Kai Simons

Solubilization of membranes by detergents

This review focuses on work that deals with the mechanisms of detergent action in membrane solubilization including properties of detergents model lipid systems and detergent-protein interactions; in addition a possible sequence of events when detergents interact with biological membranes receives attention. More specific topics include: 1) properties of detergents in aqueous solutions (use in membrane studies and physical properties of detergent solutions); 2) solubilization of lipid bilayers; 3) binding of detergents to water-soluble proteins and membrane proteins (both denaturing and mild detergents); 4) effects of detergents on biological membranes (e.g. high-affinity binding and separations of lipids and proteins); and 5) detergent removal.

Solubilization of the membrane proteins from Semliki Forest virus with Triton X100

Abstract Increasing concentrations of Triton X100 have been found to cause stepwise dissociation of the membrane of Semliki Forest virus. The final stage of the breakdown process leads to solubilization of the membrane proteins which can be separated from the membrane lipids and the viral nucleocapsid by density gradient centrifugation in the presence of 0.05% Triton X100. Two different forms of Semliki Forest virus protein have been observed with sedimentation coefficients of approximately 4 S and 23 S. The 4 S aggregate appears to consist of two polypeptide chains complexed with about 75 molecules of Triton X100. The 23 S form is a rosette-like aggregate containing about 16 polypeptide chains and about 260 molecules of Triton X100. Sucrose alters the equilibrium between the 4 S and 23 S forms: removal of sucrose leads to association of the 4 S form to the 23 S form and addition of sucrose to dissociation. A scheme for the dissociation of the Semliki Forest virus membrane is presented which is discussed with reference to other biological membranes. It is suggested that Triton X100 and deoxycholate solubilize amphipathic membrane proteins by binding to the hydrophobic segments of these proteins.

Isolation and characterization of the membrane proteins of Semliki Forest virus

Abstract Three proteins, E 1 , E 2 , and E 3 , were isolated by SDS-hydroxylapatite chromatography from the membrane of Semliki Forest virus (SFV) grown in BHK cells. All the proteins contained covalently linked carbohydrates including glucosamine, mannose, galactose, fucose, and sialic acid. The molecular weights of E 1 and E 2 were estimated to 49,000 and 52,000, respectively, by SDS-polyacrylamide gel electrophoresis. These values were independent of the acrylamide concentration of the gels. E 3 contained 45% carbohydrate and migrated anomalously in SDS gels. Its molecular weight was calculated from its chemical composition to be 10,000. The structural proteins in SFV were found to be present in about equimolar ratios. Using peptide mapping E 3 was shown to be the other cleavage product of a nonviral protein (NVP 68), earlier shown to be the precursor of E 2 in SFV-infected cells.

The lipid class composition of Semliki Forest virus and of plasma membranes of the host cells

Abstract The lipid class composition of Semliki Forest virus grown in two clones of BHK21 cells was determined. The virus contained 0.41 mg of lipid per milligram of protein; of this 31% was neutral lipid, 61% phospholipid, and 8% glycolipid. The major neutral lipid was free cholesterol; the principal phospholipids were phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and sphingomyelin. The major glycolipids were tentatively identified as sialolactosyl ceramides. The molar ratio of cholesterol to phospholipid was about 1. The lipids of Semliki Forest virus were similar to those of plasma membrane isolated from either infected or uninfected host cells. However, the virus contained much less free fatty acids and a little more cholesterol than the plasma membrane. The lipid composition of whole BHK21 cells and their endoplasmic reticulum differed clearly from that of the virus. In the virus there are about 10,000 cholesterol-phospholipid pairs, which can be arranged in a bilayer type of structure in the space available in the envelope.

Studies on the amphipathic nature of the membrane proteins in Semliki Forest virus

Abstract The membrane glycoproteins E1 and E2 of Semliki Forest virus form spikes protruding from the external surface of the virion. They have been cleaved off by thermolysin or subtilisin leaving peptide segments in the membrane of the spikeless virus particles with a molecular weight of about 5000 enriched in hydrophobic amino acids. These peptides are soluble in chloroform/methanol and are solubilized into mixed micelles with Triton X100, with sodium dodecyl sulphate and with sodium deoxycholate. Peptide mapping studies show that each membrane glycoprotein has its own lipophilic peptide segment which presumably serves to anchor these proteins to the lipid membrane. The hydrophobic segments of the glycoproteins appear to be shielded from proteolysis not only by the lipids in the intact membrane but also by Triton X100 in the detergent-protein complexes obtained when this detergent is used to remove the lipid and solubilize the proteins.

The lipids of the plasma membranes and endoplasmic reticulum from cultured baby hamster kidney cells (BHK21)

Abstract Plasma membranes and endoplasmic reticulum of two clones of cultured baby hamster kidney cells (BHK21) were prepared by the method of Wallach and Kamat (D. F. H. Wallach and V. B. Kamat , Methods Enzymol., 8 (1966) 164). The two membranes differed significantly from each other in their neutral, phospho- and sphingoglycolipid compositions.

Isolation and partial characterization of highly purified protocollagen proline hydroxylase

A procedure for the isolation of relatively stable preparations of highly purified protocollagen proline hydroxylase is reported. The most purified enzyme preparation was homogeneous in the analytical eltracentrifuge, and showed only one minor contaminant in disc electrophoresis. This enzyme preparation synthesized about 600 μg of hydroxyproline per mg protein per h under the assay conditions used, with a saturating concentration of the synthetic polytripeptide (Pro-Gly-Pro) molecular weight 6600, as substrate. The enzyme had an S20w of 6.7. However, the elution pattern in gel filtration suggested a molecular weight of about 350 000. Amino acid analysis indicated that the enzyme preparation contained relatively large amounts of aspartic acid, glutamic acid, serine, glycine and lysine. The activity of the purified enzyme was strongly inhibited by mercuribenzoate-p , and the inhibition could be reversed by dithiothreitol. N-Ethylmaleimide was a less effective inhibitor. The results suggests that free sulphydryl groups are required for the activity of the enzyme. Although partially purified preparation of the enzyme hydroxylate lysine as well as proline residues in protocollagen, the highly purified enzyme preparations did not hydroxylate lysine residues. Thus two separate enzymes, protocollagen proline hydroxylase and protocollagen lysine hydroxylase, are probably involved in the hydroxylation of proline and in protocollagen.

Characterization of the Semliki Forest virus core and envelope protein

Abstract The core and envelope proteins of Semliki Forest virus have been purified. Amino acid analysis revealed a high lysine content in the core protein, whereas the envelope protein was found to be more hydrophobic in its character.

Solubilization of the semliki forest virus membrane with sodium deoxycholate

The effects of increasing concentrations of sodium deoxycholate on Semliki Forest have been studied. Sodium deoxycholate begins to bind to the virus at less than 0.1 mM free equilibrium concentration and causes lysis of the viral membrane at 0.9 +/- 0.1 mM free equilibrium concentration when 2.2 +/- 0.2 - 103 mol of sodium deoxycholate are bound per mol of virus. Liberation of proteins from the membrane begins at 1.5 +/- 0.1 mM sodium deoxycholate and the proteins released are virtually free from phospholipid above 2.0 mM sodium deoxycholate. The overall mechanism of sodium deoxycholate solubilization of the viral membrane resembles that of Triton X-100 and sodium dodecyl sulphate except that with sodium deoxycholate the various stages of membrane disruption occur at about 10-fold higher equilibrium free detergent concentrations. At sodium deoxycholate concentrations higher than 2.3 mM the viral spike glycoproteins can be separated by sucrose gradient centrifugation or gel filtration into constituent polypeptides E1, E2 and E3. E1 carries the haemagglutinating activity of the virus.

IDENTIFICATION OF A PRECURSOR FOR ONE OF THE SEMLIKI FOREST VIRUS MEMBRANE PROTEINS

We are studying the envelope of Semliki forest virus (SFV) as a model for membrane structure and biogenesis. Semliki forest virus and other group A arboviruses acquire their envelopes by budding through the host cell plasma membrane [ 1] . The viral envelope resembles the host cell plasma membrane in its lipid composition [2] , but has a much simpler protein composition: one protein with an apparent molecular weight of about 50,000 has been found in the membrane [3]. In Sindbis virus this protein has recently been split into two bands (Er and Ez) using discontinuous sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis [4]. Both of these polypeptides contain carbohydrate. Studies on SFV protein formation are facilitated by the fact that virus infection effectively shuts off host cell protein synthesis [5]. Attempts to find out whether the structural polypeptides of the virus are translated as primary gene products or in precursor form(s) have so far been inconclusive [6-91. Our previous studies have suggested a precursor role for a non-structural glycopolypeptide (NSP68) which is found in extracts of infected cells [lo]. This polypeptide has an apparent molecular weight of 68,000 in 5% acrylamide SDS-gels. Here we show that SFV contains two envelope polypeptides, El and Ea , and that these have different primary structures. We also show that NSP68 is a precursor for the envelope polypeptide Ez, which is confirmed by the isolation of a temperaturesensitive mutant of SFV, in which precursor cleavage is blocked.