An amphipathic sequence determinant of membrane protein topology.
We developed a screen involving alkaline phosphatase gene fusions to identify mutations altering the membrane topology of a bacterial chemoreceptor (Escherichia coli Tsr). We identified three informative classes of mutations causing increased export of the protein's normally cytoplasmic carboxyl-terminal domain. The first class consisted of deletions eliminating all or most of the membrane-spanning sequence (TM2) immediately amino-terminal to the cytoplasmic domain. The second class consisted of mutations altering a highly amphipathic sequence at the beginning of the domain. The third class of mutation was a deletion of an upstream spanning sequence (TM1). The amphipathic sequence appears to be a novel determinant of membrane topology whose function is not due to its positive residue density. The amphipathic character of the sequence is relatively well-conserved in chemoreceptors and their relatives. Although deletions removing the amphipathic sequence or TM1 alone caused only partial carboxyl-terminal domain export, a double mutation removing both caused efficient export. This result suggests that the two sequences function independently to promote normal membrane insertion. The independent functioning of the two sequences may help ensure that Tsr insertion is normally a high fidelity process.
Seligman
L
L
Department of Genetics, University of Washington, Seattle 98195.
Manoil
C
C
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D002462:Cell Membrane / Q000737:chemistry; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry; D004926:Escherichia coli; D007532:Isoleucine / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011487:Protein Conformation; D017384:Sequence Deletion
D001426:Bacterial Proteins; D008565:Membrane Proteins; D009838:Oligodeoxyribonucleotides; C057635:Tsr protein, Bacteria; D007532:Isoleucine