Signal sequences with lipids

Thus, protein secretion in E. coli is dramatically affected by the physical state of the membrane lipids. Both increased and decreased fluidity inhibit secretion. It is possible that this effect arises because the signal sequence and/or the secreted protein interact with the membrane lipids, and these interactions are perturbed when the lipid fluidity is changed. However, changes in lipid fluidity also affect other membrane functions such as active transport (DiRienzo and Inouye, 1979). Thus the effect of membrane fluidity on protein secretion may be due to altered activity of a membrane-bound part of the secretory apparatus, and may not be an indication of signal sequence-membrane interaction. 

Fig. 7. The increase in surface pressure of phospholipid monolayers as a function of signal-peptide concentration for the various E. coli LamB synthetic signal sequences (from Briggs, 1986). A monolayer of egg phosphatidylethanolamine and egg phosphatidylgly-cerol (65 35) was spread from a benzene solution onto 5 mM Tris buffer, pH 7.3, yielding a hnal surface pressure of 20 dyn/cm after evaporation of the benzene. The peptide was added by injecting a concentrated solution below the lipid-water interface. The surface pressure was measured by the du Noiiy ring method with a Fisher Autotensiomat equipped with a platinum-iridium ring. The plateau values are plotted as a function of the peptide concentration for the wild-type (O), Pro— Leu pseudorevertant (A), and deletion-mutant ( ) peptides.

The experiments described in Sections VI,A,B show that two physical properties of the synthetic LamB signal peptides correlate with their in vivo export function tendency to adopt an a-helical conformation in hydrophobic environments, and tendency to insert into lipid mono-layers. These properties may be involved in the same step in the secretion process, or in different steps. An a-helical conformation may be required to generate a structure sufficiently hydrophobic to allow mono-layer insertion. Alternatively, these properties may reflect separate roles of the signal sequence in protein secretion. For instance, an a-helical conformation may be necessary for binding to a proteinaceous site, while the ability to interact with lipids may be important for another step in the secretion process. We have studied the conformations of the synthetic LamB signal peptides in phospholipid vesicles and monolayers by CD and IR spectroscopy. 

An even more striking comparison can be made between the wild-type signal peptide s conformation when adsorbed to the monolayer and its conformation in aqueous solution. In both of these environments, the peptide should be solvated by water, but its conformations are very different. The peptide is 100% )3 structure when adsorbed to the mono-layer, while it is 80% random in aqueous buffer. Thus, it appears that contact with the lipid surface induces substantial amounts of secondary structure in a molecule that takes on little structure in an aqueous environment. This finding implies that the initial binding of a signal sequence to a membrane may induce a particular structure, which may be important to the mechanism of signal-sequence function. 

Incubations with Cloned, Expressed Enzymes Individual UGT enzymes have been expressed in a wide variety of systems including insect cells (Supersomes or Baculosomes), Escherichia coli, yeast, and mammalian cells. Zakim and Dannenberg have demonstrated that the lipid membrane composition can influence activity (Zakim, 1992). There tends to be excellent protein expression insect cells transfected with baculovirus, but when activity is measured compared to mammalian cells systems, there appears to be significant amounts of inactive protein due to either poor membrane insertion or improper folding (lack of chaperones ). Bacteria do not have an ER, but alteration of the signal sequence results in active membrane bound preparations. Yeast and mammalian cells such as HEK293 or V79 cells have a more typical membrane environment and may be preferable for expression of ER proteins. 

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