The Erg28p protein is localized to the endoplasmic reticulum, where it acts as a scaffold to tether the C-4 demethylase complex involved in the sterol biosynthesis pathway of Saccharomyces cerevisiae. However, due to the challenges involved in characterizing the interactions of membrane proteins, the precise region of Erg28p that is responsible for the assembly of this enzyme complex remains unknown. To address this question, mutants with serial truncations in the C-terminus of Erg28p were constructed based on a topology prediction of its transmembrane domain. Sterol profiles demonstrated that intermediates involved in the stepwise removal of the two C-4 methyl groups from the tetracyclic sterol ring were accumulated in the ERG28Δ135-447 strain. Homologous alignment of Erg28p further identified a highly conserved 10-amino acid sequence (63LS/QARTFGT/LWT72) within the truncated region of ERG28Δ136-273. Complementation of the BY4741/erg28 strain with the ScERG28Δ175-204 plasmid resulted both in a significant growth inhibition and a reduction of ergosterol biosynthesis compared with the plasmid without the Δ175-204 truncation. Furthermore, homology modeling of the Erg28p mutant indicated that the deletion of residues 63–72 significantly disrupted the 3D structure of the four parallel helices in Erg28p. Taken together, the data indicate that the region spanning amino acids 63–72 constitutes a key consensus motif within Erg28p that is required for sterol C-4 demethylation during ergosterol biosynthesis in S. cerevisiae.
DNA phosphorothioate (PT) modifications, with the nonbridging phosphate oxygen replaced by sulfur, governed by DndABCDE or SspABCD, are widely distributed in prokaryotes and have a highly unusual feature of occupying only a small portion of available consensus sequences in a genome. Despite the presence of plentiful non-PT-protected consensuses, DNA PT modification is still employed as a recognition tag by the restriction cognate, for example, DndFGH or SspE, to discriminate and destroy PT-lacking foreign DNA. This raises a fundamental question about how PT modifications are distributed along DNA molecules to keep the restriction components in check. Here, we present two single-molecule strategies that take advantage of the nucleophilicity of PT in combination with fluorescent markers for optical mapping of both single- and double-stranded PT modifications across individual DNA molecules. Surprisingly, PT profiles vary markedly from molecule to molecule, with different PT locations and spacing distances between PT pairs, even in the presence of DndFGH or SspE. The results revealed unprecedented PT modification features previously obscured by ensemble averaging, providing novel insights into the riddles regarding unusual target selection by PT modification and restriction components.
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