Functional reconstitution of the integral membrane enzyme, isoprenylcysteine carboxyl methyltransferase, in synthetic bolalipid membrane vesicles.

Three bipolar archaeal-type diglycerophosphocholine tetraether lipids (also known as bolalipids) have been prepared to determine (1) the influence of molecular structure on the physical properties of bolalipid membranes and (2) their impact on the functional reconstitution of Ste14p, a membrane-associated isoprenylcysteine carboxyl methyltransferase from Saccharomyces cerevisiae. Three bolalipids were synthesized: C20BAS, C32BAS, and C32phytBAS. These bolalipid structures differ in that the C20BAS derivative has a short sn-1 glyceryl diether C20H40 transmembrane alkyl chain and two ether-linked sn-2 n-decyl chains, whereas the C32BAS and C32phytBAS derivatives have a longer sn-1 diether C32H64 membrane-spanning chain and two ether-linked sn-2 n-hexadecyl or phytanyl chains, respectively. Differential scanning calorimetry and temperature-dependent 31P NMR was used to determine the gel-to-liquid crystalline phase transition temperatures of the bolalipids (C32BAS Tm > 85 degrees C; C32phytBAS Tm = 14 degrees C; and C20BAS Tm = 17 degrees C). The bolalipid lateral diffusion coefficients, determined by fluorescence recovery after photobleaching at 25 degrees C, were 1.5 x 10(-8) and 1.8 x 10(-9) cm2/s for C20BAS and C32phytBAS, respectively. The mobility of C32BAS could not be measured at this temperature. Ste14p activity was monitored by an in vitro methyltransferase assay in reconstituted vesicle dispersions composed of DMPC, C20BAS/E. coli polar lipid, C20BAS/POPC, C32phytBAS/E. coli polar lipid, and C32phytBAS/POPC. Ste14p activity was lost in vesicles composed of 75-100 mol % C20BAS and 0-100 mol % C32BAS but retained in vesicles with 0-50 mol % C20BAS and 0-100 mol % C32phytBAS. Confocal immunofluorescence microscopy confirmed the presence of Ste14p in 100 mol % C20BAS and 100 mol % C32phytBAS vesicle dispersions, even though the lamellar liquid crystalline phase thickness of C20BAS is only 32 A. Because Ste14p activity was not affected by either the gel-to-liquid-crystal phase transition temperature of the lipid or the temperature of the assay, the low activity observed in 75-100 mol % C20BAS membranes can be attributed to hydrophobic mismatch between this bolalipid and the hydrophobic surface of Ste14p.