Extended Data Fig. 2: Validation of G. intestinalis GET components.
A) ATPase assays with GiGet3 (blue) & GiGet3D53N (orange). Absorbance was measured at a wavelength of 360nm at varying ATP concentrations. Experiment was done in triplicate. The lines represent the data fit using a LOESS (LOcally Estimated Scatterplot Smoothing) regression92. B) A schematic of experimental set-up which was conducted twice. ScSsa1 transfers ScBos1 with an engineered BPA crosslinking site to GiSgt2. Crosslinking is initiated by UV exposure. C) A western blot visualizing the crosslinked GiSgt2/ScBos1 complexes before and after transfer and with and without UV light treatment. C) GiGet3 and putative GiTA proteins were recombinantly expressed in E. coli then purified 2-3 times (depending on putative client) utilizing an affinity column to the Strep-tag on the TA proteins. Get3 could only be visualized if the TA protein expressed and bound to the TA protein. For western analysis, eluted samples were run on a gel and transferred to a membrane. The membrane was split in half, marked by dotted line, with the top blotted with an anti-GiGet3 antibody and the bottom by an anti-SUMO antibody. Putative TA proteins tested (GL50803_005161, GL50803_003896, GL50803_0015983, GL50803_003869, GL50803_0010803, GL50803_009849 and GL50803_0024512) are arranged by increasing TMD hydrophobicity (labeled in parentheses) using the TM tendency scale. GiGet3 was clearly identified in the eluates of GL50803_0024512 and GL50803_009849.
