title: Constitutive activity in chimeras and deletions localize sensory rhodopsin II/HtrII signal relay to the membrane-inserted domain.
abstract: Halobacterium salinarum sensory rhodopsin II (HsSRII) is a phototaxis receptor for blue-light avoidance that relays signals to its tightly bound transducer HsHtrII (H. salinarum haloarchaeal transducer for SRII). We found that disruption of the salt bridge between the protonated Schiff base of the receptor's retinylidene chromophore and its counterion Asp73 by residue substitutions D73A, N or Q constitutively activates HsSRII, whereas the corresponding Asp75 counterion substitutions do not constitutively activate Natronomonas pharaonis SRII (NpSRII) when complexed with N. pharaonis haloarchaeal transducer for SRII (NpHtrII). However, NpSRII(D75Q) in complex with HsHtrII is fully constitutively active, showing that transducer sensitivity to the receptor signal contributes to the phenotype. The swimming behaviour of cells expressing chimeras exchanging portions of the two homologous transducers localizes their differing sensitivities to the HtrII transmembrane domains. Furthermore, deletion constructs show that the known contact region in the cytoplasmic domain of the NpSRII-NpHtrII complex is not required for phototaxis, excluding the domain as a site for signal transmission. These results distinguish between the prevailing models for SRII-HtrII signal relay, strongly supporting the 'steric trigger-transmembrane relay model', which proposes that retinal isomerization directly signals HtrII through the mid-membrane SRII-HtrII interface, and refuting alternative models that propose signal relay in the cytoplasmic membrane-proximal domain.
authors.affiliation: Center for Membrane Biology, Department of Biochemistry and Molecular Biology, University of Texas Medical School, 6431 Fannin Street, Houston, TX 77030, USA.
authors.forename: Jun
authors.initials: J
authors.lastname: Sasaki
authors.forename: Toshifumi
authors.initials: T
authors.lastname: Nara
authors.forename: Elena N
authors.initials: EN
authors.lastname: Spudich
authors.forename: John L
authors.initials: JL
authors.lastname: Spudich
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D019617:Halobacterium salinarum / Q000378:metabolism; D008954:Models, Biological; D008969:Molecular Sequence Data; D019633:Natronobacterium / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D025603:Sensory Rhodopsins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
chemical_list: D011993:Recombinant Fusion Proteins; D025603:Sensory Rhodopsins