ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family

Abstract

In human cells, Ero1-Lalpha and -Lbeta (hEROs) regulate oxidative protein folding by selectively oxidizing protein disulfide isomerase. Specific protein--protein interactions are probably crucial for regulating the formation, isomerization and reduction of disulfide bonds in the endoplasmic reticulum (ER). To identify molecules involved in ER redox control, we searched for proteins interacting with Ero1-Lalpha. Here, we characterize a novel ER resident protein (ERp44), which contains a thioredoxin domain with a CRFS motif and is induced during ER stress. ERp44 forms mixed disulfides with both hEROs and cargo folding intermediates. Whilst the interaction with transport-competent Ig-K chains is transient, ERp44 binds more stably with J chains, which are retained in the ER and eventually degraded by proteasomes. ERp44 does not bind a short-lived ribophorin mutant lacking cysteines. Its overexpression alters the equilibrium of the different Ero1-Lalpha redox isoforms, suggesting that ERp44 may be involved in the control of oxidative protein folding.

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

“…A determining factor for the Ero1α shift appears to be the Ero1α oxidation state. Characteristically, Ero1α appears in two oxidized states, OX1 and OX2, which are sensitive to incubation with DTT (Anelli et al 2002). We detected a partial, temporary shift of the Ero1α oxidation state to the reduced form after 30 min of hypoxia (Fig.…”

“…We hypothesized that Ero1α, being an ER luminal protein, would exhibit intra-ER motility. To test this idea, we first treated HEK 293 cells with reducing agents known to lead to secretion of over-expressed, fully reduced Ero1α via a disruption of the Ero1α ERp44 retention mechanism (Anelli et al 2002(Anelli et al , 2003. As expected, treatment of cells with 1 mM 2-mercaptoethanol (2ME) and 1 mM dithiothreitol (DTT) for 2 h led to a shift of the Ero1α signal on the Optiprep gradient that monitors the localization of Ero1α among the MAM, the rER, the Golgi, and the plasma membrane.…”

“…Thus, we incubated HEK 293 cells in a hypoxic chamber containing 1% oxygen. Since reducing agents such as 2ME or DTT cause a shift of the oxidation state of Ero1α from the fully oxidized OX1 and OX2 forms to the reduced form, we first aimed to determine whether hypoxia could have a similar effect (Anelli et al 2002). Indeed, the incubation in hypoxic atmosphere temporarily transformed a portion of Ero1α from the fully oxidized OX2 to the fully reduced form after 30 min of hypoxia (Fig.…”

ERp44 C160S/C212S mutants regulate IP3R1 channel activity

“…It has been commonly believed that ERp44 is an ER luminal protein and plays an important role in protein folding, thiolmediated retention (Anelli et al, 2002(Anelli et al, , 2003 and secreted molecules polymerizing (Alloza et al, 2006;Fraldi et al, 2008; Wang et al, 2008). Recent studies have shown that ERp44 is the first identified regulatory protein of IP 3 R 1 function from the ER lumen side (Higo et al, 2005).…”

“…ERp44 is firstly characterized as an ER resident protein functioning in protein folding, thiol-mediated retention (Anelli et al, 2002(Anelli et al, , 2003, secreted molecules polymerizing (Alloza et al, 2006;Wang et al, 2008), and identified as the first regulatory protein of IP 3 Rs from the ER lumen side. It has been reported that ERp44 affects calcium transient (Higo et al, 2005;Pan et al, 2011) and gene transcription (Pan et al, 2011) by binding to and inhibiting IP 3 receptor subtype 1 (IP 3 R 1 ).…”