Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1996 Nov;178(22):6475–6478. doi: 10.1128/jb.178.22.6475-6478.1996

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

B Perazzona 1, E N Spudich 1, J L Spudich 1
PMCID: PMC178533  PMID: 8932303

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 approximately 20% of the cytoplasmic portion of the protein.

Full Text

The Full Text of this article is available as a PDF (431.2 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aatsinki J. T., Lakkakorpi J. T., Pietilä E. M., Rajaniemi H. J. A coupled one-step reverse transcription PCR procedure for generation of full-length open reading frames. Biotechniques. 1994 Feb;16(2):282-4, 286-8. [PubMed] [Google Scholar]
  2. Bourret R. B., Borkovich K. A., Simon M. I. Signal transduction pathways involving protein phosphorylation in prokaryotes. Annu Rev Biochem. 1991;60:401–441. doi: 10.1146/annurev.bi.60.070191.002153. [DOI] [PubMed] [Google Scholar]
  3. Chen X., Koshland D. E., Jr The N-terminal cytoplasmic tail of the aspartate receptor is not essential in signal transduction of bacterial chemotaxis. J Biol Chem. 1995 Oct 13;270(41):24038–24042. doi: 10.1074/jbc.270.41.24038. [DOI] [PubMed] [Google Scholar]
  4. Chervitz S. A., Falke J. J. Lock on/off disulfides identify the transmembrane signaling helix of the aspartate receptor. J Biol Chem. 1995 Oct 13;270(41):24043–24053. doi: 10.1074/jbc.270.41.24043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Chervitz S. A., Falke J. J. Molecular mechanism of transmembrane signaling by the aspartate receptor: a model. Proc Natl Acad Sci U S A. 1996 Mar 19;93(6):2545–2550. doi: 10.1073/pnas.93.6.2545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Feinberg A. P., Vogelstein B. "A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity". Addendum. Anal Biochem. 1984 Feb;137(1):266–267. doi: 10.1016/0003-2697(84)90381-6. [DOI] [PubMed] [Google Scholar]
  7. Jung K. H., Spudich J. L. Protonatable residues at the cytoplasmic end of transmembrane helix-2 in the signal transducer HtrI control photochemistry and function of sensory rhodopsin I. Proc Natl Acad Sci U S A. 1996 Jun 25;93(13):6557–6561. doi: 10.1073/pnas.93.13.6557. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Krah M., Marwan W., Oesterhelt D. A cytoplasmic domain is required for the functional interaction of SRI and HtrI in archaeal signal transduction. FEBS Lett. 1994 Oct 24;353(3):301–304. doi: 10.1016/0014-5793(94)01068-4. [DOI] [PubMed] [Google Scholar]
  9. Krebs M. P., Hauss T., Heyn M. P., RajBhandary U. L., Khorana H. G. Expression of the bacterioopsin gene in Halobacterium halobium using a multicopy plasmid. Proc Natl Acad Sci U S A. 1991 Feb 1;88(3):859–863. doi: 10.1073/pnas.88.3.859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Krebs M. P., Spudich E. N., Khorana H. G., Spudich J. L. Synthesis of a gene for sensory rhodopsin I and its functional expression in Halobacterium halobium. Proc Natl Acad Sci U S A. 1993 Apr 15;90(8):3486–3490. doi: 10.1073/pnas.90.8.3486. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nieuwlandt D. T., Daniels C. J. An expression vector for the archaebacterium Haloferax volcanii. J Bacteriol. 1990 Dec;172(12):7104–7110. doi: 10.1128/jb.172.12.7104-7110.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Rudolph J., Oesterhelt D. Chemotaxis and phototaxis require a CheA histidine kinase in the archaeon Halobacterium salinarium. EMBO J. 1995 Feb 15;14(4):667–673. doi: 10.1002/j.1460-2075.1995.tb07045.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Spudich E. N., Spudich J. L. The photochemical reactions of sensory rhodopsin I are altered by its transducer. J Biol Chem. 1993 Aug 5;268(22):16095–16097. [PubMed] [Google Scholar]
  14. Spudich J. L., Bogomolni R. A. Mechanism of colour discrimination by a bacterial sensory rhodopsin. Nature. 1984 Dec 6;312(5994):509–513. doi: 10.1038/312509a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Spudich J. L. Color sensing in the Archaea: a eukaryotic-like receptor coupled to a prokaryotic transducer. J Bacteriol. 1993 Dec;175(24):7755–7761. doi: 10.1128/jb.175.24.7755-7761.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Spudich J. L. Protein-protein interaction converts a proton pump into a sensory receptor. Cell. 1994 Dec 2;79(5):747–750. doi: 10.1016/0092-8674(94)90064-7. [DOI] [PubMed] [Google Scholar]
  17. Summers W. C. A simple method for extraction of RNA from E. coli utilizing diethyl pyrocarbonate. Anal Biochem. 1970 Feb;33(2):459–463. doi: 10.1016/0003-2697(70)90316-7. [DOI] [PubMed] [Google Scholar]
  18. Yao V. J., Spudich E. N., Spudich J. L. Identification of distinct domains for signaling and receptor interaction of the sensory rhodopsin I transducer, HtrI. J Bacteriol. 1994 Nov;176(22):6931–6935. doi: 10.1128/jb.176.22.6931-6935.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Yao V. J., Spudich J. L. Primary structure of an archaebacterial transducer, a methyl-accepting protein associated with sensory rhodopsin I. Proc Natl Acad Sci U S A. 1992 Dec 15;89(24):11915–11919. doi: 10.1073/pnas.89.24.11915. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES