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The molecular networks that control endoplasmic reticulum (ER) redox conditions in mammalian cells are incompletely understood. Here, we show that after reductive challenge the ER steady-state disulphide content is restored on a time scale of seconds. Both the oxidase Ero1α and the oxidoreductase protein disulphide isomerase (PDI) strongly contribute to the rapid recovery kinetics, but experiments in ERO1-deficient cells indicate the existence of parallel pathways for disulphide generation. We find PDI to be the main substrate of Ero1α, and mixed-disulphide complexes of Ero1 primarily form with PDI, to a lesser extent with the PDI-family members ERp57 and ERp72, but are not detectable with another homologue TMX3. We also show for the first time that the oxidation level of PDIs and glutathione is precisely regulated. Apparently, this is achieved neither through ER import of thiols nor by transport of disulphides to the Golgi apparatus. Instead, our data suggest that a dynamic equilibrium between Ero1- and glutathione disulphide-mediated oxidation of PDIs constitutes an important element of ER redox homeostasis.

The orientation of signal–anchor proteins in the endoplasmic reticulum membrane is largely determined by the charged residues flanking the apolar, membrane-spanning domain and is influenced by the folding properties of the NH2-terminal sequence. However, these features are not generally sufficient to ensure a unique topology. The topogenic role of the hydrophobic signal domain was studied in vivo by expressing mutants of the asialoglycoprotein receptor subunit H1 in COS-7 cells. By replacing the 19-residue transmembrane segment of wild-type and mutant H1 by stretches of 7–25 leucine residues, we found that the length and hydrophobicity of the apolar sequence significantly affected protein orientation. Translocation of the NH2 terminus was favored by long, hydrophobic sequences and translocation of the COOH terminus by short ones. The topogenic contributions of the transmembrane domain, the flanking charges, and a hydrophilic NH2-terminal portion were additive. In combination these determinants were sufficient to achieve unique membrane insertion in either orientation.