ATP‐Dependent and drug‐inhibited vesicle enlargement reconstituted using synthetic lipids and recombinant proteins

Abstract

A recombinant ECTO-NOX (tNOX) and a recombinant plasma membrane associated AAA-ATPase (ATPase Associated with Different Cellular Activities) were combined in stoichiometric proportions into liposomes together with albumin as a source of protein thiols. Large lamellar vesicles were formed from phosphatidylcholine, cholesterol and dicetyl phosphate in a molar ratio of 50:45:5, where the phosphatidylcholine was a 2:1 mixture of synthetic dimyristoyl and dipalmitoyl phosphatidylcholines. The lipids were dried to a film and reconstituted into vesicles by resuspension in buffer containing the recombinant proteins in equimolar ratios of 0.04 nmoles/mg lipid. In the presence of ATP, these vesicles enlarged in an ATP-dependent manner based on light-scattering measurements. Because the drug-inhibited ECTO-NOX protein, tNOX was utilized, the enlargement was inhibited by capsaicin, a quinone site tNOX inhibitor specific for tNOX. With the lipid vesicle systems, the recombinant ECTO-NOX, the recombinant AAA-ATPase, a source of protein thiols and ATP all were required. In control experiments, no ATP-dependent vesicle enlargement was observed with the AAA-ATPase or the ECTO-NOX protein alone. Also addition of ATP was without any effect when only the single proteins were incorporated into the lipid vesicles. A model has been developed whereby the plasma membrane AAA-ATPase is linked via disulfide bonds, formed and broken by the ECTO-NOX protein, to membrane structural proteins. Binding of ATP and subsequent hydrolysis and release of ADP would advance the ATPase hexamer ratchet thereby both thinning the membrane and increasing the vesicle surface.

Identification of the constitutive ultradian oscillator of the circadian clock (ENOX1) in <i>Saccharomyces cerevisiae</i>

“…As yeast enlarge by tip growth, or budding [25,26], the role of ENOX1 in physical membrane displacements essential to cell enlargement [27][28][29] is apparently unessential such that deletion mutants could be used to identify the ENOX1 of S. cerevisiae as YML117W. The activity of the YML117W ENOX1 protein is protease and heat (80˚C) resistant in keeping with data from mammalian sources [30][31][32].…”

Ultradian oscillators of the circadian clock in <i>Saccharomyces</i> <i>cerevisiae</i>

“…This attribute is probably due to the difference in the mechanism of enlargement. Plant and mammalian cells enlarge by physical membrane displacement [37][38][39] while yeast enlarge by tip growth, or budding [40,41]. In mammalian cells, the cell enlargement function of ENOX1 is central to its activity and essential for cell viability.…”

Cancer Site-Specific Isoforms of ENOX2 (tNOX), A Cancer-Specific Cell Surface Oxidase

“…This transport of protons and electrons involves a membrane quinone, coenzyme Q [3], and the overall process is thought to be essential to energize cell enlargement [4].…”