Giant bilayer vesicles were reconstituted from several lipids and lipid/cholesterol (CHOL) mixtures: stearolyloleoylphosphatidylcholine (SOPC), bovine sphingomyelin (BSM), diarachidonylphosphatidylcholine (DAPC), SOPC/CHOL, BSM/CHOL, DAPC/CHOL, and extracted red blood cell (RBC) lipids with native cholesterol. Single-walled vesicles were manipulated by micropipette suction and several membrane material properties were determined. The properties measured were the elastic area compressibility modulus K, the critical areal strain alpha c, and the tensile strength tau lys, from which the failure energy or membrane toughness Tf was calculated. The elastic area expansion moduli for these lipid and lipid/cholesterol bilayers ranged from 57 dyn/cm for DAPC to 1,734 dyn/cm for BSM/CHOL. The SOPC/CHOL series and RBC lipids had intermediate values. The results indicated that the presence of cholesterol is the single most influential factor in increasing bilayer cohesion, but only for lipids where both chains are saturated, or mono- or diunsaturated. Multiple unsaturation in both lipid chains inhibits the condensing effect of cholesterol in bilayers. The SOPC/CHOL system was studied in more detail. The area expansion modulus showed a nonlinear increase with increasing cholesterol concentration up to a constant plateau, indicating a saturation limit for cholesterol in the bilayer phase of approximately 55 mol% CHOL. The membrane compressibility was modeled by a property-averaging composite theory involving two bilayer components, namely, uncomplexed lipid and a lipid/cholesterol complex of stoichiometry 1/1.22. The area expansion modulus of this molecular composite membrane was evaluated by a combination of the expansion moduli of each component scaled by their area fractions in the bilayer. Bilayer toughness, which is the energy stored in the bilayer at failure, showed a maximum value at approximately 40 mol% CHOL. This breakdown energy was found to be only a fraction of the available thermal energy, implying that many molecules (approximately 50-100) may be involved in forming the defect structure that leads to failure. The area expansion modulus of extracted RBC lipids with native cholesterol was compared with recent measurements of intact RBC membrane compressibility. The natural membrane was also modeled as a simple composite made up to a compressible lipid/cholesterol matrix containing relatively incompressible transmembrane proteins. It appears that the interaction of incompressible proteins with surrounding lipid confers enhanced compressibility on the composite structure.

The 30-kDa movement protein (MP) is essential for cell-cell spread of tobacco mosaic virus in planta. To explore the structural properties of MP, the full-length recombinant MP gene was expressed in Escherichia coli, and one-step purification from solubilized inclusion bodies was accomplished by using anion exchange chromatography. Soluble MP was maintained at >4 mg͞ml without aggregation and displayed Ϸ70% ␣-helical conformation in the presence of urea and SDS. A trypsin-resistant core domain of the MP had tightly folded tertiary structure, whereas 18 aa at the C terminus of the monomer were rapidly removed by trypsin. Two hydrophobic regions within the core were highly resistant to proteolysis. Based on results of CD spectroscopy, trypsin treatment, and MS, we propose a topological model in which MP has two putative ␣-helical transmembrane domains and a proteasesensitive carboxyl terminus.T he plus-sense, 6.4-kb single-stranded (ss) RNA genome of tobacco mosaic virus (TMV) encodes a 17.5-kDa coat protein, a 30-kDa movement protein (MP), and proteins of 126 kDa and 183 kDa that function in virus replication (1). The MP is essential for cell-cell spread of infection (2, 3).Many proteins, including several plant virus movement proteins (3), are associated with intracellular and intercellular channels that permit passage of water, ions, metabolites, and signaling molecules into and between cells and cell compartments. Prokaryotic examples include porins (4), potassium channels (5), and aquaporins (6). Eukaryotic examples include aquaporins (7), gap junctions (8), translocation channels of the endoplasmic reticulum (ER; ref. 9), nuclear pore complexes (10), ryanodine receptors (11), and the acetylcholine receptor (12). In higher plants, the channels that mediate intercellular communication are termed plasmodesmata (3). Plasmodesmata allow passive transport of proteins of at least 50 kDa in young tobacco leaf tissues, but the size exclusion limit decreases as these tissues mature (13). TMV infection results in a temporary increase in the size exclusion limit of plasmodesmata from Ϸ0.4 kDa to Ϸ20 kDa in mature leaf epidermis and mesophyll tissues (14). Although the MP is required for this dramatic and transient increase in intercellular permeability, the mechanisms responsible are unclear (3).Many viruses replicate in association with ER membranes, and some viruses associate with the cytoskeleton of the host (15-22). MP behaves as an intrinsic membrane protein, promotes the formation of ER aggregates, and probably facilitates establishment of TMV replication complexes that contain viral RNA, replicase, and MP (16,20,[22][23][24]. Many recombinant viral MPs expressed in Escherchia coli bind ss nucleic acids in vitro without nucleotide sequence specificity (25-29). Thus, it was proposed that the MP functions as an intracellular and intercellular carrier of TMV RNA, at least in part by association with the cytoskeleton and ER membranes (15)(16)(17)22).Recombinant viral MPs typically form insoluble inclusion bodies (25,...

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