The Pivotal Role of Erp44 in Patrolling Protein Secretion Tiziana Tempio1,2 and Tiziana Anelli1,2,*

REVIEW The pivotal role of ERp44 in patrolling protein secretion Tiziana Tempio1,2 and Tiziana Anelli1,2,*

ABSTRACT et al., 2001; Pagani et al., 2000). Oxidized Ero1α recharges itself by Interactions between protein ligands and receptors are the main electron transfer to molecular oxygen (Tu and Weissman, 2002), language of intercellular communication; hence, how cells select which generates one molecule of H2O2 for every disulfide bond that is proteins to be secreted or presented on the plasma membrane is a formed in the nascent proteins. central concern in cell biology. A series of checkpoints are located The tenet of quality control (QC) is that only when a given step in along the secretory pathway, which ensure the fidelity of such protein their structural maturation process is complete can proteins move to signals (quality control). Proteins that pass the checkpoints operated downstream compartments. Two theories have been proposed to in the endoplasmic reticulum (ER) by the binding immunoglobulin explain the selectivity of protein secretion: cargo selection and bulk protein (BiP; also known as HSPA5 and GRP78) and the calnexin– flow. The first entails that export is promoted by cargo receptors, such – calreticulin systems, must still overcome additional scrutiny in the ER- as the lectins ER Golgi intermediate compartment (ERGIC) 53 kDa Golgi intermediate compartment (ERGIC) and the Golgi. One of the protein (ERGIC-53; also known as LMAN1), the ERGIC-53-like main players of this process in all metazoans is the ER-resident protein (ERGL; also known as LMAN1L) and the VIP-36-like protein 44 (ERp44); by cycling between the ER and the Golgi, ERp44 protein (VIPL; also known as LMAN2L) for glycoproteins (Breuza controls the localization of key enzymes designed to act in the ER but et al., 2004; Kwon et al., 2016; Neve et al., 2003; Nufer et al., 2003; that are devoid of suitable localization motifs. ERp44 also patrols the Yerushalmi et al., 2001; Zhang et al., 2009). According to the second secretion of correctly assembled disulfide-linked oligomeric proteins. model, proteins can proceed to downstream organelles unless Here, we discuss the mechanisms driving ERp44 substrate retained in, or retrieved to, a given compartment (Barlowe and recognition, with important consequences on the definition of ‘thiol- Helenius, 2016). mediated quality control’. We also describe how pH and zinc Bulk flow relies on the activity of QC systems to efficiently gradients regulate the functional cycle of ERp44, coupling quality recognize proteins that are not properly folded or assembled, to control and membrane trafficking along the early secretory prevent their transport (Fig. 1). In the ER, the same chaperones compartment. devoted to protein folding and maturation, such as the binding immunoglobulin protein (BiP; also known as HSPA5 and GRP78), KEY WORDS: ERp44, Protein folding, Quality control, Secretory calnexin (CNX; also known as CANX) and calreticulin (CRT; also pathway, Zinc known as CALR), act as controllers, recognizing hydrophobic stretches (Marcinowski et al., 2013) or sugar moieties (Tannous et al., Introduction 2015) in aberrant polypeptides (proximal QC). Additional QC steps Proteins that reside in the secretory pathway, as well as proteins occur in the ERGIC and in the Golgi (distal QC), in which the destined to traffic to the extracellular milieu or the plasma membrane, assembly of multimeric proteins is supervised (Anelli and Sitia, 2018; begin their journey in the endoplasmic reticulum (ER). There, they Shibuya et al., 2015; Sun and Brodsky, 2019). This QC mechanism undergo post-translational modifications, including N-glycosylation, was first described in 1990 to control the secretion of polymeric glycosylphosphatidylinositol (GPI) anchor addition and disulfide immunoglobulin M (IgM) (Fra et al., 1993; Sitia et al., 1990). A bond formation, which are controlled by specific enzymes (Braakman cysteine residue was shown to act as a three-way switch, mediating and Hebert, 2013). The insertion of disulfide bonds is crucial to the assembly, retention and degradation of unpolymerized subunits ensure the stability of polypeptides destined to the oxidizing (Fra et al., 1993). Post-ER QC steps also control the assembly of extracellular environment (Darby and Creighton, 1995). Certain multimeric receptors. Unassembled subunits of multimeric catalytic or allosteric disulfides also regulate protein function and membrane proteins, such as the major histocompatibility complex activity (Cook and Hogg, 2013). In mammalian cells, oxidative (MHC) class I and II, or the T-cell receptor are in fact recognized in folding is catalyzed by a large array of oxidoreductases belonging to the Golgi and retrieved to the ER (Dusseljee et al., 1998; Hughes the protein disulfide isomerase (PDI) family (Appenzeller-Herzog et al., 1997; Yamamoto et al., 2001) as a result of the activity of and Ellgaard, 2008). Most of these proteins have two cysteine different receptors (Briant et al., 2017; Yamasaki et al., 2014). residues in the active site (CXXC) that mediate disulfide interchange One of the main players in post-ER QC is the ER-resident protein reactions. When reduced, they act as reductases or isomerases; when 44 (ERp44) (Anelli et al., 2007). ERp44 belongs to the PDI protein oxidized, they transfer their disulfide bonds to nascent proteins family, although it has peculiar characteristics. First, it lacks the (Wang et al., 2015). In eukaryotic cells, the reduced PDI is recharged resolving cysteine residue in its active site (CRFS) (Anelli et al., primarily by the ER oxidoreductin 1 enzymes (Ero1α and Ero1β in 2002), a characteristic shared only with a few other PDI homologs, mammals) (Cabibbo et al., 2000; Frand and Kaiser, 1999; Mezghrani such as anterior gradient 2 (AGR2) and 3 (AGR3), and thioredoxin- related transmembrane protein 5 (TMX5; also known as

1Division of Genetics and Cell Biology, Vita-Salute San Raffaele University, TXNDC15); this excludes a role as a disulfide donor (Kozlov Milan 20132, Italy. 2IRCCS San Raffaele Scientific Institute, Milan 20132, Italy. et al., 2010), suggesting instead it could act as an isomerase. Second, and differently from most other family members, which reside in the *Author for correspondence ([email protected]) ER (reviewed in Hatahet and Ruddock, 2007), ERp44 is primarily

T.A., 0000-0002-9159-9164 localized in the ERGIC and cis Golgi (Anelli et al., 2007). Journal of Cell Science

1 REVIEW Journal of Cell Science (2020) 133, jcs240366. doi:10.1242/jcs.240366

Endoplasmic reticulum ERES ERGIC Golgi Fig. 1. QC steps along the secretory Secretion pathway. The structure of the secretory pathway is perfectly suited for timing the different modifications that complex Folding proteins must undergo to attain their (i) native state. Monomeric proteins (blue oval) (i) can proceed to the Golgi only if they have reached their native status. Oligomeric proteins stabilized by non- Assembly covalent interactions or buried disulfides (blue and violet dimer) (ii) can reach the (ii) downstream compartments only if they Cargo are properly folded and assembled. enrichment These steps are supervised by the

and selection proximal QC in the ER – unfolded

at ERES SS SS

(iii) SS proteins are thus retained and cannot

SS SS SS

Assembly SS HS SH SS leave the ER. Correctly folded proteins HS SH are enriched at ERES and transported to HS S H the ERGIC and the cis-Golgi. Some HS SSSH oligomeric soluble proteins, whose subunits are linked by exposed disulfide (iv) HSSSSS Stop SS HSSSSS bonds (orange ovals) (iii) undergo SH SS SH ERp44-dependent QC in post-ER compartments (distal QC). Likewise, some oligomeric transmembrane Assembly Transport proteins (light violet) (iv) are also to the PM scrutinized in post-ER compartments by distal QC, provided that all subunits Stop passed the proximal QC. It is not clear where protein oligomerization occurs, whether in the cis-Golgi or during the travel of the monomers from the ER to the Retain Retrieve Golgi. Only completely assembled structures can proceed and reach their Proximal QC: Distal QC: final destination; assembly intermediates correct folding and correct will be instead stopped and retrieved non-covalent assembly? oligomerization? to the ER.

Key Transmembrane Oligomeric protein Monomeric protein Oligomeric protein protein subunit

In this Review, we describe recent mechanistic evidence on how elegans (Box 1). This protein is abundant in professional secretory ERp44 escorts and regulates protein secretion, defining the set of cells (Human Protein Atlas) and its levels increase during B proteins derived from each cell that are secreted into the extracellular lymphocyte differentiation into plasma cells (van Anken et al., space (secretome), and at the same time controlling the composition 2003), concomitantly with the onset of IgM secretion. These results of the early exocytic organelles. In addition, we propose that ERp44 suggested that ERp44 could play a role in ER redox control, as it substrates can be grouped into clients, which are multimeric proteins covalently interacts with Ero1α (Anelli et al., 2002), as well as in that need the activity of ERp44 to be secreted in the correct protein folding, given it is overexpressed together with many other conformation, or partners, which in turn are ER-resident enzymes, folding enzymes during B to plasma cell differentiation (van Anken such as peroxiredoxin 4 (Prx4; also known as PRDX4) (Kakihana et al., 2003). et al., 2013), Ero1α (Anelli et al., 2003; Otsu et al., 2006), The generation of antibodies against ERp44 was crucial for us to endoplasmic reticulum aminopeptidase 1 (ERAP1) (Hisatsune understand that, while the overexpressed protein massively et al., 2015) and sulfatase modifying factor 1 (SUMF1) (Fraldi accumulates in the ER (Anelli et al., 2002), endogenous ERp44 et al., 2008). The partners require ERp44 for their intracellular also localizes to the ERGIC and cis-Golgi (Anelli et al., 2007). localization. The discussion about the mechanisms regulating ERp44 was observed to co-fractionate with the Golgi enzyme ERp44 substrate recognition, requires a revisiting of the concept mannosidase II in a proteomic analysis of secretory sub of thiol-mediated QC, which has implications in the mechanisms of compartments of the secretory pathway (Gilchrist et al., 2006) and covalent oligomerization. it was enriched in coat protein complex I (COPI) vesicles (Adolf et al., 2019). ERp44 localization is partially dependent on ERp44 intracellular localization interactions with ERGIC-53 (Anelli et al., 2007), an hexameric ERp44 was first identified in co-immunoprecipitation assays aimed transmembrane lectin that acts at the ER exit sites (ERES) as a cargo at isolating new Ero1α interactors (Anelli et al., 2003); its name receptor for a select glycoprotein clientele, such as the coagulation derives from the fact that it accumulates in the ER when factors V and VIII (Hauri et al., 2000; Zhang et al., 2009). ERGIC- overexpressed (Anelli et al., 2002). ERp44 appears to be 53 continuously shuttles between the ER and the Golgi, binding its 2+ conserved during evolution, first appearing in Caenorhabditis substrates in the ER, as a result of the high Ca concentration and Journal of Cell Science

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Box 1. ERp44 during evolution

Site II Zn2+ binding Site I Zn2+ binding H277 H281 H299 H328 H332 Caenorhabditis_elegans_F42G8.7 LLKLAPVMADGKVLTAVLRHFNKGLDDLPFLLIDQFTHSFPSPWKGNEIFAEGNIKQFVADLFNDNHHRKLHEKLNEL Caenorhabditis_elegans_C06A6.5 RSAINPLLADGHKFAHPLKHLGKTKEDLPVLAIDSFQHMYLFPDM-TQMNIPGKLREFVMDLHSGKLHKDFHENLDQR Caenorhabditis_elegans_C30H7.2 RKAINCLVGDGTIFKHPLSHLGKSESDLPVIAIDSFRHMYLFKNF-EDVNVPGKLREFVLDLHSGKLHREFHHGPDPV Drosophila_melanogaster KQNVNFLTADGKRFAHPLHHLGKSEDDLPLIAIDSFKHMYLFPHF-SDMYSPGKLKQFLQDLYSGKLHREFHYGPDPS Hippocampus_abdominalis KGSINFLHADCDKFRHPLLHIQKTPAECPVIAIDSFRHMYVFPDY-QDLNIPGKLKQFVLDLHSGKLHREFHHGPDPT Danio_rerio KGSINFLHADCDKFRHPLLHIQKTPADCPVIAIDSFRHMYVFPEF-SDLAVPGKLRQFVLDLHSGKLHREFHHGPDPT Mus_musculus KGTINFLHADCDKFRHPLLHIQKTPADCPVIAIDSFRHMYVFGDF-KDVLIPGKLKQFVFDLHSGKLHREFHHGPDPT Homo_sapiens KGTINFLHADCDKFRHPLLHIQKTPADCPVIAIDSFRHMYVFGDF-KDVLIPGKLKQFVFDLHSGKLHREFHHGPDPT Macaca_fascicularis KGTINFLHADCDKFRHPLLHIQKTPADCPVIAIDSFRHMYVFGDF-KDVLIPGKLKQFVFDLHSGKLHREFHHGPDPT Xenopus_tropicali KGTINFLHADCEKFRHPLLHIQKTPADCPVIAIDSFRHMYVFSDF-KDLSISGKLKQFVLDLHSGKLHREFHHGPDPT Xenopus_laevis KGTINFLHADCEKFRHPLLHIQKTPADCPVIAIDSFRHMYVFPDF-KDLS------: : .* : * *: * : *.: **.* * : ::

C-terminal tail Caenorhabditis_elegans_F42G8.7 IQKIVTETEEI--EKQASEEKVEKPTEKLEKHESVFNKLKPASTRYSFAKEEL--- Caenorhabditis_elegans_C06A6.5 MIELAKAKAARGITDDHEAQAPSTRPIDTTPPPSVFKELKPSDKRYSILQKSE--- Caenorhabditis_elegans_C30H7.2 ------TGN------QAPDTEPPPSTFEKLKPASSRYTILDKTEL-- Drosophila_melanogaster ------NDIEPDPH--TGKGTSPPESKFKELGPSKHRYTLLEKDEL-- Hippocampus_abdominalis ------DSTPGQEEFGGDGASKPPQSSFQKLAPSETRYTILGRDRDEL Danio_rerio ------DSTPGQQEENREVPSNPPESSFQKLAPSETRYTILR-DRDEL Mus_musculus ------DTAPGEQD--QDVASSPPESSFQKLAPSEYRYTLLRD-RDEL Homo_sapiens ------DTAPGEQA--QDVASSPPESSFQKLAPSEYRYTLLRD-RDEL Macaca_fascicularis ------DIAPGEQA--QDVASSPPESSFQKLAPSEYRYTLLRD-RDEL Xenopus_tropicali ------DVAPDQPT--EDIISNPPESSFQKLAPSEHRYTILRRDRDEL Xenopus_laevis ------

ERp44 is not present in bacteria, yeast or plants, but it is highly conserved in higher eukaryotes (Drosophila melanogaster 51% identity, Xenopus laevis 79%, Xenopus tropicalis 82%, Danio rerio 77%, Hippocampus abdominalis 76%, Mus musculus 93%, Macaca fascicularis 99%). The first and second Zn2+- binding sites (H299, H328 and H332 in magenta, and H277 and H281 in green, respectively) are maintained throughout metazoans, whereas residues in Zn2+-binding site III (H333 and Y78) are less conserved. Also, the terminal 20 amino acids of the ERp44 tail (in light blue in the alignment) are quite conserved, according to our BLAST analysis, suggesting common regulatory mechanisms among different species. Interestingly, we also found that Caenorhabditits elegans has three ERp44 homologs, C30H7, C06A6 and F42G8, which share 45%, 41% and 29% of identity with human ERp44, respectively. F42G8 and C06A6 have a proline instead of an arginine residue in the active site, just after the active cysteine (CPFS) and a very long C-terminal tail. In human ERp44, R30 forms a hydrogen bond with the tail in the closed conformation (Watanabe et al., 2017). Furthermore, R30 also interacts with substrates via hydrogen bonds (Yang et al., 2016). The presence of a proline instead of an arginine residue could lower the reactivity of the active cysteine (Poole, 2015), affecting substrate binding. Interestingly, C. elegans F42G8 also differs as to the residues surrounding the active site (ASWCPFS instead of conserved ADWCRFS). It would be of interest to unravel the substrate specificity of these ERp44 isoforms in C. elegans, where only Ero1 is present among the known ERp44 partners. In yeast, Ero1 has a long tail that ensures ER residency and is essential for activity (Pagani et al., 2001). In metazoans, the localization of both Ero1α and Ero1β largely depends on ERp44 (Anelli et al., 2003; Otsu et al., 2006), suggesting the primary function of the latter is the regulation of redoxstasis along the secretory pathway. To satisfy the increasing demand for QC, higher eukaryotes might have exploited the cycling properties of ERp44.

the neutral pH, and releasing them in the Golgi, because of a lower signals thought to regulate vesicular trafficking (Cancino et al., Ca2+ concentration and acidic pH (Itin et al., 1996; Appenzeller- 2014; Capitani and Sallese, 2009; Sallese et al., 2009). Herzog et al., 2004). The movements of ERGIC-53 are regulated by Not only is ERp44-ΔRDEL massively secreted, but it also exits the interactions of the amino acids of its C-terminal cytosolic tail the cell faster than a PDI lacking its KDEL sequence (Anelli et al., with COPII (for anterograde movements) and COPI (for retrograde 2007). This suggests that, in contrast to what occurs for other ER- movements) (Itin et al., 1995). ERp44 colocalizes and interacts non- resident enzymes, ERp44 has a lower affinity for the ‘ER matrix’,a covalently with ERGIC-53 (Anelli et al., 2007). This could be supramolecular complex in the ER involved in protein folding and important for ERp44 trafficking from the ER to the Golgi; indeed, composed of ER chaperones and folding enzymes (Ma and downregulation of ERGIC-53 or overexpression of an ER-localized Hendershot, 2004; Reddy et al., 1996). ERp44 could also exploit ERGIC-53 mutant (Itin et al., 1995) favors ERp44 accumulation in cargo receptors that favor its exit from the ER. the ER (Anelli et al., 2007). These studies described a new protein of the secretory pathway, The deletion of the C-terminal ERp44-retrieval RDEL sequence enriched in the ERGIC and in the cis-Golgi, which was regarded as causes massive ERp44 secretion, implying that ERp44 is actively a new player in protein folding and QC. brought back to the ER from the Golgi by KDEL receptors (KDELRs) (Anelli et al., 2003). KDELRs are Golgi-localized Structure and function of ERp44 transmembrane receptors that recognize soluble ER-resident The ERp44 active site proteins bearing C-terminal KDEL-like sequences (Lewis and ERp44 is composed of three thioredoxin (trx)-like domains (a, b and Pelham, 1990; Pelham, 1991). KDELRs play a key role in shaping b′) with a C-terminal tail ending with the RDEL motif (Fig. 2A). The the early secretory compartment, capturing substrates in the acidic redox active site (CRFS) is placed in the a domain (Anelli et al., milieu of the Golgi and retrieving them to the ER (Brauer et al., 2002). A key cysteine residue (C29) forms disulfide bonds with client

2019; Capitani and Sallese, 2009). KDELR activity generates proteins, whereas another cysteine in the a domain (C63) might be Journal of Cell Science

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A B Fig. 2. ERp44 structure. (A) Schematic diagram of ERp44. b ERp44 is composed of three thioredoxin (trx)-like domains ′ SH SH (a, b and b ) and a C-terminal tail. A leader sequence is responsible for the entering of ERp44 in the secretory C29 C63 C160 C212 C272 C289 RDEL a bЈ Leader ab bЈ C-tail pathway. The a domain contains two cysteine residues, C29 sequence C29 in the active site and C63, while two disulfides stabilize b T369 and b′ (Anelli et al., 2003). (B) Schematic representation of ERp44 3D structure in the closed conformation. At neutral Tail pH, ERp44 presents as a clover leaf, with the C-terminal tail C (in light blue) covering the active site (Wang et al., 2008a,b; b Watanabe et al., 2017). The positively charged region surrounding C29 in the active site is depicted in orange, the hydrophobic region present in the b′ domain is in violet. The Zn2+ T369 in the C-terminal tail can form a hydrogen bond with bЈ C29, which controls the tail movements (Vavassori et al., 2013). The C-terminal RDEL is depicted as a dark blue anchor. (C) Zn2+-induced binding conformational changes in ERp44. The top left image depicts the structure of ERp44 a at pH 7.2. C29 (depicted as orange balloons) forms hydrogen bonds with T369 in the tail (light blue) and is C-tail thereby poorly accessible to substrates. Upon binding of 2+ Metal-free monomer Zn2+-bound monomer Zn (red circle) via histidine residues 323, 328 and 332 (site I), ERp44 monomers undergo conformational changes that expose the C29 active site (top right image). Further Zn2+ binding favors ERp44 dimerization via H271 1and H277 (site II). An ERp44 dimer can bind up to five Zn2+ ions 2+ Zn (bottom image), two of which are bound to site III (formed by C29 on one molecule and 78 on the other molecule in the dimer). The two forms (open Zn2+ bound and dimeric Zn2+ bound) are likely in equilibrium (PDB 5XWM; the panel has been kindly provided by Satoshi Watanabe and Kenji Inaba, Tohoku University, Japan).

Zn2+-mediated non-covalent dimer involved in the release, as its deletion stabilizes the ERp44-client A partially open ERp44 conformation covalent complexes (Anelli et al., 2003; Wang et al., 2008a). Four The previously described structure (Wang et al., 2008a) shows additional cysteines are present in the molecule, forming two ERp44 in a closed conformation, with C29 hidden by the tail. structural disulfide bonds in b and b′ domains (Anelli et al., 2003). More recently, we found that the pH gradient between the ER and the Golgi (Wu et al., 2001) regulates ERp44 function; ERp44 is The closed ERp44 conformation inactive at the neutral pH of the ER (7.2), but active in the The first ERp44 crystals were obtained in 2008 through a more acidic pH of the Golgi (6.5) (Vavassori et al., 2013). In vitro crystallization process at a pH 7.5 (Wang et al., 2008a). In them, analyses correlated the pH level with the accessibility and the three trx-like domains were organized in a clover leaf reactivity of ERp44 active site. Accordingly, the neutralization configuration (Fig. 2B), with the long C-terminal tail covering the of post-ER compartments through downregulation of the hydrophobic region in the b′ domain, and the partially positively Golgi pH Regulator (GPHR) channel, responsible for Golgi charged residues around the C29 in the client-binding region. This acidification (Maeda et al., 2008), dramatically lowers the ability arrangement immediately suggested that the tail acted as a gate for of ERp44 to interact covalently with its substrates (Vavassori substrates. An unstructured loop is present between the b′ domain et al., 2013). and the tail, and is likely involved in movements of the tail itself. When the structures of ERp44 were solved at a higher resolution, Accordingly, mutants lacking the entire tail or only the terminal at pH 7.2 (corresponding to the ER) and at pH 6.5 (corresponding to part, facing C29, covalently bind a wider set of protein substrates the Golgi) (Watanabe et al., 2017), it became clear that, at pH 6.5, (Vavassori et al., 2013; Wang et al., 2008a). The threonine T369 in protonation of five conserved histidines (H157, H299, H328, H332 the terminal part of the tail forms a hydrogen bond with C29, which and H333) induces remarkable conformational changes (Fig. 2C), is important for partially controlling tail opening (Vavassori et al., namely the rotation of the b and b′ domains by 7° and 10°, 2013). When T369 is replaced by cysteine residue, a disulfide bond respectively, compared to the conformation at pH 7.2, and the is formed with C29, and ERp44 is unable to interact with its movement of the C-terminal tail. Altogether, these changes expose substrates and is constitutively secreted (Vavassori et al., 2013). positively charged regions in the a and b′ domains, important for the Thus, tail opening is crucial both for interaction with substrates and electrostatic interaction with ERp44 clients. This explains the for the exposure of the C-terminal RDEL and its interaction with the relevance of the pH gradient in regulating ERp44 activity

KDELR. (Watanabe et al., 2017). Journal of Cell Science

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The zinc-dependent dimer enzymes involved in protein folding and processing in the early Detailed analysis of the ERp44 structures suggests that the highly secretory pathway (a job shared finally with ERp44); instead, ERp44 conserved histidine residues in the b′ domain form a pocket to clients only require ERp44 activity to be correctly assembled and then accommodate zinc (Zn2+). Indeed, biochemical analyses showed secreted. Moreover, ERp44 has been proposed to interact non- that ERp44 binds Zn2+ with submicromolar affinity and this induces covalently with ERGIC-53 (Anelli et al., 2007; Cortini and Sitia, conformational changes resulting in the exposure of hydrophobic 2010) and with the ER-resident calcium channel inositol 1,4,5- surfaces and dimerization of the molecule (Watanabe et al., 2019). trisphosphate receptor type 1 (IP3R1) (Higo et al., 2005). Owing to the Hence, a new structure of ERp44 was solved in the presence of Zn2+ space restriction, we will not discuss the interaction between ERp44 and (Fig. 2C). When Zn2+ is available, ERp44 forms non-covalent IP3R1 in this Review. Below, we will focus on the covalent interactions homodimers. Monomers show a completely open conformation, of ERp44 with its known substrates, both clients and partners. with the tail released from the a domain. Each ERp44 monomer binds one Zn2+ ion via H299, H328 and H332 at the end of the b′ ERp44 clients domain with high affinity, constituting the Zn2+-binding site I. The While proximal QC prevents the premature exit of folding other three Zn2+-binding sites are present at the interface between intermediates and incompletely assembled proteins from the ER the two ERp44 monomers: site II (H277 and H281 of each (Adams et al., 2019; Ellgaard and Helenius, 2003; Sitia and monomer) and site III (C29, Y78 of molecule A and H333 of Braakman, 2003), ERp44 is involved in one of the post-ER QC molecule B). Thus, two ERp44 molecules can coordinate five Zn2+ steps, acting on protein subunits that have already passed the atoms (Watanabe et al., 2019). The binding of Zn2+ to site I proximal QC (Anelli et al., 2007). It supervises the correct assembly significantly increases the Zn2+ affinity of site II, facilitating the of multimeric proteins linked by disulfide bonds, such as IgM formation of the Zn2+-bridged dimer; deleting site III has minor but (Anelli et al., 2007), adiponectin (Wang et al., 2007), interleukin-23 detectable effects on the capacity of ERp44 to dimerize (Watanabe (IL-23) (Meier et al., 2019) and interleukin-12 (IL-12) (Alloza et al., et al., 2019). The physiological role of site III is currently under 2006) (Fig. 3). investigation. Therefore, ERp44 is a pH and Zn2+-dependent chaperone. It Immunoglobulin M might also serve as a resource for storing of Zn2+ in the secretory IgM biogenesis entails at least two sequential and independently pathway, similar to the role of metallothionein 1 and 2 in the cytosol regulated steps. The first is the assembly of two heavy (µ) and two (Kimura and Kambe, 2016). light chains (L) to produce µ2L2 monomers (Hendershot and Sitia, 2005). This step is common to other immunoglobulin isotypes and How Zn2+ drives ERp44 localization and activity is supervised by BiP and other ER-resident chaperones (Hendershot 2+ Considering that many secretory proteins require Zn , it is not et al., 1987; Hendershot and Sitia, 2005) (Fig. 3A). µ2L2 molecules surprising that numerous Zn2+ transporters are located along the released from the proximal QC are ready for oligomerization into 2+ exocytic pathway. At least four transporters, Zn transporter 4 hexamers (µ2L2)6 or pentamers (µ2L2)5-J chain (Hendershot and (ZnT4), ZnT5, ZnT6, ZnT7 (also known as SLC30A4–SLC30A7), Sitia, 2005). Their assembly requires µ2L2 subunits to form inter- that import this metal from the cytosol reside in the Golgi, while monomer covalent bonds between the penultimate cysteine of the molecules acting in the opposite way, the Zn2+ importer proteins secretory µ chain (C575) (Li et al., 2020; Pasalic et al., 2017). ERp44 (ZIPs), are present in the ER (Kambe et al., 2017; Suzuki et al., 2020; regulates IgM oligomerization with ERGIC-53 (Anelli et al., 2007); Tsuji et al., 2017). This distribution generates a steep Zn2+ gradient ERGIC-53 recognizes two conserved N-glycans in the µ chains between the Golgi and the ER, with profound implications in the (Giannone et al., 2017), providing an hexameric platform for IgM ERp44 functional cycle. Accordingly, genetic or pharmacologic Zn2+ polymerization (Anelli et al., 2007; Cortini and Sitia, 2010), ERp44 depletion induces the secretion of ERp44 and its clients, while Zn2+ forms disulfide bonds with unassembled IgM subunits preventing excess induces ERp44 accumulation in the ER (Watanabe their secretion (Anelli et al., 2007). ERp44 might also play an active et al., 2019). Thus, the Zn2+-induced conformational changes role in polymerization in cooperation with ERGIC-53 or other lectins. simultaneously expose the substrate and KDELR sites, modulating Accordingly, ERp44 and ERGIC-53 increase their expression, with the activity and localization of ERp44 (Watanabe et al., 2019). similar kinetics, during the differentiation of B lymphocytes to A crucial issue is whether and how Zn2+ and pH levels are plasma cells (Anelli et al., 2007), which are the cells responsible for co-regulated in the secretory pathway; ZnTs are Zn2+–protons the production of polymeric IgM (Anelli and van Anken, 2013). exchangers (Chao and Fu, 2004; Gati et al., 2017; Ohana et al., 2009; Shusterman et al., 2014), which may link the two modes of Adiponectin regulation. Altering pH would be expected to affect the activity of Adiponectin is a protein involved in the homeostasis of circulating Zn2+ transporters and, vice versa, alteration of their activity would glucose, which assembles into different higher-order complexes, be expected to affect the pH of the compartment in which they are with distinct biological properties (Pajvani and Scherer, 2003; working. Pajvani et al., 2004; Wang et al., 2008b). Starting from a minimal ensemble of trimers [low molecular mass (LMM)], inter-trimeric ERp44 functions and interactors disulfide bonds via cysteine 36 yield hexamers [middle molecular ERp44 substrates can be grouped into clients and partners. The mass (MMM)] and higher order assemblies [high molecular mass former include multimeric soluble proteins, assembled via disulfide (HMM)] with greater anti-diabetic activity (Pajvani et al., 2004). bonds, for which ERp44 has an essential role in controlling assembly Unlike in the case of IgM, for which IgM assembly intermediates (Alloza et al., 2006; Anelli et al., 2007; Wang et al., 2007; Meier are never secreted, all three adiponectin assembly structures are et al., 2019), whereas the ER enzymes that lack the ER-retention physiologically secreted and can be detected in circulation (Wang motif and rely on ERp44 for their intracellular localization are et al., 2008b). considered ERp44 partners (Anelli et al., 2003; Otsu et al., 2006; ERp44 covalently binds to the C36 of the fully oxidized forms of

Kakihana et al., 2013). We define them as partners as they are LMM and of MMM adiponectin, selectively preventing their Journal of Cell Science

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ER pH 7.2 ERGIC pH 6.7 cis-Golgi pH 6.5 A

BiP Loading on ERp44 release ERGIC-53 Oligomerization Release from BiP ERGIC-53 Secretion

+ L ERp44 binding μ μ μ L 2L2 to assembly

intermediates

S--S

Assembly S--S Immunoglobulin M M Immunoglobulin

ERGIC-53 KDELR Retrieval KDELR binding B

Adiponectin H SH SH S SH monomers SH SH Secretion

SH SH SH S--S Trimer SHSH Oxidation S--S SH assembly

Adiponectin LMM

LMM MMM HMM S--S Binding to ADN inhibited Ero1α ERp44 binding

KDELR binding Retrieval

S--S S--S Secretion + IL-12β SH SH α SH SH SH BiP

IL-23 S

S-- S H

SH BiP release

SH S

IL-23α - S-

Interleukin 23 S ERp44 H BiP +ERp44 binding

release SH

KDELR binding Degradation Retrieval

Fig. 3. ERp44 as the secretome gatekeeper. (A) Different steps in IgM assembly and ERp44-mediated QC. During IgM biogenesis, μ2L2 assembly is assisted by ER-resident chaperones, primarily the immunoglobulin heavy chain-binding protein (BiP) (red circle) and the protein disulfide isomerase (PDI) (not shown). The mid-blue repsresents the immunogluobulin domains of the heavy chain; L is depicted in green. These μ2L2 ‘monomers’ are assembled via disulfide bonds involving C575 in the highly conserved C-terminal μs tailpiece (shown in purple). ERGIC-53 hexamers (in yellow) provide a platform for the assembly of planar polymers, receiving μ2L2 subunits that have already passed the BiP-dependent checkpoint. In post-ER compartments, ERp44 recognizes C575 in incomplete polymers preventing their secretion. Only completely oligomerized IgM can be secreted. The complexes between ERp44 and IgM assembly intermediates are instead retrieved to the ER thanks to the KDELR. (B) Assembly and ERp44-mediated QC of adiponectin. Transport-competent trimers of adiponectin (LMM) can be secreted as such or further assembled into disulfide-linked hexamers (MMM) and oligomers (HMM). All forms can be released by cells. In the ERGIC–cis-Golgi, active ERp44 preferentially captures adiponectin intermediates (LMM and MMM) that contain oxidized cysteine residues (indicated in the figure with S–S). Then, the covalent complex between ERp44 and adiponectin assembly intermediates is retrieved to the ER by KDELRs. In the ER, Ero1α can compete with adiponectin for ERp44, favoring secretion of LMM isoforms (Hampe et al., 2015). As a result, Ero1α and ERp44 coordinately dictate the size of the adiponectin complexes secreted by adipocytes. (C) QC steps in IL-23 assembly. The IL-23α subunit is recognized by BiP during early steps of IL-23 biogenesis. ERp44 acts downstream of BiP, recognizing cysteine residues that are free in the native conformation of IL-23α. ERp44 retrieves IL-23α from the ERGIC–cis- Golgi compartment to the ER. BiP and ERp44 cooperate to maintain IL-23α in an assembly-competent state. The assembly with IL-12β inhibits chaperone interaction and results in secretion of the heterodimeric IL-23 complex, connected by a disulfide bond. During the first steps, if IL-23α does not assemble properly, it is targeted for degradation and ERp44 binding competes with this event. Journal of Cell Science

6 REVIEW Journal of Cell Science (2020) 133, jcs240366. doi:10.1242/jcs.240366 release and transforming them in reduced trimers, ready to be metalloenzymes (with better-known extracellular functions) are inserted in HMM (Hampe et al., 2015; Pajvani and Scherer, 2003; ERp44 partners, the ER-resident aminopeptidase I (ERAP1) Wang et al., 2007, 2008b) (Fig. 3B). A proportion of the reduced (Hisatsune et al., 2015) and SUMF1 (Fraldi et al., 2008). In the oligomeric LMM and MMW adiponectin forms can be secreted ER, ERAP1 regulates peptide trimming for class I antigen (Hampe et al., 2015). The combined activity of Ero1α and ERp44 presentation and, when secreted, it controls blood pressure via axis ultimately determines the size of adiponectin complexes, as angiotensin 2 cleavage (Hisatsune et al., 2015). SUMF1 in the ER Ero1α releases ERp44 from these complexes, thus favoring activates sulfatases (Dierks et al., 2005), but it can also be secreted adiponectin trimers insertion in HMM structures (Wang et al., and taken up by distant cells (Zito et al., 2007). 2007). The high affinity of Ero1α for ERp44 (Anelli et al., 2003) therefore creates a subtle regulatory mechanism that influences the ERp44 and redox partners – Ero1 and Prx4 fate of adiponectin and of other ERp44 substrates (Anelli et al., Both Ero1α and Prx4 interact weakly with different ER-resident 2003). oxidoreductases [ERp72, ERp46 or P5; also known as PDIA4, TXNDC5 and PDIA6 (or TXNDC7), respectively] (Araki et al., Interleukins IL-12 and IL-23α 2013) but only PDIA1 (also known as P4HB) and ERp44 are The heterogeneous ERp44 clientele also includes pro-inflammatory efficient in mediating their intracellular localization (Anelli et al., secreted cytokines, such as interleukin 12 (IL-12) (Alloza et al., 2003; Otsu et al., 2006; Kakihana et al., 2013). 2006) and interleukin 23α subunit (IL-23α) (Meier et al., 2019). Ero1α exists in two redox isoforms, an ‘active’ form OX1 and an IL-12 consists of a 35 kDa α subunit and a 40 kDa β subunit, and ‘inactive’ OX2 (Appenzeller-Herzog et al., 2008; Baker et al., has a crucial role in the differentiation of naïve T cells into interferon 2008). The selective binding of ERp44 to the OX1 form favors the α-producing T helper type 1 (TH1) cells (Hunter, 2005). IL-12α and accumulation of OX2, likely controlling the working cycle of Ero1α IL-12β monomers fold in the ER, where they transiently associate and the redox homeostasis in the ER (Anelli et al., 2002). with CRT (Alloza et al., 2006). Folded IL-12 subunits covalently Accordingly, covalent ERp44–Ero1α complexes are particularly interact with ERp44 (Alloza et al., 2006) and are assembled to form resistant to reducing agents (Anelli et al., 2002, 2003; Otsu et al., a secretable disulfide-linked heterodimer (Reitberger et al., 2017). 2006), suggesting that ERp44 not only retrieves Ero1α but can also Thus, CRT and ERp44 are involved in the sequential control of modulate its activity. IL-12 production; CRT assists the folding of IL-12 subunits, while Prx4 can covalently bind to ERp44 when oxidized by H2O2 ERp44 controls their assembly (Alloza et al., 2006). (Yang et al., 2016). The ERp44–Prx4 complex is then attacked by IL-23 is a heterodimeric cytokine composed of an IL-12β subunit PDIA1, which emerges in its oxidized state, ready to transfer its (shared with IL-12) and an IL-23α subunit. The assembly with the bond to incoming cargoes. At this stage, reduced ERp44 and Prx4 β-subunit and the formation of a disulfide bond between IL-12β and are ready for another cycle (Yang et al., 2016). As a result, ERp44 IL-23α results in the secretion of the heterodimeric IL-23 (Meier functions as backup retention machinery for Prx4, acting after et al., 2019; Oppmann et al., 2000). If IL-12β is absent, IL-23α is PDIA1 (Kakihana et al., 2013). degraded; its degradation is slowed down by the binding of ERp44 (Fig. 3C) (Meier et al., 2019). This provides an example of ERp44 and metalloenzymes – ERAP1 and SUMF1 competition between protein folding, degradation and export from In the ER, ERAP1 is essential for trimming peptides to the length of the ER, in which ERp44 has a key role. In immune cells, different nine residues, which is the optimal size for class I antigen presentation ER QC systems can monitor the assembly of IL-23 versus that of IL- (López de Castro, 2018). Like other aminopeptidases, ERAP1 needs 12, thereby distinguishing between these cytokines to direct Zn2+ to be active (Kochan et al., 2011). Considering the low immune responses; indeed, IL-12, but not IL-23, stimulates concentration of Zn2+ in the ER, it is likely that ERAP1 acquires Zn2+ differentiation of TH1 cells (reviewed in Teng et al., 2015). in downstream compartments. Here, Zn2+ exposes the client and Intriguingly, it was found that Zn2+ has immunoregulatory KDELR-binding sites of ERp44, which can now retrieve active properties (Haase and Rink, 2014) and upregulates IL-23α ERAP1 to the ER. However, a portion of ERAP1 molecules can transcription (Doi et al., 2015). Hence, Zn2+ levels might also proceed along the secretory pathway and be secreted (Hisatsune et al., influence IL-23 assembly by modulating the activity of ERp44. 2015). Overexpression or silencing of ERp44 decreases and increases These data demonstrate that, by acting at the ERGIC and cis- ERAP1 secretion, respectively (Hisatsune et al., 2015; Watanabe Golgi level, ERp44 has a key role in controlling the complete and et al., 2019). Thus, the levels of ERp44 control ERAP1 secretion and correct assembly of disulfide-linked oligomeric substrates. ultimately its function (Hisatsune et al., 2015; Watanabe et al., 2019). SUMF1, also known as formylglycine-generating enzyme ERp44 partners (FGE), is another ERp44 partner; its localization is modulated The group of ERp44 partners includes several enzymes that exert cooperatively by PDIA1, ERGIC-53 and ERp44 (Fraldi et al., most of their known activities in the ER, but lack KDEL-like motifs 2008). SUMF1 is essential to activate all sulfatases as they enter into (Fig. 4A). Among these, Ero1α–Ero1β and Prx4 are both involved the ER, by modifying a cysteine residue into a formyl-glycine in oxidative protein folding (Zito et al., 2010; Zito, 2013). Prx4, (Dierks et al., 2005). Its absence causes the loss of sulfatase activity, together with glutathione peroxidase 7 and 8, scavenges H2O2 with consequences in lysosomal catabolic pathways and in the molecules that might accumulate in the ER as a byproduct of metabolic pathway of several steroid molecules; this causes a oxidative folding or NADPH oxidase 4 (NOX4) activity (Ramming devastating disease called multiple sulfatase deficiency (Cosma and Appenzeller-Herzog, 2013; Tavender and Bulleid, 2010). The et al., 2003; Diez-Roux and Ballabio, 2005). In the ER, the covalent reason these enzymes lack a KDEL remains an open question; the interaction with PDIA1 modulates SUMF1 activity in a redox- hypothesis is that they could exert extracellular functions. As to dependent manner (Fraldi et al., 2008). For activation, SUMF1 Ero1 enzymes, yeast Ero1p has a tail, which is needed to keep it needs a cupric ion (Cu2+) (Knop et al., 2017), a rare metal in the ER; associated with membranes (Pagani et al., 2001); this tail is lost in thus, the interaction with ERGIC-53 promotes the export of SUMF1 2+ higher eukaryotes when ERp44 appears (Box 1). Moreover, two from the ER. Once in the Golgi, SUMF1 acquires Cu , binds to Journal of Cell Science

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A pH 7.2

Endoplasmic reticulum ERGIC Golgi Extracellular space

Extracellular redox Oxidative Ero1α blood coagulation protein Ero1β Retrieval folding Prx4 Extracellular redox?

Loading Control of blood with Zn2+ Peptide trimming pressure via for antigen ERAP1 angiotensin II presentation cleavage Loading Processing with Cu2+? SUMF1 Transcellular of sulfatases activity?

B Anterograde movement (via ERGIC-53 or another GPHR ZnTs cis-Golgi pH 6.5 ER pH ≥7 transporter?) Partially H+ H+ Zn open H+ Binding state H+ Zn Zn Closed ? Zn state ERGIC-53 SH His Zn SH Zn His Site1 Zn substrate Zn Zn Zn Site1 substrateSubstrate Zn Open Substrate state Zinc- release: Zn dependent Zn reductase? S--S homodimers bstrate su Retrieval Binding to KDELR

2+ Low Zn2+ KDELR High Zn ZIPs

Fig. 4. Regulating the composition of the early secretory pathway. (A) ERp44 dictates the ultimate localization of a series of ER enzymes that lack ER-retention motifs. Their retrieval may occur at different sites along the secretory pathway. The retrieval of redox enzymes (Ero1α,Ero1β in blue and Prx4 in green) occurs soon after exiting the ER. It is possible that the interaction occurs also in the ER, possibly depending on the redox state of the enzymes. The retrieval of ERAP1 (in purple) and SUMF1 (in yellow) might occur downstream, possibly to allow their binding to Zn2+ and Cu2+, respectively. Consistently with Cu2+ transporters being located distally with respect to Zn2+, SUMF1 may bind also to O-glycosylated ERp44 later in the Golgi compartment (medial and trans Golgi). (B) ERp44 cycle of action. ERp44 cycles continuously between the ER and the cis-Golgi. In the ER, the pH is almost neutral and the Zn2+ concentration is low, due to the activity of Zn2+ exporters, namely the Zrt- and Irt-like protein ZIP. Here, ERp44 is in a closed conformation with low affinity for its substrates. Interactions with ERGIC-53 or other cargo receptors may favor its exit from the ER. In the Golgi, the pH is more acidic due to the activity of the anion channel Golgi pH regulator (GPHR) and the Zn2+ concentration increases, due to the activity of Zn2+ transporters (ZnTs), acting as importers. This promotes ERp44 activation; ERp44 opens its tail and captures its substrates, via C29 (see Fig. 2). The ERp44–substrate complex is then recognized and retrieved by the KDELR to the ER, where the complex dissociates. In the Golgi, ERp44 also can form Zn2+-bridged homodimers, which dissociate upon an excess of substrates; it may also act as Zn2+-storage devices in the secretory pathway, akin to metallothionein 1 (MT1) and 2 (MT2) in the cytoplasm. In the ER, ERp44 also dissociates from the substrate, but the source of reducing power needed for this release is still not known.

ERp44 and is retrieved to the ER (Fraldi et al., 2008). The as to where the interaction with the substrate occurs (Sannino et al., observation that SUMF1 can also be secreted and taken up from the 2014); if substrate binding occurs before the site where O-glycans are medium in order to reach the ER to exert its role might allow added, ERp44 will be retrieved in a non-glycosylated form. By replacement therapies for multiple sulfatase deficiency (Zito et al., contrast, substrates binding in downstream compartments will cause 2007). Cells depleted of ERp44 show massive secretion of SUMF1 the accumulation of O-glycosylated ERp44. This is the case when and have lower sulfatase activity (Fraldi et al., 2008). This suggests SUMF1 is overexpressed, which suggests that the interaction between that ERp44 is essential for SUMF1 function (Fraldi et al., 2008). ERp44 and SUMF1 occurs after the medial-Golgi (Sannino et al., Hence, ERp44 patrols the intracellular localization of different 2014) (Fig. 4A). It is tempting to speculate that late retrieval is needed ER enzymes whose activity can also be required extracellularly. for SUMF1 to bind copper (Knop et al., 2017) in the trans-Golgi compartment (Lutsenko, 2016). Moreover, the ‘client-induced ERp44–substrate interaction along the secretory pathway retrieval’ mechanism also suggests that ERp44 could interact with ERp44 mutants unable to bind Zn2+ are secreted, unless binding of a highly abundant substrates with high affinity in the ER, as the substrate exposes their RDEL motifs. Since ERp44 can undergo presence of the substrate can facilitate the opening of the tail (Sannino

O-glycosylation, this ‘client-induced retrieval’ can yield information et al., 2014). Journal of Cell Science

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by the C-tail, likely represent ER-localized ERp44 (Wang et al., Box 2. How does ERp44 select its substrates? 2008a,b; Watanabe et al., 2017) (Fig. 4B). In the neutral pH of the The charges surrounding substrate cysteine residues and the cysteine ER, the presence of ZiP7 and absence of ZnTs (Kambe et al., 2017) oxidation state play fundamental roles in client selection by ERp44. keep the Zn2+ concentration low, preventing premature ERp44 A matter of charge activation. In downstream compartments, the more acidic pH could ERp44 preferentially recognizes negatively charged regions on its suffice to favor partial ERp44 opening (Watanabe et al., 2017). substrates (Watanabe et al., 2017; Yang et al., 2016), which interact with 2+ the positively charged substrate-binding surface around ERp44 C29. In Moreover, the much higher concentration of Zn in the downstream Ero1α, the cysteine residues involved in binding with ERp44 are located compartments, guaranteed by Znt5, ZnT6 and ZnT7 (Kambe et al., in negatively charged loops (Watanabe et al., 2017), as is the case for 2017), induces complete ERp44 activation (Watanabe et al., 2019). Prx4 (Yang et al., 2016). In addition, SUMF1 and ERAP1 have negatively In the Golgi, ERp44 can also form Zn2+-bridged homodimers, charged regions in their putative ERp44-binding sites (Kochan et al., which dissociate upon binding to substrates that are in excess 2011). Although IgM monomers cannot be secreted, adiponectin can be (Watanabe et al., 2019). secreted as LMM and MMM complexes. According to our analysis, more hydrophobic or partially positively charged amino acids are located ERp44 activity along the secretory pathway is thus the result of a 2+ around adiponectin C36 (…LPKGACTGWMA…). The partial negative coordinated effect of Zn and pH gradient between the ER and the charge given by the C-terminal tyrosine residue in the µ chain Golgi, to ensure client binding in the best-suited region of the (…TAGTCY) is important for IgM QC (Pasalic et al., 2017). However, secretory pathway. the presence of a negatively charged residue upstream of the C-terminal α λ cysteine residues in the Ig- and Ig- chains was shown to impact the Conclusions and future perspectives stringency of IgM retention and retrieval (Guenzi et al., 1994; Reddy et al., 1996). The data obtained so far depict ERp44 as a multifunctional 2+ A matter of oxidation molecule, regulated by pH and Zn , which incessantly cycles in the Glutathione disulfide (GSSG) or other electron acceptors would be early secretory pathway. ERp44 activity is crucial to maintain needed for reduced ERp44 to covalently bind reduced clients. However, secretome fidelity and to shape the molecular composition of the recent data suggest that ERp44 interacts preferentially with oxidized main secretory compartments. However, many questions are still Prx4 (Yang et al., 2016). Indeed, the negatively charged regions in the open regarding the mechanisms of action of ERp44. binding site are exposed only when the disulfide bond C87–C208 is present in Prx4, which can then bind ERp44 (Yang et al., 2016). In the case of adiponectin, C36 is proximal to an aromatic tryptophan residue How does ERp44 exit the ER? that controls its oxidation state (Hampe et al., 2015) promoting ERp44 To efficiently retrieve substrates from downstream binding. As for Ero1α, cysteine residues recognized by ERp44 are compartments, ERp44 should outnumber them; thus, it should involved in intramolecular disulfide bonds (Inaba et al., 2010). SUMF1 be actively sorted out of the ER. Proteins exit the ER by forms covalent dimers (Zito et al., 2005), but it is unclear which form specifically interacting with transporters that associate with coat ERp44 recognizes. ERp44 interacts with IL-23 via cysteine residues that protein complex II (COPII) proteins at ERES (Ma et al., 2017). are reduced in the native protein (Meier et al., 2019), but we cannot exclude that binding occurs when the two residues form a non-native The accumulation of exogenous ERp44 in the ER may reflect the disulfide that is attacked by ERp44. The preference for oxidized clients shortage of these transporters and thus the delayed exiting of this would avoid the need for oxidative sources, needed instead to catalyze protein from the ER (Anelli et al., 2002). A candidate could be the binding between reduced ERp44 and a reduced client. ERGIC-53, which binds and cooperates with ERp44 in IgM biogenesis and whose silencing leads endogenous ERp44 to accumulates in the ER (Anelli et al., 2007; Cortini and Sitia, 2010). It is still not clear whether the cooperation of ERp44 with ERp44 prefers oxidized cysteine residues in a negatively charged ERGIC-53 is limited to a few substrates or whether ERGIC-53, stretch in its substrates (Box 2). This indicates that ERp44 cannot be regulating its movement, is more broadly involved in ERp44 considered a canonical chaperone. The surfaces of recognition activity. between other enzymes of the PDI family and their substrates are hydrophobic (Hatahet and Ruddock, 2007), and typical chaperones Is there a possible extracellular role for ERp44? disfavor negative residues (Houben et al., 2020; Koopman and Most cell types efficiently retain ERp44; endogenous ERp44 Rudiger, 2020). This confirms that ERp44 has a different function secretion is induced only by inhibiting RDEL exposure via and that it recognizes folded proteins, in line with its role in the distal mutations or via Zn2+ chelation (Vavassori et al., 2013; secretory compartment. Watanabe et al., 2019). However, platelets and endometrial Thus, ERp44 acts at post-ER regions, interacting with its cells constitutively secrete endogenous ERp44 (Holbrook et al., substrates in different locations along the secretory pathway, 2010; Sannino et al., 2014). Neither the mechanisms nor the depending on the pH and Zn2+ gradient as well as on its affinity physiological relevance is known. ERp44-secreting cells might for the substrates. experience environmental changes in the ERGIC or cis-Golgi, such as a low Zn2+ concentration in these compartments, or From the structure to the cycle of action insufficient KDELR activity. Moreover, it is not clear which is As discussed above, ERp44 preferentially interacts with its the physiologically ERp44 secreted form; we assume it is the substrates in the ERGIC and the cis-Golgi. Accordingly, data Zn2+-free monomer. It will be of particular interest to determine from in vitro and cellular studies indicate that acidic pH and high whether secreted ERp44 plays novel signaling role(s). Zn2+ concentration induce conformational changes that eventually Secreted ERp44 could also act as a transporter for partners open the ERp44 tail to expose C29 and the surrounding positively that would be expected to serve extracellular roles, such as charged region; this allows interaction with the substrate (Vavassori Ero1α or ERAP1. et al., 2013; Watanabe et al., 2019). The different 3D structures The role of Zn2+ in regulating ERp44 dynamics and activity obtained likely represent snapshots of ERp44 in its functional cycle. (Watanabe et al., 2019) is also crucial to link QC in the secretory 2+ 2+ The closed structures at neutral pH, in which the active site is hidden pathway to Zn homeostasis. It is tempting to speculate that Zn Journal of Cell Science

9 REVIEW Journal of Cell Science (2020) 133, jcs240366. doi:10.1242/jcs.240366 imbalances could affect distal QC (and hence secretion and protein Roberto Sitia for criticisms, suggestions and discussions. We also thank Kenji Inaba composition of the ER) with wide-reaching effects on the entire and Satoshi Watanabe for their help in preparing Fig. 2C. organism. Conversely, modulating Zn2+ homeostasis could offer a Competing interests way to induce selective secretion of specific ER chaperones under The authors declare no competing or financial interests. ERp44 control. Funding Back to the ER – how can ERp44 release its substrates? The authors thank Fondazione Banca del Monte di Lombardia, Fondazione Another open question is how ERp44 releases its substrates, once in Telethon (grant no. GGP15059), Ministero dell’Istruzione, dell’Universitáe della Ricerca (MIUR)-PRIN (grant no. 2017XA5J5N) and Associazione Italiana per la the ER. Here, the higher pH allows the release of ERp44 from Ricerca sul Cancro (AIRC) IG 2019 (grant no. 23285) for financial support. KDELR, but it is still unknown which reaction favors the dissociation of the ERp44–substrate complex, a step that requires References reducing equivalents. Reduced molecules of the PDI family or Adams, B. M., Oster, M. E. and Hebert, D. N. (2019). Protein quality control in the reduced glutathione could act as reductase of the disulfide bond in endoplasmic reticulum. Protein J. 38, 317-329. doi:10.1007/s10930-019-09831-w – Adolf, F., Rhiel, M., Hessling, B., Gao, Q., Hellwig, A., Bethune, J. and Wieland, ERp44 substrate complexes, as demonstrated for the interaction F. T. (2019). Proteomic profiling of mammalian COPII and COPI vesicles. Cell with Prx4 (Yang et al., 2016). C63 of ERp44 could also be involved Reports 26, 250-265.e5. doi:10.1016/j.celrep.2018.12.041 in the release of the substrates. In addition, Zn2+ would be expected Alloza, I., Baxter, A., Chen, Q., Matthiesen, R. and Vandenbroeck, K. (2006). to be released from ERp44 in the ER; then, the almost neutral pH Celecoxib inhibits interleukin-12 alphabeta and beta2 folding and secretion by a novel COX2-independent mechanism involving chaperones of the endoplasmic reduces the reactivity of C29 (Vavassori et al., 2013), and both the reticulum. Mol. Pharmacol. 69, 1579-1587. doi:10.1124/mol.105.020669 neutral pH and the lower Zn2+ concentration should favor the closed Anelli, T. and Sitia, R. (2018). Mechanisms of oxidative protein folding and Thiol- conformation of ERp44. dependent quality control: tales of cysteines and cystines. In Oxidative Folding of Proteins: Basic Principles, Cellular Regulation and Engineering (ed. M. Feige), pp. 249-266 The Royal Society of Chemistry. Novel questions on post-ER QC Anelli, T. and Van Anken, E. (2013). Missing links in antibody assembly control. The recent data on ERp44–substrate interactions requires a new Intl. J. Cell Biol. 2013, 606703. doi:10.1155/2013/606703 interpretation of the so-called thiol-mediated retention (Sitia et al., Anelli, T., Alessio, M., Mezghrani, A., Simmen, T., Talamo, F., Bachi, A. and 1990). In its first definition, thiol-mediated retention was a Sitia, R. (2002). ERp44, a novel endoplasmic reticulum folding assistant of the thioredoxin family. EMBO J. 21, 835-844. doi:10.1093/emboj/21.4.835 mechanism able to recognize exposed thiols in assembly Anelli, T., Alessio, M., Bachi, A., Bergamelli, L., Bertoli, G., Camerini, S., intermediates (Fra et al., 1993; Sitia et al., 1990). This process Mezghrani, A., Ruffato, E., Simmen, T. and Sitia, R. (2003). Thiol-mediated was named ‘retention’ as its substrates do not acquire a mannose-6- protein retention in the endoplasmic reticulum: the role of ERp44. EMBO J. 22, 5015-5022. doi:10.1093/emboj/cdg491 phosphate modification (occurring in the Golgi); this suggested that Anelli, T., Ceppi, S., Bergamelli, L., Cortini, M., Masciarelli, S., Valetti, C. and they do not exit from the ER (Isidoro et al., 1996). Then it became Sitia, R. (2007). Sequential steps and checkpoints in the early exocytic clear that ERp44 is a key player in thiol-mediated retention, compartment during secretory IgM biogenesis. EMBO J. 26, 4177-4188. doi:10. cycling along the early secretory pathway (Anelli et al., 2003, 1038/sj.emboj.7601844 Appenzeller-Herzog, C. and Ellgaard, L. (2008). The human PDI family: versatility 2007; Watanabe et al., 2019). More recent studies indicate that packed into a single fold. Biochim. Biophys. Acta 1783, 535-548. doi:10.1016/j. ERp44 prefers certain disulfide bonds on its substrates, likely bbamcr.2007.11.010 recognizing them as ‘non-native’ (Box 2). Based on these Appenzeller-Herzog, C., Roche, A. C., Nufer, O. and Hauri, H. P. (2004). pH- observations, we should redefine this process as ‘cysteine- induced conversion of the transport lectin ERGIC-53 triggers glycoprotein ’ release. J. Biol. Chem. 279, 12943-12950. doi:10.1074/jbc.M313245200 mediated retrieval . Along these lines of discussion, the Appenzeller-Herzog, C., Riemer, J., Christensen, B., Sorensen, E. S. and mechanisms of interaction with other known ERp44 substrates, Ellgaard, L. (2008). A novel disulphide switch mechanism in Ero1alpha balances such as IgM assembly intermediates, ERAP1 and IL-23α, should ER oxidation in human cells. EMBO J. 27, 2977-2987. doi:10.1038/emboj. be better analyzed in the future. 2008.202 Araki, K., Iemura, S.-i., Kamiya, Y., Ron, D., Kato, K., Natsume, T. and Nagata, K. In addition, understanding how many QC checkpoints exist along (2013). Ero1-α and PDIs constitute a hierarchical electron transfer network of the secretory pathway represents a new emerging question. Clearly endoplasmic reticulum oxidoreductases. J. Cell Biol. 202, 861-874. doi:10.1083/ different post-ER QC steps exist for soluble or membrane multimeric jcb.201303027 proteins (Sicari et al., 2019; Sun and Brodsky, 2019). Focusing on Baker, K. M., Chakravarthi, S., Langton, K. P., Sheppard, A. M., Lu, H. and Bulleid, N. J. (2008). Low reduction potential of Ero1α regulatory disulphides ERp44 and its ability to bind different substrates in different sub ensures tight control of substrate oxidation. EMBO J. 27, 2988-2997. doi:10.1038/ compartments along the secretory pathway (Sannino et al., 2014) emboj.2008.230 (Fig. 4A) clearly suggests that a gradient of binding conditions Barlowe, C. and Helenius, A. (2016). Cargo capture and bulk flow in the early occurs, to ensure the best QC efficiency for each substrate. secretory pathway. Annu. Rev. Cell Dev. Biol. 32, 197-222. doi:10.1146/annurev- cellbio-111315-125016 A better understanding of the regulation of ERp44 activity could Braakman, I. and Hebert, D. N. (2013). Protein folding in the endoplasmic have broad implications. On the one hand, from a cellular point of reticulum. Cold Spring Harbor Perspect. Biol. 5, a013201. doi:10.1101/ view, ERp44 regulates the ER composition by controlling the cshperspect.a013201 secretion of ER enzymes. On the other hand, from a systemic point Brauer, P., Parker, J. L., Gerondopoulos, A., Zimmermann, I., Seeger, M. A., Barr, F. A. and Newstead, S. (2019). Structural basis for pH-dependent retrieval of view, data published until now indicate that an adequate ERp44 of ER proteins from the Golgi by the KDEL receptor. Science 363, 1103-1107. activity is important for maintaining glucose homeostasis and for doi:10.1126/science.aaw2859 controlling immune responses, by acting both on IgM and on IL- Breuza, L., Halbeisen, R., Jeno, P., Otte, S., Barlowe, C., Hong, W. and Hauri, 12 and IL-23. Whether ERp44 is related to diabetes or pathologies H. P. (2004). 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