Dynamics of chemoreceptor trimers of dimers: In vivo crosslinking studies

STUDDERT CA AND PARKINSON JS

Boca Ratón, Florida, USA

Congreso; VIIIth BLAST (Bacterial Locomotion and Signal Transduction) Meeting; 2005

Transmembrane chemoreceptors of the methyl-accepting chemotaxis protein (MCP) family form signaling complexes through binding interactions with the cytoplasmic CheA  and CheW proteins. In E. coli the receptor signaling complexes typically reside in tight clusters at the poles of the cell. In a previous study of cysteine-directed receptor crosslinking in vivo, we suggested that trimers of receptor dimmers are important building blocks of receptor clusters. We found that receptors of different detection specificities could join the same trimer, with probabilities proportional to their relative abundance in the receptor population. Moreover, trimer formation did not depend on the presence of CheA, CheW, or any other soluble component of the chemotaxis signaling pathway. In the present work we extended our in vivo crosslinking methods to assess the contributions of CheA and CheW to the stability of the receptor trimers of dimers. We used two different receptors (Tar and Tsr) with suitable cysteine reporters and a trifunctional maleimide reagent (TMEA) to capture two or three receptor subunits, ostensibly from the same trimer of dimers. The cells expressed the chromosomally-expressed Tar reporter constitutively, whereas expression of the plasmid-borne Tsr reporter was induced at the start of the experiment. At various times thereafter, the cells were treated with TMEA to follow the kinetics of appearance of mixed dimer- and trimer-sized receptor crosslinking products. We found that in the presence of both CheA and CheW, pre-formed trimers were very stable, as evidenced by slow incorporation of newly-synthesized receptors into mixed crosslinking products over the course of several cell generations. In the absence of ongoing protein synthesis, the proportion of mixed crosslinking products did not increase, even after long incubation times. In contrast, cells lacking CheA and/or CheW showed rapid appearance of mixed crosslinking products. Evidently, under these conditions, newly-synthesized receptors can freely mix with old ones to generate a population of trimers of dimers whose compositions reflect the overall proportions of the two receptor types. These findings suggest that receptors within polar clusters, which have formed ternary signaling complexes through CheA/CheW binding interactions, are somehow prevented from exchanging members with newly-made receptor molecules. Conceivably, in forming signaling teams, CheA/CheW bind to and stabilize receptor trimers of dimers, blocking further exchanges with the pool of receptor dimers. Alternatively, or in addition, the receptor cluster may physically shield many of its component signaling teams from encounters with newly-synthesized receptor molecules, which might only have access to receptors at the periphery of the cluster.