The acridine orange derivative, l0N-nonyl acridine orange, is an appropriate marker of the inner mitochondrial membrane in whole cells. We use membrane model systems to demonstrate that 10N-nonyl acridine orange binds to negatively charged phospholipids (cardiolipin, phosphatidylinositol and phosphatidylserine). The stoichiometry has been found to be 2 mol 10N-nonyl acridine orange/ mol cardiolipin and 1 mol dye/mol phosphatidylscrine or phosphatidylinositol, while, with zwitterionic phospholipids, significant binding could not be detected. The affinity constants were 2 x 1 O6 M ~ for cardiolipin-I ON-nonyl-acridine-orange association and only 7 x lo4 M ~ ' for that of phosphatidylserine and phosphatidylinositol association. The high affinity of the dye for cardiolipin may be explained by two essential interactions; firstly an electrostatic interaction betwcen the quaternary ammonium of nonyl acridine orange and the ionized phosphate residues of cardiolipin and secondly, hydrophobic interactions between adjacent chromophores. A linear relationship was demonstrated between the cardiolipin content of model membranes and the incorporated dye. Consequently, a convenient and rapid method for cardiolipin quantification in membranes was established and applied to the cardiolipin-containing organelle, the mitochondrion.The 10N-nonyl acridine orange (NAO), which is specifically incorporated into the inner mitochondrial membrane [l], plays a prominent role in the study of mitochondria in whole cells [2, 31. It enables monitoring of mitochondria in different situations, such as the cell cycle [4] and cell ageing [5] and also discrimination between different subpopulations of a heterogenous cell population, according to thcir mitochondrial contents [6, 71. Nevertheless, up to now the membrane molccular species which are specifically recognized by NAO, have not been determined.The large number of inner-mitochondrial-membrane enzymes implicated in oxidative phosphorylation, differing in their conformation and biological properties require a similar lipid environment for their activity. Cardiolipin, one of the three major phospholipids present in the inner membrane [8 -121, has been reported to be essential for the activity of the ADP/ATP carrier protein [I 31, for the phosphate transport protein [14] and for various other enzyme complexes [15,16]. This phospholipid has also been reported to be associated with the F1-FO ATPase [17]. Consequently, the NAO inhibition of the inner-mitochondrial-membrane enzymes [ 181 may be due to the interaction between the positively charged dye and cardiolipin, the main acidic phospholipid present in the inner mitochondrial membrane.The purpose ofthis work was to establish, with reference to different model membranes, that lON-nonyl acridine orange intcracts with acidic phospholipids and, more particularly, with cardiolipin. The absorbance spectra of NAO incubated with liposomes and measurements of the degree of saturation allowed us to determine the specificity and the stoichiometry of NAO ...

No abstract

The distribution of cardiolipin across the inner mitochondrial membrane was directly determined by using the ability of the fluorescent dye 1 O-N-nonyl-3,6-bis(dimethylamino)acridine (10-N-nonyl acridine orange) to form dimers when it interacts with the diacidic phospholipid. Two independent methods were employed : (a) a spectrophotometric measurement of 10-N-nonyl acridine orange binding to isolated rat liver mitochondria, mitoplasts and inside-out submitochondrial particles, and (b) a flow-cytometric analysis of specific red fluorescence, emitted when two dye molecules are bound to one membrane cardiolipin ; the stoichiometry of 10-N-nonyl acridine orange binding to phosphatidylserine and phosphatidylinositol, 1 mol dye/mol phospholipid, prevented dye dimerisation and subsequent red-fluorescence appearance. 57% total cardiolipin was present in the outer leaflets of inner membranes of isolated organelles, a distribution confirmed by saturation measurements for mitoplasts and inside-out submitochondrial particles. The same asymmetry was directly observed in situ with mitochondrial membranes of quiescent L1210 cells, and with mitochondrial membranes of respiring yeasts. Nevertheless, alterations in ATP synthesis and inhibition of mitochondrial protein synthesis revealed that cardiolipin distribution was apparently tightly correlated with mitochondrial membrane assembly and activity.

The presence of an alpha4-fucosyltransferase in plants was first deduced from the characterization of Lewis-a glycoepitopes in some N-glycans. The first plant gene encoding an alpha4-fucosyltransferase was recently cloned in Beta vulgaris. In the present paper we provide evidence for the presence of an alpha4-fucosyltransferase in A. thaliana by measurement of this glycosyltransferase activity from a purified microsomal preparation and by immunolocalization of Le(a) epitopes on glycans N-linked to glycoproteins located to the Golgi apparatus and on the cell surface. The corresponding gene AtFT4 (AY026941) was characterized. A unique copy of this gene was found in A. thaliana genome, and a single AtFT4 transcript was revealed in leaves, in roots, and at a lower extent in flowers. The coding sequence of AtFT4 gene is interrupted by two introns spanning 465 bp and 84 bp, respectively. The putative 393-amino-acid protein (44 kDa, pI: 6.59) contains an N-terminal hydrophobic region and one potential N-glycosylation site, but AtFT4 has poor homology (less than 30%) to the other alpha3/4-fucosyltransferases except for motif II. When expressed in COS 7 cells the protein is able to transfer Fuc from GDP-Fuc to a type 1 acceptor substrate, but this transferase activity is detected only in the culture medium of transfected cells

Transmembrane asymmetry of cardiolipin in yeast was monitored during the switch from fermentative to gluconeogenic growth and the reverse. As soon as cells used ethanol as an electron donor to produce ATP by oxidative phosphorylation, rapid and abundant cardiolipin synthesis was observed on the matrix side of the inner mitochondrial membrane followed by a transverse rearrangement between the two leaflets. The cardiolipin distribution changed from about 20:80 (in/out) to 70:30 (in/out), and after translocation towards the outer leaflet it finally became 37:63 (in/out). At the same time, cytochrome c oxidase activity remained stable, then increased as a possible result of the topographical rearrangement. During the reverse process from gluconeogenic to fermentative growth, the amount of cardiolipin rapidly decreased by half, its bilayer distribution apparently changing to a monolayer organization before the 20:80 (in/out) asymmetry of repressed cells was re-established. Experimental impairment of cardiolipin topography by antibiotic inhibition of gene expression or in situ dissipation of mitochondrial membrane potential produced data that prove that the amount and transmembrane distribution of the phospholipid are two specific parameters of the mitochondrial inner membrane organization in both fermentative (2.2 fmol/cell and 20:80, in/out) and gluconeogenic (4.2 fmol/cell and 37:63, in/out) growing yeast cells. Finally, the inner mitochondrial membrane topography of cardiolipin appeared to be closely associated with the transmembrane redox potential.