and auto-inhibition, and providing a basis for structure-based drug development. Coarse-grained molecular dynamics simulations in a lipid bilayer suggest lipid-mediated contacts between monomers and a substantial protein-induced membrane deformation that could act as a proton-attracting funnel.

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