Signal sequences isolated

The results described above in no way diminish the probability that various proteins are necessary for protein secretion, and that some of these proteins interact with signal sequences. Isolated signal sequences and precursor proteins have been shown to bind to proteins in the RER and to inhibit the translocation and processing of secretory proteins. 

The gene for this protein has also been isolated from A. faecalis and sequenced and expressed in Escherichia coli cells (Yamamoto et al., 1987). Like azurin, it contains an amino-terminal signal sequence, suggesting that it is secreted into the periplasm in A. faecalis, although not all of the blue protein is found in this fraction. Mutants of this protein are being made and characterized in T. Beppu s laboratory (Univ. of Tokyo) (personal communication, 1989). 

A cDNA clone encoding miraculin was isolated and sequenced. The encoded precursor of miraculin was composed of 220 amino acid residues, including a possible signal sequence of 29 amino acids.95 96 Attempts have been made to express these proteins in E. coli, yeast, and tobacco. While it was confirmed that these proteins were expressed in these organisms, miraculin expressed in E. coli and yeast showed no activity.97 The perception of 0.1 moll-1 of citrate after taking 1 pmoll-1 of miraculin corresponds to the sweetness of 0.4 mol l-1 of sucrose, which means that it is 400 000 times sweeter than sucrose on a molar basis. Interestingly, a mixture of miraculin with citrate did not elicit sweetness.97 Miraculin was denied approval for this purpose by the FDA. Miraculin also has no legal status in the European Union. 

A. Studies of Precursor Proteins and Isolated Signal Sequences.152... 

Hence, bacterial genetic results provide many suggestions of structure-function correlations in signal sequences. These results serve as a point of departure for studies that determine the physical properties of isolated signal sequences as a means of elucidating their mode of action (see Section VI). 

The signal hypothesis postulates the existence of several proteins necessary for secretion. These include the components of the SRP, which is proposed to bind to the signal sequence and block further translation of the mRNA coding for the mature protein SRP receptor, or docking protein, which relieves the translation block imposed by the SRP ribophorins, which bind the ribosome to the ER membrane signal peptidase and signal-peptide peptidase, discussed above and other proteins, which form a pore or transport apparatus in the membrane. Some of these proteins, the SRP, SRP receptor, signal peptidase, and ribophorins, have been isolated from eukaryotic cell extracts and characterized. 

The gene prlA was first identified as part of the E. coli secretion apparatus because mutations in the gene can restore export of signal-sequence mutants of the X-receptor protein (Emr et al., 1981). Subsequently, Ito et al. (1983) isolated a temperature-sensitive mutant, secY, that was pleiotropically defective in protein secretion. The prlA and secY genes are almost certainly identical (Ito et al., 1984). 

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