Sequences determining the cytoplasmic localization of a chemoreceptor domain.
The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.
Seligman
L
L
Department of Genetics, University of Washington, Seattle 98195, USA.
Bailey
J
J
Manoil
C
C
D000469:Alkaline Phosphatase / Q000235:genetics; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000648:ultrastructure*; D001483:Base Sequence; D002462:Cell Membrane / Q000235:genetics / Q000648:ultrastructure*; D016764:Cell Polarity; D004252:DNA Mutational Analysis; D004926:Escherichia coli / Q000235:genetics / Q000648:ultrastructure*; D008565:Membrane Proteins / Q000235:genetics / Q000648:ultrastructure*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000235:genetics / Q000648:ultrastructure*; D011993:Recombinant Fusion Proteins / Q000648:ultrastructure; D017384:Sequence Deletion; D012694:Serine / Q000378:metabolism; D013329:Structure-Activity Relationship
D001426:Bacterial Proteins; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; C057635:Tsr protein, Bacteria; D012694:Serine; D000469:Alkaline Phosphatase