Fig. 2. Inter-receptor crosslinking tests with proximal reporter sites.

A. Schematic representation of a crosslinking experiment in which two different receptors bear cysteine reporters at the same or closely adjacent levels. Individual dimers represent Tsr (light gray) or Tar (dark gray) molecules; small circles represent cysteine replacements. The three dimers form trimers through interactions between their tips (dark region). Upon TMEA treatment, cells expressing both receptor types can form pure 3-subunit crosslinking products (Tsr-only, left; Tar-only, right) and may also form mixed crosslinking products (center), if the reporter sites are sufficiently close to one another.

B. Cross section views of mixed trimers bearing reporters in adjacent levels. Left: Tar 436C (level II), Tsr 440C (level III). Right: Tar 430C (level II), Tsr 440C (level III). Cross section images were made with Pymol, viewing the trimer of dimers from the cytoplasm towards the membrane. Residues between the cytoplasmic tip and the corresponding level are hidden. Tsr subunits are light gray, with the cysteine reporters black. Tar subunits are dark gray, with the cysteine reporters white. A schematic representation of the TMEA crosslinking reagent (center) is shown to scale in the center of each trimer.

C. UU1581 cells carrying pCS12 Tsr-E440C and pPA818 Tar-6his-E436C or pPA818 Tar-6his-V430C were grown to mid-log phase and treated with TMEA. Tar expression was induced at 10 µM IPTG and Tsr expression at 0.15 (+), 0.30 (++), or 0.60 (+++) µM sodium salicylate. Cells expressing each receptor individually were also included to show the mobility of the pure crosslinking products. Samples were analyzed by SDS-PAGE in long, 9% acrylamide gels, and visualized by immunoblotting with an anti-Tsr antibody that reacts with both receptors. Cartoons on the left indicate the region of the gel where 2- and 3-subunit crosslinking products are found. Mixed crosslinking products are indicated with asterisks.