In order to analyze the information content of a chloroplast transit sequence, we have constructed and analyzed by in vitro assays seven substitution and 20 deletion mutants of the ferredoxin transit sequence. The N-terminal part and the C-terminal part are important for targeting, and in addition the C-terminal region is required for processing. A third region is important for translocation but not for the initial interaction with the envelope. A fourth region is less essential for in vitro import. Purified precursors were tested for their ability to compete for the in vitro import of radiolabeled wild-type precursor, which confirmed the important role in chloroplast recognition of both the N- and the C-terminal domain of the transit sequence. Monolayer experiments showed that the N terminus was mainly involved in the insertion into mono-galactolipid-containing lipid surfaces whereas the C terminus mediates the recognition of negatively charged lipids. A sequence comparison to other transit sequences suggests that the domain structure of the ferredoxin transit sequence can be extended to these sequences and thus reveals a general structural design of transit sequences.
A nisin-resistant (NIS r) variant of Listeria monocytogenes Scott A was isolated by stepwise exposure to increasing concentrations of nisin in brain heart infusion (BHI) broth. The NIS r strain was about 12 times more resistant to nisin than was the wild-type (WT) strain. Accordingly, higher nisin concentrations were required to dissipate both components of the proton motive force in the NIS r strain than in the WT strain. Comparison of the membrane fatty acyl composition of the sensitive strain with that of its NIS r derivative revealed no significant differences. From phospholipid head group composition analysis and phospholipid biosynthesis measurements during growth in the absence and presence of nisin, it could be inferred that the NIS r strain produces relatively more phosphatidylglycerol (PG) and less diphosphatidylglycerol (DPG) than the parent strain does. Monolayer studies with pure lipid extracts from both strains showed that nisin interacted more efficiently with lipids derived from the WT strain than with those derived from the NIS r strain, reflecting qualitative differences in nisin sensitivity. Involvement of the cell wall in acquisition of nisin resistance was excluded, since the WT and NIS r strains showed a comparable sensitivity to lysozyme. Recently, it has been demonstrated that nisin penetrates more deeply into lipid monolayers of DPG than those of other lipids including PG, phosphatidylcholine, phosphatidylethanolamine, monogalactosyldiacylglycerol, and di
This survey gives a short overview of the various reagents and procedures that can be used for pre-, post- and on-column derivatization in capillary electrophoresis. First there is an introduction about capillary electrophoresis as an analytical technique; this is followed by a discussion of the pros and cons of the various modes of derivatization and a comparison with liquid chromatography. In the following paragraphs the reagents for a number of functional groups are discussed. The emphasis is on derivatization of the amino group. Most of the information on the reagents and derivatization procedures is listed in tables together with information on the detection mode, analytes, sensitivity and samples. In addition to the amino group, information is given on labeling of aldehyde, keto, carboxyl, hydroxyl and sulfhydryl groups.
In order to get insight into the functioning of transit sequences in chloroplast protein transport, the import of the full-length transit peptide of ferredoxin (trfd) was investigated. trfd rapidly associated with chloroplasts under import conditions and becomes protected against externally added proteases. Import of radiolabeled trfd is inhibited equally efficiently by nonlabeled trfd as well as by the intact precursor of ferredoxin. This strongly suggests that trfd enters the general import pathway of proteins into chloroplasts. trfd import was stimulated by ATP, which is the first demonstration that ATP is involved in membrane translocation of a targeting signal. Imported trfd was membrane-associated but was also partially degraded by internal proteases, most likely present in the stroma, indicating that the membrane-associated fraction of trfd is en route to its functional localization. The degradation products are exported out of the organelle. In contrast to the import of the precursor of ferredoxin, the import of trfd was independent of protease-sensitive components on the chloroplast surface, indicating that the initial binding of precursor proteins may be facilitated by transit sequence-lipid interactions.
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