The N-terminal Cytoplasmic Tail of the Aspartate Receptor Is Not Essential in Signal Transduction of Bacterial Chemotaxis

Xiaomin Chen,Daniel E Koshland

doi:10.1074/jbc.270.41.24038

Xiaomin Chen, Daniel E Koshland

Open Access

https://doi.org/10.1074/jbc.270.41.24038

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Abstract

To determine the role in transmembrane signaling of the N-terminal peptide of the first transmembrane region of the aspartate receptor, it was subjected to extensive mutagenesis. Drastic changes did not alter the chemotactic ability of the receptor to aspartate significantly. Thus the cytoplasmic N terminus of the first transmembrane region does not play an essential role in transmembrane signaling, and the entire signal that is transmitted to the cytoplasmic domain must be sent through the second transmembrane region. This eliminates the models requiring an interaction of this N-terminal peptide with the remaining cytoplasmic portion of the receptor.

Highlights

Each aspartate receptor subunit has a periplasmic ligand binding domain, two transmembrane segments (transmembrane (TM)1 1 and TM 2), and one cytoplasmic domain
There might be some interactions between the cytoplasmic domain and the TM 1, probably through the cytoplasmic extension of TM 1, a six-residue tail at the Nterminal end of the receptor sequence, and this interaction might be important for transmembrane signaling
In order to optimize the overexpression of the aspartate receptor, an EcoRI restriction site was placed immediately upstream of the tars start codon so that the tars gene could be subcloned into another vector that has a strong promoter

Summary

MATERIALS AND METHODS

Bacterial Strains and Plasmids—Escherichia coli strain CJ236 (dut ung) was from BioRAD. Swarm Assay—The plasmids listed in Table II were used to transform E. coli strain RP8611, which has none of the four chemotactic receptor genes. Cells were grown from single colonies in a 1-ml Luria broth with 100 ␮g/ml ampicillin (LBϩAmp) at 30 °C overnight. They were inoculated onto the center of the minimal, aspartate, or tryptone plates, using a sealed Pasteur pipet tip. Cells containing the class B plasmids were grown in 50 ml of LBϩAmp over night at 37 °C. Cells containing the class C plasmids were grown in 10 ml of LBϩAmp overnight at 37 °C. The membrane prepared from RP3808 with pEMBL18 was TABLE I Comparison of the N-terminal amino acid sequences of some chemotactic receptors

Amino acid sequence

MFNIR MR

RESULTS

Mutant nH

DISCUSSION