Dynamic Retention of Ero1α and Ero1β in the Endoplasmic Reticulum by Interactions with PDI and ERp44

Abstract

Disulfide bonds are formed in the endoplasmic reticulum (ER) by sequential interchange reactions: Ero1alpha and Ero1beta transfer oxidative equivalents to protein disulfide isomerase (PDI), which in turn oxidizes cargo proteins. Neither Ero1alpha nor Ero1beta contains known ER localization motif(s), raising the question of how they are retained in this organelle. Here the authors show that, unlike endogenous molecules, overexpressed Ero1alpha and Ero1beta are secreted by HeLa transfectants, suggesting saturation of their normal retention mechanism(s). Co-expression of either PDI or ERp44 prevents Ero1 secretion in a KDEL/RDEL dependent way. Covalent interactions between ERp44 and Ero1 are essential for retention. In contrast, a mutant PDI lacking the four cysteines in the two active sites still inhibits secretion, albeit less efficiently. PDI and ERp44 compete for Ero1 binding. PDI also prevents Ero1 aggregation and dimerization, thus chaperoning its own oxidase. This dynamic retention mechanism of Ero1 may be important for fine-tuning the regulation of ER redox homeostasis and quality control.

Ero1α requires oxidizing and normoxic conditions to localize to the mitochondria-associated membrane (MAM)

“…Both yeast and rice Ero1 proteins require association with the ER membrane for their functioning (Onda et al 2009;Pagani et al 2001). Human Ero1α requires the interaction with PDI and another oxidoreductase, ERp44, for its retention within the ER (Anelli et al 2003;Otsu et al 2006). …”

Crystal structure of human ERp44 shows a dynamic functional modulation by its carboxy‐terminal tail

“…However, the absence of the second cysteine allows longer-lived mixed disulphides to be formed, a characteristic compatible with its role in quality control. ERp44 contributes to retain Ero1a and Ero1b intracellularly, the two rate-limiting factors in PDI oxidation (Mezghrani et al, 2001), by forming mixed disulphides through Cys 29 in its CRFS motif (Otsu et al, 2006). Similarly, ERp44 forms transient disulphides with immunoglobulin M subunits (Anelli et al, 2002(Anelli et al, , 2003(Anelli et al, , 2007, adiponectin (Wang et al, 2007) or formylglycine-generating enzyme (FGE; Mariappan et al, 2008), regulating their transport.…”

ERp44 C160S/C212S mutants regulate IP3R1 channel activity

“…2), suggesting that ERp44 C-tail is not required for the interaction between ERp44 and IP 3 Rs. Although ERp44 interacts with substrates through mixed disulphide bonds (Otsu et al, 2006), hydrophobic interactions are important in aligning Cys29 to target cysteines in the client proteins (Wang et al, 2008). In the present study, we showed that the inhibition of IICR by ERp44 was not affected by ERp44 C29S (Fig.…”

“…3), suggesting that the intermolecular disulfide bond between ERp44 and IP 3 R 1 is not involved in the inhibition of IICR by ERp44. Luminal-side regulation has been proposed to involve conformational changes of IP 3 R that affect the channel activity (Thrower et al, 2000;Otsu et al, 2006). Another possibility is that the intermolecular disulfide bond causes conformational change and generates a new molecular surface of IP 3 R 1 that can function as a novel binding site for other regulating proteins (Kang et al, 2008;Cortini and Sitia, 2010).…”