The rotational diffusion of chloroplast phosphate translocator and of lipid molecules in bilayer membranes

Abstract

The rotational mobility of the phosphate translocator from the chloroplast envelope and of lipid molecules in the membrane of unilamellar azolectin liposomes has been investigated. The rotational dynamics of the liposome membrane were investigated by measuring the rotational diffusion of eosin-5-isothiocyanate(EITC)-labeled L-adipalmitoylglycerophosphoethanolamine (Pam2GroPEtn) in the lipid phase of the vesicles, either in the presence or absence of the reconstituted phosphate translocator.The temperature dependence of the anisotropy decay showed that above 25 "C the main contribution to the anisotropy decay was caused by uniaxial anisotropic rotation of the labelled lipid molecules around the axis normal to the membrane plane. The rate of rotation of the labelled lipid molecules was strongly dependent on the viscosity of the medium (ql). Extrapolation to q 1 = 0 Pa . s yielded a correlation time of $ = 20 5 ns, t = 30T, for lipid rotation with respect to the membrane normal. The rotational diffusion coefficient of the lipid molecules was calculated to be D, = 2.0 x lo9 rad' . s-' and the apparent microviscosity in the vesicle membrane, as derived from the rotational correlation time, was q2 z 12 mPa . s.The rotational correlation time of the phosphate translocator in the membrane was only slightly dependent on the viscosity of the medium. The temperature dependence of the protein rotation also indicated that the rotation of the protein in the membrane was largely restricted and occurred mainly about the axis normal to the membrane plane. Measurements at a medium viscosity of q 1 = 1 mPa . s yielded a value of $r z 450 ns corresponding to D, = 8.8 x lo7 rad' . s -' for protein rotation with respect to the membrane normal. From this value and the data of the lipid rotation, the cross-sectional area of the protein part embedded in the membrane was calculated to be z 9 nm2. This cross-sectional area is large enough to include at most 14 membrane-spanning helices.Our results also indicated that at lipid/protein molar ratios 2 1.5 x lo4: 1 aggregation occurred in the model membranes below 30°C. However, above 30°C and at a high dilution of the protein in the membrane it appeared that the membrane viscosity monitored by lipid and protein rotational diffusion were identical.The triose phosphate-3-phosphoglycerate-phosphate translocator (phosphate translocator) from the inner membrane of the chloroplast envelope catalyzes the export of fixed carbon (triose phosphates) from the chloroplast into the cytosol. This process plays a key role in the regulation of plant carbon metabolism (for review see [I, 21).The apparent molecular mass of the protein is 29 kDa and hydrodynamic studies in detergent solution indicate that the active unit is a dimer [3]. The phosphate translocator seems to function as a strict antiporter in a gated-pore fashion as proposed for the ADP/ATP carrier from the inner membrane of mitochondria [4].When reconstituted into liposomes the activity of the phosphate translocator was shown to be regulated by ...

Membrane Fluidity and Lipid Order in Ternary Giant Unilamellar Vesicles Using a New Bodipy-Cholesterol Derivative

“…Accordingly, we used both the average and slow rotational time constants to calculate the corresponding rotational diffusion coefficient and membrane microviscosity. Assuming that Bdp-Chol is a sphere with a radius of 0.54 nm, the apparent microviscosity of the DOPC fluid domain is 49 AE 1 cP (i.e., fluidity of~2.1 P À1 at room temperature) based on the average rotational time, which agrees with values in other studies (60)(61)(62)(63) in which different lipid types and fluorescent analogs were used. However, in using the average rotational time, the fast rotational components may be too fast to sample Bdp-Chol friction with other lipid molecules in the bilayer, thus underestimating the microviscosity.…”

Molecular aspects of plastid envelope biochemistry

“…The phosphate translocator, like other membrane proteins will probably consist of a number of membrane-spanning helices with connecting loops. Interestingly, the cross-sectional area of the protein part embedded in the membrane was calculated to be 9 nm2 [240]. This cross-sectional area is large enough to include at most 14 membrane-spanning helices.…”

Functional genomics of phosphate antiport systems of plastids

“…All TPTs are nuclear‐encoded and possess N‐terminal transit peptides that direct the adjacent protein to the chloroplasts. In its functional state, the TPT forms a homodimer and belongs to the group of translocators with a 6 + 6 helix‐folding pattern, similar to mitochondrial carrier proteins (Wagner et al 1989, Walker and Runswick 1993). It could also be shown that the recombinant TPT protein, overexpressed in a heterologous system (yeast cells), exhibited transport characteristics almost identical to those of the authentic chloroplast protein (Loddenkötter et al 1993).…”