Deletion mapping of the sites on the HtrI transducer for sensory rhodopsin I interaction

Abstract

The phototaxis receptor sensory rhodopsin I (SRI) transmits signals through a membrane-bound transducer protein, HtrI. The genes for the receptor and transducer, sopI and htrI, respectively, are normally cotranscribed; however, previous work has established that fully functional interacting proteins are produced when htrI is expressed from the chromosome and sopI is expressed from a different promoter on a plasmid. In this report we show that in the membrane, concentrations of SRI from plasmid expression of wild-type sopI are negligible in the absence of HtrI protein in the cell. This requirement for HtrI is eliminated when sopI is extended at the 5-end with 63 nucleotides of the bop gene, which encodes the N-terminal signal sequence of the bacteriorhodopsin protein. The signal is cleaved from the chimeric protein, and processed SRI is stable in the HtrI-free membrane. These results suggest a chaperone-like function for HtrI that facilitates membrane insertion or proper folding of the SRI protein. Six deletion constructs of HtrI were examined to localize the interaction sites for its putative chaperone function and for HtrI control of the SRI photocycle, a phenomenon described previously. The smallest HtrI fragment identified, which contained interaction sites for both SRI stability and photocycle control, consisted of the N-terminal 147 residues of the 536-residue HtrI protein. The active fragment is predicted to contain two transmembrane helices and the first ϳ20% of the cytoplasmic portion of the protein.The integral membrane protein HtrI (22) transmits signals from the membrane-embedded phototaxis receptor sensory rhodopsin I (SRI) (18) to a cytoplasmic signal transduction pathway (14) that controls the flagellar motors of Halobacterium salinarium cells (for reviews, see references 13, 16, and 19). The primary sequence of HtrI predicts two transmembrane helices (TM1 and TM2) within the N-terminal ϳ60 residues, and these are followed by a hydrophilic cytoplasmic domain of ϳ480 residues. The C-terminal ϳ260 residues are homologous to the adaptation and signaling regions of eubacterial chemotaxis receptors (2). SRI photoreactions involving proton transfer reactions in the SRI photoactive site are altered when HtrI is genetically deleted from the membrane (15,17). This effect indicates that SRI and HtrI are physically associated in the cell, which is strongly supported by the isolation of a molecular complex of purified SRI and HtrI from H. salinarium membranes (14a). In the present work we report an additional influence of HtrI on SRI, namely, the facilitation of SRI insertion in a stable form in the membrane.The effects of HtrI on the SRI photocycle are readily measured by flash spectroscopy, providing an assay for receptortransducer interaction. In particular, HtrI-free SRI exhibits a strongly pH-dependent photocycle, which becomes pH insensitive in the SRI-HtrI complex (15). The pH sensitivity of the photocycle and the newly reported stabilization effects are exploited in this communication to determine from...

Five Residues in the HtrI Transducer Membrane-proximal Domain Close the Cytoplasmic Proton-conducting Channel of Sensory Rhodopsin I

“…This is a much shorter region than the 94 cytoplasmic residues found sufficient in a previous study (24), the key advantage here being the use of SRI-HtrI fusions that allowed much shorter HtrI fragments to be stabilized. (ii) The final 5 residues (62-66) are nearly completely responsible for the closure effect, and (iii) channel closure by this membrane-proximal domain requires the membrane-embedded domain of HtrI, which alone interacts with SRI altering to a small extent its properties, but without significantly closing the channel.…”

“…C-terminal deletion analysis showed that the N-terminal 147 residues of HtrI (full-length HtrI contains 536 residues) are sufficient to close the channel of SRI in the native H. salinarum membrane (24). Shorter fragments either were not expressed well and/or folded improperly in the H. salinarum cell.…”

Functional Importance of the Interhelical Hydrogen Bond between Thr204 and Tyr174 of Sensory Rhodopsin II and Its Alteration during the Signaling Process

“…Ϫ lacking the four archaeal rhodopsins (BR, HR, SRI, and SRII) as well as the two transducer proteins (HtrI and HtrII) was used for transformation (18) according to the protocol described previously (19). To obtain a high expression level, the stronger bop promoter was used instead of the native promoter (19).…”

Evidence for Post-translational Membrane Insertion of the Integral Membrane Protein Bacterioopsin Expressed in the Heterologous Halophilic Archaeon Haloferax volcanii

“…In ninaA mutants, rhodopsin is retained within the endoplasmic reticulum, and its levels are reduced by more than 100-fold (48). The membrane insertion of sensory rhodopsin I (SopI) found in H. salinarum is dependent on a chaperon-like function of its signal transducer, HtrI, which facilitates membrane insertion and protein folding of SopI (49). It was also shown that the chaperonin GroEL can promote post-translational membrane insertion of the multispanning membrane protein lactose permease in vitro (50).…”