Transbilayer transport of phosphatidic acid in response to transmembrane pH gradients

Abstract

Preliminary studies have shown that asymmetric transbilayer distributions of phosphatidic acid (PA) can be induced by transmembrane pH gradients (delta pH) in large unilamellar vesicles [Hope et al. (1989) Biochemistry 28, 4181-4187]. Here the mechanism of PA transport is examined employing TNS as a fluorescent probe of lipid asymmetry. It is shown that the kinetics of PA transport are consistent with the transport of the uncharged (protonated) form. Transport of the neutral form can be rapid, exhibiting half-times for transbilayer transport of approximately 25 s at 45 degrees C. It is also shown that PA transport is associated with a large activation energy (28 kcal/mol) similar to that observed for phosphatidylglycerol. The maximum induced transbilayer asymmetry of PA corresponded to approximately 95% on the inner monolayer for vesicles containing 5 mol % PA.

Phospholipids in animal eukaryotic membranes: transverse asymmetry and movement

“…If the analogues are partially water-soluble, their spontaneous insertion in the membrane corresponds to a partition between the aqueous buffer and the hydrophobic bilayer. Their distribution between the two monolayers with time is determined either by selective destruction of the probe in the outer leaflet or by selective extraction of the analogue from the external monolayer using Kornberg and McConnell, 1971Yuann and Morse, 1991Cabral et al, 1987Homan and Pownall, 1988Rothman and Dawidowicz, 1975de Kruijff et al, 1978Eastman et al, 1991Rothman et al, 1976Daleke and Huestis, 1985Middelkoop et al, 1986Bergmann et al, 1984Bitbol and Devaux, 1988Morrot et al, 1989Crain and Zilversmit, 1980Sandra and Pagano, 1978Lipka et al, 1991Barsukov et al, 1986 back-exchange into acceptor structures such as liposomes or bovine serum albumin (Bergmann et al, 1984;Biitikofer et al, 1990;Calvez et al, 1988;Connor et al, 1990;Daleke and Huestis, 1989). In the case of erythrocytes, whose shape is sensitive to the relative amount of lipid in each of the two membrane leaflets according to the bilayer-couple hypothesis (Deuticke, 1968;Sheetz and Singer, 1974), these changes ( Figure 3) have also been used to follow the transmembrane motion of lipids (Daleke and Huestis, 1985;.…”

“…This is true for fatty acids, phosphatidic acid and phosphatidylglycerol (Table 3). In contrast, even if they can exist as uncharged molecules, phosphatidylinositol and cardiolipin have a slow diffusion across bilayers (Biutikofer et al, 1990;Eastman et al, 1991), probably because they maintain an important hydration shell around their polar head groups, which prevents them from crossing the hydrophobic core of the bilayer.…”

“…To reach such a situation, there has to be a local environment able to trap a lipid in the leaflet where it is to be accumulated. Cullis's group has published several reports (Eastman et al, 1991;Hope and Cullis, 1987;Redelmeier et al, 1990) showing how to generate an asymmetric distribution of lipids which are weak acids or weak bases. It was noted above that such molecules (phosphatidic acid, for instance) diffuse very quickly through a bilayer when in their neutral form, which leads to a symmetrical distribution of the neutral molecules.…”

A Novel Pathway for Transport and Metabolism of a Fluorescent Phosphatidic Acid Analog in Yeast

“…So, it remains possible that NBD-PtdOH and NBD-PtdCho may share a similar mechanism of non-endocytic import. However, Eastman et al (28) suggest that, in contrast with other phospholipids, when PtdOH becomes protonated it can spontaneously flip across the bilayer. This spontaneous transbilayer flip/flop of PtdOH across synthetic bilayers is very sensitive to temperature (28), whereas spontaneous movement of NBD-DAG across bilayers occurs readily even at low temperature (7).…”

“…However, Eastman et al (28) suggest that, in contrast with other phospholipids, when PtdOH becomes protonated it can spontaneously flip across the bilayer. This spontaneous transbilayer flip/flop of PtdOH across synthetic bilayers is very sensitive to temperature (28), whereas spontaneous movement of NBD-DAG across bilayers occurs readily even at low temperature (7). The characteristics of NBD-PtdOH import in yeast are consistent with those described for spontaneous flip/flop, suggesting it may play a role in internalization.…”