Chen H, Kendall D A
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06269, USA.
J Biol Chem. 1995 Jun 9;270(23):14115-22. doi: 10.1074/jbc.270.23.14115.
Transmembrane segments of proteins generally consist of a long stretch of hydrophobic amino acids, which can function to initiate membrane insertion (start-stop sequences), initiate translocation (signal-anchor sequences), or stop further translocation of the following polypeptide chain (stop-transfer sequences). In this study, we have taken Escherichia coli alkaline phosphatase, a transported and water-soluble protein, and examined the requirements for converting it into a transmembrane protein with particular orientation. Since the wild type enzyme is transported, there is no predisposition against membrane translocation, yet it is not a membrane protein, so it does not possess any intrinsic membrane topogenic preferences. A series of potential transmembrane segments was introduced into an internal position of the enzyme to test the ability of each to initiate translocation, stop translocation, and adopt a particular orientation. For this purpose, cassette mutagenesis was used to incorporate new structural segments composed of polymers of alanines and leucines. The threshold value of hydrophobicity required to function as a stop-transfer sequence was determined. For a transmembrane segment of typical length (21 residues), this value is equivalent to the hydrophobicity of 16 alanines and 5 leucines. Interestingly, much shorter segments will also suffice to stop translocation, but these must be composed of more highly hydrophobic residues (e.g. 11 leucines). When the wild type amino-terminal signal peptide is deleted or made dysfunctional, sufficiently hydrophobic internal segments can initiate translocation of the following polypeptide and function as a signal anchor. Furthermore, in so doing, the orientation of the protein is changed from N(out)-C(in) to N(in)-C(out).
