SEQUENCES DETERMINING THE CYTOPLASMIC LOCALIZATION OF A CHEMORECEPTORDOMAIN

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a si mple topology consisting of two membrane-spanning sequences (TM1 and T M2) separating a large periplasmic domain from N-terminal and C-termin al cytoplasmic regions. We analyzed the contributions of several seque nce elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions, The principa l findings were as follows, (i) The cytoplasmic localization of the C- terminal domain depended on TM2 but was quite tolerant of mutations pa rtially deleting or introducing charged residues into the sequence. (i i) The basal level of C-terminal domain export was significantly highe r in proteins with the wild-type periplasmic domain than in derivative s 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 positiv ely charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is a n overdetermined process directed by an interplay of sequences promoti ng and opposing establishment of the normal structure.