Functional protein synthesis was observed in cell-sized lipid vesicles following encapsulation of a gene-expression system. Expression of rsGFP (red-shifted green fluorescent protein) within individual vesicles was observed by fluorescence microscopy. Interestingly, at the early stage of the reaction, the expression efficiency inside the vesicle was remarkably higher than that in the solution outside. The synthesized rsGFP in individual vesicles is safe from attack by proteinase K added to the external aqueous solution. Studies on cell-sized vesicles expressing protein should contribute to a fundamental understanding of certain aspects of living systems and will be useful for practical applications, such as the construction of microreactors.

To gain some insight into the structural and functional roles of sterols in higher plant cells, various plant sterols have been incorporated into soybean phosphatidyicholine (PtdCho) bilayers and tested for their ability to regulate water permeability and acyl chain ordering. Sitosterol was the most efficient sterol in reducing the water permeability of these vesicles and stigmasterol appeared to have no significant effect.Vesicles containing 24fmethylcholesterol exhibited an intermediate behavior, similar to that of vesicles containing cholesterol. Cycloartenol, the first cyclic biosynthetic precursor of plant sterols, reduced the water permeability in a very effective way. Oftwo unusual plant sterols, 246-methylpollinastanol and 14a,24Udimethylcholest-8-en-3fi-ol, the former was found to be functionally equivalent to sitosterol and the latter was found to be relatively inefficient. 2H NMR experiments have been performed with oriented bilayers consisting of soybean PtdCho with sitosterol, stigmasterol, or 24-methylpoll nastanol. The results provided clear evidence that sitosterol and 24k-methylpolinastanol exhibit a high efficiency to order PtdCho acyl chains that closely parallels their ability to reduce water permeability. By contrast, stigmasterol shows a low efficiency for both functions. These results show that sitosterol and stigmasterol, two major 24-ethylsterols differing only by the absence or presence of the A22 double bond in the side chain, probably play different roles in regulating plant membrane properties; they also may explain why 9P,19-cyclopropylsterols behave as good surrogates of sitosterol.In mammalian cells, the bulk of free cholesterol is localized in the plasma membrane where it participates in the regulation of membrane fluidity and in the activity of many membrane-bound enzymes (1). Changes in cholesterol content have been shown to induce modifications in cellular functions, which depend on the dynamics of the cell-surface membrane such as the recognition of an external signal (1). In higher plant cells, the three typical phytosterols: sitosterol, and 24F-methylcholesterol [the mixture of 24R (i.e., campesterol) and 24S (i.e., 22-dihydrobrassicasterol) epimers] are also mainly concentrated in the plasma membrane (2). To investigate whether the different plant sterols play similar roles in higher plant cells, we started a study with well-defined model membrane systems prepared from soybean phosphatidylcholine (PtdCho), a major representative plant phospholipid, and various sterols in different molar ratios. Because of their high content in polyunsaturated fatty acyl chains (3), soybean PtdCho bilayers can be considered a valuable model of higher plant membrane. The present work deals with the effect of some plant sterols on water permeability and acyl chain ordering of these bilayers. Permeability changes were monitored by measuring the swelling rates of large unilamellar vesicles (LUVs) following an osmotic shock in a stopped-flow spectrophotometer. This method had been develo...

Background: Aureochromes in stramenopiles are thought to function as light-regulated transcription factors, although the molecular mechanism is unknown. Results: Monomeric AUREO1 is present in reduced conditions and undergoes dimerization upon illumination. Conclusion: Blue light-induced dimerization enhances the affinity for the target sequence. Significance: AUREO1 is useful for understanding the blue light responses of stramenopiles, and for optogenetics and biophysical analyses.

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