To further examine the qualities of the heme interactions of these mutants, we added exogenous imidazole (a structural mimic of histidine that can displace transient heme ligands to each purified HCCS cocomplex and obtained UV vis absorption spectra.
Accordingly, interactions of these mutants with Nup93 and Nup205, which bind the N-terminal part of Nup53 (Fahrenkrog et al, 2000; Hawryluk-Gara et al, 2008), were unaffected as shown by GST pull downs (Supplementary Figure S1A).
Nevertheless, the phenotypic effects we see in pcm mutants, or as a result of pacman knockdown, and the genetic interactions of these mutants with other genes in the caspase pathway, are entirely consistent with protein being expressed from the increased reaper and hid mRNAs.
In the latter case, dFMRP granule formation induced by ΔRGG mutants in Schneider cells is likely due both to self-interactions of these mutants and to their interplay with endogenous FMRP (via the PP domain), which through its LC sequence likely contributes to FMRP granules formation.
Similar experiments using the IV 252/253 and KK 278/280 mutant forms of full length Pc2 also revealed a decrease in the interaction of these Pc2 mutants with nc-SUMO3 (Figure 4C).
We then use these metrics to generate databases in order to enable the quantitative phenotypic characterisation of various mutants, of interactions between these mutants, and of the extent of rescue by the forced expression of a gene in different mutant contexts.
Due to poor expression and stability of the Tob W93A mutant, we were unable to test the interaction of this mutant with CNOT7 in vitro, although the mutant expressed in mammalian cells for subsequent interaction assays in vivo.
The disassembly is driven not by direct interaction of the mutant BtubB with protofilaments, but by its sequestering wild type BtubA into inactive mutant dimers.
In order to further elucidate the interaction of this mutant with the enzyme's substrates, we carried out ITC binding studies.
Based on the in vitro transcription assays, we conclude that the adverse effect of the R60E mutation on trxB transcription is likely caused by either a weaker interaction of the mutant Spx with the trxB promoter, RNAP, or both.
