Mol. Cell, published online 16 May 2013; doi:10.1016/j.molcel.2013.04.016

Quality control mechanisms in the early secretory pathway facilitate protein folding and oligomeric assembly. For example, ERp44 is an oxidoreductase that prevents orphan subunits of disulfide-linked oligomers from being secreted. However, it was unclear how cycling of ERp44 between the endoplasmic reticulum and cis-Golgi was related to thiol-mediated client retention. Vavassori et al. use 1-anilinonaphthalene-8-sulfonate–binding fluorescence spectroscopy and a maleimide-based probe to show pH-dependent accessibility of the reactive cysteine (Cys29) of ERp44 in vitro. In cells, if the normal decrease in pH in the early secretory pathway compared to the endoplasmic reticulum was neutralized, ERp44-client interactions, which are normally enhanced at this location, were inhibited. In silico analyses based on a previously published crystal structure predicted that Cys29 would be deprotonated and occluded by the C-terminal tail at neutral pH but protonated and accessible at low pH. Mutation of the residues that putatively stabilize protonated Cys29 abolished the pH sensitivity of ERp44-client interactions in vitro and rendered these interactions constitutive in cells. Taken together, these data support a model in which ERp44 engages clients in the early secretory pathway, shuttles them back to the endoplasmic reticulum and releases them, owing in part to a pH-dependent conformational change leading to occlusion of Cys29 by the C-terminal tail.