Screening for transmembrane association in divisome proteins using TOXGREEN, a high-throughput variant of the TOXCAT assay

Abstract

TOXCAT is a widely used genetic assay to study interactions of transmembrane helices within the inner membrane of the bacterium Escherichia coli. TOXCAT is based on a fusion construct that links a transmembrane domain of interest with a cytoplasmic DNA-binding domain from the Vibrio cholerae ToxR protein. Interaction driven by the transmembrane domain results in dimerization of the ToxR domain, which, in turn, activates the expression of the reporter gene chloramphenicol acetyl transferase (CAT). Quantification of CAT is used as a measure of the ability of the transmembrane domain to self-associate. Because the quantification of CAT is relatively laborious, we developed a high-throughput variant of the assay, TOXGREEN, based on the expression of super-folded GFP and detection of fluorescence directly in unprocessed cell cultures. Careful side-by-side comparison of TOXCAT and TOXGREEN demonstrates that the methods have comparable response, dynamic range, sensitivity and intrinsic variability both in LB and minimal media. The greatly enhanced workflow makes TOXGREEN much more scalable and ideal for screening, since hundreds of constructs can be rapidly assessed in 96 well plates. Even for small scale investigations, TOXGREEN significantly reduces time, labor and cost associated with the procedure. We demonstrate applicability with a large screening for self-association among the transmembrane domains of bitopic proteins of the divisome (FtsL, FtsB, FtsQ, FtsI, FtsN, ZipA and EzrA) belonging to 11 bacterial species. The analysis confirms a previously reported tendency for FtsB to self-associate, and suggests that the transmembrane domains of ZipA, EzrA and FtsN may also possibly oligomerize.

The CD28 Transmembrane Domain Contains an Essential Dimerization Motif

“…To determine whether the CD28 TM domain can mediate protein dimerization, we used the Tox-Luc system, which was derived from the Tox-CAT system originally developed by Engelman and colleagues in 1999 (77, 82) ( Figure 2). The Tox-CAT system (and its derivatives with different reporters in place of CAT) has been widely used and validated to study the efficiency of TM interaction motifs in a wide variety of TM domain containing proteins (83)(84)(85)(86). In this bacterial expression assay, inclusion of a TM dimerization motif drives the activation of the ToxR transcription factor, which in turn induces levels of luciferase expression that is proportional to the efficiency of dimerization (Figure 2A).…”

Identifying ionic interactions within a membrane using BLaTM, a genetic tool to measure homo- and heterotypic transmembrane helix-helix interactions

“…These genetic tools convert the interaction of TMDs in a natural cell membrane into reporter gene expression. In particular, the ToxR-based assays in E. coli , which include ToxR 18 , TOXCAT 19 , as well as the recently developed dsTβL 20 and TOXGREEN 21 have provided insights into the TMD-driven homodimerization of single-span membrane proteins, where high-resolution structures are especially lacking. In these assays, TMDs of interest are fused to a cytoplasmic ToxR domain.…”

Transmembrane domain–driven PD-1 dimers mediate T cell inhibition

“…The TMDs of the 58 members of the human RTK family mediate a wide range of degrees of homodimerization, a property that has been quantified with the bacterial TOX R transcription factor TMD dimerization (TOXGREEN) assay (Fig. 3C) ( 33 , 37 ). In the TOXGREEN assay, a TMD of interest is expressed between a periplasmic maltose binding protein and a cytoplasmic TOX R dimerization–dependent transcription factor.…”

“…The TOXGREEN assay for TMD dimerization in Escherichia coli was performed at stationary phase as described ( 37 ). Briefly, TMDs were PCR-amplified with Nhe I and Bam HI flanking restriction sites and cloned into the pccGFPKAN plasmid (Addgene plasmid #73649).…”