A New Role for Annexin A11 in the Early Secretory Pathway via Stabilizing Sec31A Protein at the Endoplasmic Reticulum Exit Sites (ERES)

Exit of cargo molecules from the endoplasmic reticulum (ER) for transport to the Golgi is the initial step in intracellular vesicular trafficking. The coat protein complex II (COPII) machinery is recruited to specialized regions of the ER, called ER exit sites (ERES), where it plays a central role in the early secretory pathway. It has been known for more than two decades that calcium is an essential factor in vesicle trafficking from the ER to Golgi apparatus. However, the role of calcium in the early secretory pathway is complicated and poorly understood. We and others previously identified Sec31A, an outer cage component of COPII, as an interacting protein for the penta-EF-hand calcium-binding protein ALG-2. In this study, we show that another calcium-binding protein, annexin A11 (AnxA11), physically associates with Sec31A by the adaptor function of ALG-2. Depletion of AnxA11 or ALG-2 decreases the population of Sec31A that is stably associated with the ERES and causes scattering of juxtanuclear ERES to the cell periphery. The synchronous ER-to-Golgi transport of transmembrane cargoes is accelerated in AnxA11- or ALG-2-knockdown cells. These findings suggest that AnxA11 maintains architectural and functional features of the ERES by coordinating with ALG-2 to stabilize Sec31A at the ERES.     

ALG-2 and peflin regulate COPII targeting and secretion in response to calcium signaling

ER-to-Golgi transport is the first step in the constitutive secretory pathway, which, unlike regulated secretion, is believed to proceed nonstop independent of Ca²⁺ flux. However, here we demonstrate that penta-EF hand (PEF) proteins ALG-2 and peflin constitute a hetero-bifunctional COPII regulator that responds to Ca²⁺ signaling by adopting one of several distinct activity states. Functionally, these states can adjust the rate of ER export of COPII-sorted cargos up or down by ∼50%. We found that at steady-state Ca²⁺, ALG-2/peflin hetero-complexes bind to ER exit sites (ERES) through the ALG-2 subunit to confer a low, buffered secretion rate, while peflin-lacking ALG-2 complexes markedly stimulate secretion. Upon Ca²⁺ signaling, ALG-2 complexes lacking peflin can either increase or decrease the secretion rate depending on signaling intensity and duration—phenomena that could contribute to cellular growth and intercellular communication following secretory increases or protection from excitotoxicity and infection following decreases. In epithelial normal rat kidney (NRK) cells, the Ca²⁺-mobilizing agonist ATP causes ALG-2 to depress ER export, while in neuroendocrine PC12 cells, Ca²⁺ mobilization by ATP results in ALG-2-dependent enhancement of secretion. Furthermore, distinct Ca²⁺ signaling patterns in NRK cells produce opposing ALG-2-dependent effects on secretion. Mechanistically, ALG-2-dependent depression of secretion involves decreased levels of the COPII outer shell and increased peflin targeting to ERES, while ALG-2-dependent enhancement of secretion involves increased COPII outer shell and decreased peflin at ERES. These data provide insights into how PEF protein dynamics affect secretion of important physiological cargoes such as collagen I and significantly impact ER stress.     

Structure and function of ALG-2, a penta-EF-hand calcium-dependent adaptor protein

ALG-2 (a gene product of PDCD6) is a 22-kD protein containing five serially repetitive EF-hand structures and belongs to the penta-EF-hand (PEF) family, including the subunits of typical calpains. ALG-2 is the most conserved protein among the PEF family members and its homologs are widely found in eukaryotes. X-ray crystal structures of various PEF proteins including ALG-2 have common features: presence of eight α-helices and dimer formation via paired EF5s that are positioned in anti-parallel orientation. ALG-2 forms a homodimer and a heterodimer with its closest paralog peflin. Like calmodulin, a well-known four-EF-hand protein, ALG-2 interacts with various proteins in a Ca²⁺-dependent fashion, but the binding motifs are completely different. With some exceptions, ALG-2-interacting proteins commonly contain Pro-rich regions, and ALG-2 recognizes at least two distinct Pro-containing motifs: PPYP(X)nYP (X, variable; n=4 in ALIX and PLSCR3) and PXPGF (represented by Sec31A). A shorter alternatively spliced isoform, lacking two residues and designated ALG-2^ΔGF122, does not bind ALIX but maintains binding capacity to Sec31A. X-ray crystal structural analyses have revealed that binding of calcium ions induces the configuration of the side chain of R125 so that it opens Pocket 1, which accepts PPYP, but Pocket 1 remains closed in the case of ALG-2^ΔGF122. ALG-2 dimer has two ligand-binding sites, each in a monomer molecule, and appears to function as a Ca²⁺-dependent adaptor protein to either stabilize a preformed complex or to bridge two proteins on scaffolds in systems of the endosomal sorting complex required for transport (ESCRT) and ER-to-Golgi transport.     

The Ca2+-binding protein ALG-2 is recruited to endoplasmic reticulum exit sites by Sec31A and stabilizes the localization of Sec31A

The formation of transport vesicles that bud from endoplasmic reticulum (ER) exit sites is dependent on the COPII coat made up of three components: the small GTPase Sar1, the Sec23/24 complex, and the Sec13/31 complex. Here, we provide evidence that apoptosis-linked gene 2 (ALG-2), a Ca(2+)-binding protein of unknown function, regulates the COPII function at ER exit sites in mammalian cells. ALG-2 bound to the Pro-rich region of Sec31A, a ubiquitously expressed mammalian orthologue of yeast Sec31, in a Ca(2+)-dependent manner and colocalized with Sec31A at ER exit sites. A Ca(2+) binding-deficient ALG-2 mutant, which did not bind Sec31A, lost the ability to localize to ER exit sites. Overexpression of the Pro-rich region of Sec31A or RNA interference-mediated Sec31A depletion also abolished the ALG-2 localization at these sites. In contrast, depletion of ALG-2 substantially reduced the level of Sec31A associated with the membrane at ER exit sites. Finally, treatment with a cell-permeable Ca(2+) chelator caused the mislocalization of ALG-2, which was accompanied by a reduced level of Sec31A at ER exit sites. We conclude that ALG-2 is recruited to ER exit sites via Ca(2+)-dependent interaction with Sec31A and in turn stabilizes the localization of Sec31A at these sites.     

VPS37 Isoforms Differentially Modulate the Ternary Complex Formation of ALIX,ALG-2, and ESCRT-I

The endosomal sorting complex required for transport (ESCRT) system comprises a series of protein complexes that play essential roles in multivesicular body (MVB) sorting of ubiquitylated membrane proteins, enveloped RNA virus budding, and cytokinesis in mammalian cells. The complex, named ESCRT-I, consists of four subunits (TSG101, VPS28, VPS37, and MVB12). There are four VPS37 isoforms. We have reported that ALIX (an ALG-2-interacting protein and accessory protein in the ESCRT system) is physically linked with TSG101 by ALG-2 in a Ca²⁺-dependent manner, but the role of ALG-2 as an adaptor protein for the ESCRT-I complex remains unknown. To characterize this adaptor function, initially we investigated the binding of ALG-2 to ESCRT-I complexes containing each one of the four different VPS37 isoforms by two approaches: first, Far-Western blot analysis with biotin-labeled ALG-2 probe, and second, a pulldown assay to determine the binding of the four recombinant ESCRT-I complexes to Strep-tagged ALG-2 after co-expression in HEK293T cells. VPS37B and VPS37C appeared to interact with ALG-2 in a stronger manner than TSG101 does. The results of in vitro binding assays using purified recombinant proteins indicated that ALG-2 functions as a Ca²⁺-dependent adaptor protein that bridges ALIX and ESCRT-I to form a ternary complex, ESCRT-I/ALIX/ALG-2.     

Involvement of the penta-EF-hand protein Pef1p in the Ca2+-dependent regulation of COPII subunit assembly in Saccharomyces cerevisiae

Although it is well established that the coat protein complex II (COPII) mediates the transport of proteins and lipids from the endoplasmic reticulum (ER) to the Golgi apparatus, the regulation of the vesicular transport event and the mechanisms that act to counterbalance the vesicle flow between the ER and Golgi are poorly understood. In this study, we present data indicating that the penta-EF-hand Ca(2+)-binding protein Pef1p directly interacts with the COPII coat subunit Sec31p and regulates COPII assembly in Saccharomyces cerevisiae. ALG-2, a mammalian homolog of Pef1p, has been shown to interact with Sec31A in a Ca(2+)-dependent manner and to have a role in stabilizing the association of the Sec13/31 complex with the membrane. However, Pef1p displayed reversed Ca(2+) dependence for Sec13/31p association; only the Ca(2+)-free form of Pef1p bound to the Sec13/31p complex. In addition, the influence on COPII coat assembly also appeared to be reversed; Pef1p binding acted as a kinetic inhibitor to delay Sec13/31p recruitment. Our results provide further evidence for a linkage between Ca(2+)-dependent signaling and ER-to-Golgi trafficking, but its mechanism of action in yeast seems to be different from the mechanism reported for its mammalian homolog ALG-2.     

ALG-2 directly binds Sec31A and localizes at endoplasmic reticulum exit sites in a Ca2+-dependent manner

Intracellular localization of the penta-EF-hand Ca2+-binding protein ALG-2 in HeLa cells was investigated by immunofluorescent confocal microscopy using a polyclonal antibody. In addition to its presence in the nucleus, ALG-2 was found to be distributed in a punctate pattern in the cytoplasm, where it was partly co-stained with an endoplasmic reticulum (ER) exit site marker p125. In vitro GST pull down analysis demonstrated that ALG-2 and its alternatively spliced isoform interact with the COPII component Sec31A in a Ca2+-dependent manner, and a biotin-labeled ALG-2 overlay assay revealed direct binding of ALG-2 to Sec31A. Biochemical and immunofluorescent microscopic analyses showed that ALG-2 was enriched at the Sec31A-localizing membrane compartments upon stimulation with the Ca2+ ionophore A23187. In contrast, treatment of cells with the membrane-permeant Ca2+ chelator BAPTA-AM led to a dispersion of ALG-2 throughout the cells and to a significant loss of Sec31A in the perinuclear region. These findings establish Sec31A as a novel target for ALG-2 and provide a framework for studies on the roles of ALG-2 in ER-Golgi transport.     

Sec16 defines endoplasmic reticulum exit sites and is required for secretory cargo export in mammalian cells

The selective export of proteins and lipids from the endoplasmic reticulum (ER) is mediated by the coat protein complex II (COPII) that assembles onto the ER membrane. In higher eukaryotes, COPII proteins assemble at discrete sites on the membrane known as ER exit sites (ERES). Here, we identify Sec16 as the protein that defines ERES in mammalian cells. Sec16 localizes to ERES independent of Sec23/24 and Sec13/31. Overexpression, and to a lesser extent, small interfering RNA depletion of Sec16, both inhibit ER-to-Golgi transport suggesting that Sec16 is required in stoichiometric amounts. Sar1 activity is required to maintain the localization of Sec16 at discrete locations on the ER membrane, probably through preventing its dissociation. Our data suggest that Sar1-GTP-dependent assembly of Sec16 on the ER membrane forms an organized scaffold defining an ERES.     

Apoptosis-linked Gene-2 (ALG-2)/Sec31 Interactions Regulate Endoplasmic Reticulum (ER)-to-Golgi Transport: A POTENTIAL EFFECTOR PATHWAY FOR LUMINAL CALCIUM*

Luminal calcium released from secretory organelles has been suggested to play a regulatory role in vesicle transport at several steps in the secretory pathway; however, its functional roles and effector pathways have not been elucidated. Here we demonstrate for the first time that specific luminal calcium depletion leads to a significant decrease in endoplasmic reticulum (ER)-to-Golgi transport rates in intact cells. Ultrastructural analysis revealed that luminal calcium depletion is accompanied by increased accumulation of intermediate compartment proteins in COPII buds and clusters of unfused COPII vesicles at ER exit sites. Furthermore, we present several lines of evidence suggesting that luminal calcium affected transport at least in part through calcium-dependent interactions between apoptosis-linked gene-2 (ALG-2) and the Sec31A proline-rich region: 1) targeted disruption of ALG-2/Sec31A interactions caused severe defects in ER-to-Golgi transport in intact cells; 2) effects of luminal calcium and ALG-2/Sec31A interactions on transport mutually required each other; and 3) Sec31A function in transport required luminal calcium. Morphological phenotypes of disrupted ALG-2/Sec31A interactions were characterized. We found that ALG-2/Sec31A interactions were not required for the localization of Sec31A to ER exit sites per se but appeared to acutely regulate the stability and trafficking of the cargo receptor p24 and the distribution of the vesicle tether protein p115. These results represent the first outline of a mechanism that connects luminal calcium to specific protein interactions regulating vesicle trafficking machinery.     

Penta-EF-hand protein ALG-2 functions as a Ca-dependent adaptor that bridges Alix and TSG101

Alix and TSG101, known to physically interact with each other, have Pro-rich regions that are bound by ALG-2 Ca²⁺-dependently. We investigated the role of ALG-2 in the Alix-TSG101 association by pulldown assays using Strep-tagged Alix and its various mutants. The ALG-2-binding site was required for the Ca²⁺-dependent pulldown of TSG101 using HEK293T cells, whereas the PSAP sequence, a binding motif for the UEV domain of TSG101, was dispensable. Alix-TSG101 association was not observed using ALG-2-knockdown cells but became detectable by addition of the purified recombinant ALG-2 protein in the assay mixtures. Exogenous expression of mGFP-fused ALG-2 also restored the pulldown capability of Strep-Alix, but an alternatively spliced shorter ALG-2 isoform and a dimerization-defective mutant were incompetent. Based on the X-ray crystal structure model showing the presence of one ligand-binding site in each molecule of an ALG-2 dimer, we propose that Ca²⁺-loaded ALG-2 bridges Alix and TSG101 as an adaptor protein.     

Co-chaperone Specificity in Gating of the Polypeptide Conducting Channel in the Membrane of the Human Endoplasmic Reticulum

In mammalian cells, signal peptide-dependent protein transport into the endoplasmic reticulum (ER) is mediated by a dynamic polypeptide-conducting channel, the heterotrimeric Sec61 complex. Previous work has characterized the Sec61 complex as a potential ER Ca²⁺ leak channel in HeLa cells and identified ER lumenal molecular chaperone immunoglobulin heavy-chain-binding protein (BiP) as limiting Ca²⁺ leakage via the open Sec61 channel by facilitating channel closing. This BiP activity involves binding of BiP to the ER lumenal loop 7 of Sec61α in the vicinity of tyrosine 344. Of note, the Y344H mutation destroys the BiP binding site and causes pancreatic β-cell apoptosis and diabetes in mice. Here, we systematically depleted HeLa cells of the BiP co-chaperones by siRNA-mediated gene silencing and used live cell Ca²⁺ imaging to monitor the effects on ER Ca²⁺ leakage. Depletion of either one of the ER lumenal BiP co-chaperones, ERj3 and ERj6, but not the ER membrane-resident co-chaperones (such as Sec63 protein, which assists BiP in Sec61 channel opening) led to increased Ca²⁺ leakage via Sec6 complex, thereby phenocopying the effect of BiP depletion. Thus, BiP facilitates Sec61 channel closure (i.e. limits ER Ca²⁺ leakage) via the Sec61 channel with the help of ERj3 and ERj6. Interestingly, deletion of ERj6 causes pancreatic β-cell failure and diabetes in mice and humans. We suggest that co-chaperone-controlled gating of the Sec61 channel by BiP is particularly important for cells, which are highly active in protein secretion, and that breakdown of this regulatory mechanism can cause apoptosis and disease.     

Dynamic organization of COPII coat proteins at endoplasmic reticulum export sites in plant cells

Protein export from the endoplasmic reticulum (ER) is mediated by the accumulation of COPII proteins such as Sar1, Sec23/24 and Sec13/31 at specialized ER export sites (ERES). Although the distribution of COPII components in mammalian and yeast systems is established, a unified model of ERES dynamics has yet to be presented in plants. To investigate this, we have followed the dynamics of fluorescent fusions to inner and outer components of the coat, AtSec24 and AtSec13, in three different plant model systems: tobacco and Arabidopsis leaf epidermis, as well as tobacco BY-2 suspension cells. In leaves, AtSec24 accumulated at Golgi-associated ERES, whereas AtSec13 showed higher levels of cytosolic staining compared with AtSec24. However, in BY-2 cells, both AtSec13 and AtSec24 labelled Golgi-associated ERES, along with AtSec24. To correlate the distribution of the COPII coat with the dynamics of organelle movement, quantitative live-cell imaging analyses demonstrated that AtSec24 and AtSec13 maintained a constant association with Golgi-associated ERES, irrespective of their velocity. However, recruitment of AtSec24 and AtSec13 to ERES, as well as the number of ERES marked by these proteins, was influenced by export of membrane cargo proteins from the ER to the Golgi. Additionally, the increased availability of AtSec24 affected the distribution of AtSec13, inducing recruitment of this outer COPII coat component to ERES. These results provide a model that, in plants, protein export from the ER occurs via sequential recruitment of inner and outer COPII components to form transport intermediates at mobile, Golgi-associated ERES.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

ALG-2 oscillates in subcellular localization, unitemporally with calcium oscillations

A variety of stimuli can trigger intracellular calcium oscillations. Relatively little is known about the molecular mechanisms decoding these events. We show that ALG-2, a Ca2+-binding protein originally isolated as a protein associated with apoptosis, is directly linked to Ca2+ signalling. We discovered that the subcellular distribution of a tagged version of ALG-2 could be directed by physiological external stimuli (including ATP, EGF, prostaglandin, histamine), which provoke intracellular Ca2+ oscillations. Cellular stimulation led to a redistribution of ALG-2 from the cytosol to a punctate localization in an oscillatory fashion unitemporally with Ca2+ oscillations, whereas a Ca2+-binding deficient mutant of ALG-2 did not redistribute. Using tagged ALG-2 as bait we identified its novel target protein Sec31A and based on the partial colocalization of endogenous ALG-2 and Sec31A we propose that ALG-2 temporarily binds to the COPII vesicles providing a link between Ca2+ signalling and ER to Golgi trafficking.     

Interaction of Pseudomonas aeruginosa Exotoxin A with the human Sec61 complex suppresses passive calcium efflux from the endoplasmic reticulum

According to live-cell calcium-imaging experiments, the Sec61 complex is a passive calcium-leak channel in the human endoplasmic reticulum (ER) membrane that is regulated by ER luminal immunoglobulin heavy chain binding protein (BiP) and cytosolic Ca²⁺-calmodulin. In single channel measurements, the open Sec61 complex is Ca²⁺ permeable. It can be closed not only by interaction with BiP or Ca²⁺-calmodulin, but also with Pseudomonas aeruginosa Exotoxin A which can enter human cells by retrograde transport. Exotoxin A has been shown to interact with the Sec61 complex and, thereby, inhibit ER export of immunogenic peptides into the cytosol. Here, we show that Exotoxin A also inhibits passive Ca²⁺ leakage from the ER in human cells, and we characterized the N-terminus of the Sec61 α-subunit as the relevant binding site for Exotoxin A.     

Vesicular Calcium Regulates Coat Retention,Fusogenicity, and Size of Pre-Golgi Intermediates

The significance and extent of Ca²⁺ regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca²⁺ with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca²⁺ in the early secretory pathway. Specific depletion of luminal Ca²⁺ in living normal rat kidney cells using cyclopiazonic acid (CPA) resulted in the extreme expansion of vesicular tubular cluster (VTC) elements. Consistent with this, a suppressive role for vesicle-associated Ca²⁺ in COPII vesicle homotypic fusion was demonstrated in vitro using Ca²⁺ chelators. The EF-hand–containing protein apoptosis-linked gene 2 (ALG-2), previously implicated in the stabilization of sec31 at endoplasmic reticulum exit sites, inhibited COPII vesicle fusion in a Ca²⁺-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca²⁺ on homotypic fusion. Immunoisolation established that Ca²⁺ chelation inhibits and ALG-2 specifically favors residual retention of the COPII outer shell protein sec31 on pre-Golgi fusion intermediates. We conclude that vesicle-associated Ca²⁺, acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca²⁺-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.     

ULK1 phosphorylates Sec23A and mediates autophagy-induced inhibition of ER-to-Golgi traffic

Background

Autophagy is an inducible autodigestive process that allows cells to recycle proteins and other materials for survival during stress and nutrient deprived conditions. The kinase ULK1 is required to activate this process. ULK1 phosphorylates a number of target proteins and regulates many cellular processes including the early secretory pathway. Recently, ULK1 has been demonstrated to phosphorylate Sec16 and affects the transport of serotonin transporter at the ER exit sites (ERES), but whether ULK1 may affect the transport of other cargo proteins and general secretion has not been fully addressed.

Results

In this study, we identified Sec23A, a component of the COPII vesicle coat, as a target of ULK1 phosphorylation. Elevated autophagy, induced by amino acid starvation, rapamycin, or overexpression of ULK1 caused aggregation of the ERES, a region of the ER dedicated for the budding of COPII vesicles. Transport of cargo proteins was also inhibited under these conditions and was retained at the ERES. ULK1 phosphorylation of Sec23A reduced the interaction between Sec23A and Sec31A. We identified serine 207, serine 312 and threonine 405 on Sec23A as ULK1 phosphorylation sites. Among these residues, serine 207, when changed to phospho-deficient and phospho-mimicking mutants, most faithfully recapitulated the above-mentioned effects of ULK1 phospho-regulation.

Conclusion

These findings identify Sec23A as a new target of ULK1 and uncover a mechanism of coordinating intracellular protein transport and autophagy.

     

Leucine-rich repeat kinase 2 regulates Sec16A at ER exit sites to allow ER–Golgi export

Leucine-rich repeat kinase 2 (LRRK2) has been associated with Parkinson’s disease (PD) and other disorders. However, its normal physiological functions and pathogenic properties remain elusive. Here we show that LRRK2 regulates the anterograde ER–Golgi transport through anchoring Sec16A at the endoplasmic reticulum exit sites (ERES). LRRK2 interacted and co-localized with Sec16A, a key protein in the formation of ERES. Lrrk2 depletion caused a dispersion of Sec16A from ERES and impaired ER export. In neurons, LRRK2 and Sec16A showed extensive co-localization at the dendritic ERES (dERES) that locally regulate the transport of proteins to the dendritic spines. A loss of Lrrk2 affected the association of Sec16A with dERES and impaired the activity-dependent targeting of glutamate receptors onto the cell/synapse surface. Furthermore, the PD-related LRRK2 R1441C missense mutation in the GTPase domain interfered with the interaction of LRRK2 with Sec16A and also affected ER–Golgi transport, while LRRK2 kinase activity was not required for these functions. Therefore, our findings reveal a new physiological function of LRRK2 in ER–Golgi transport, suggesting ERES dysfunction may contribute to the pathogenesis of PD.     

Calmodulin regulation of the calcium-leak channel Sec61 is unique to vertebrates

In eukaryotes, protein transport into the endoplasmic reticulum (ER) is facilitated by a protein-conducting channel, the Sec61 complex. The presence of large, water-filled pores with uncontrolled ion permeability, such as those formed by Sec61 complexes in the ER membrane, would interfere with the regulated release of calcium from the ER lumen into the cytosol, an essential mechanism of intracellular signaling. We identified a calmodulin (CaM) binding motif in the cytosolic N-terminus of Sec61α from Canis familiaris that binds CaM, but not Ca²⁺-free apo-CaM, with nanomolar affinity and sequence specificity. In single channel lipid bilayer measurements, CaM potently mediated Sec61-channel closure in a Ca²⁺-dependent manner. No functional CaM binding motif was identified in the corresponding region of Sec61p from Saccharomyces cerevisiae, and no channel closure occurred in the presence of CaM and Ca²⁺. Therefore, CaM binding to the cytosolic N-terminus of Sec61α is involved in limiting Ca²⁺-leakage from the ER in C. familiaris but not S. cerevisiae.     

A Novel Endoplasmic Reticulum Export Signal: PROLINE AT THE +2-POSITION FROM THE SIGNAL PEPTIDE CLEAVAGE SITE*

NUCB1 (nucleobindin 1) is a Golgi-localized soluble protein with a signal peptide and multiple functional domains. We reported recently that NUCB1 is a negative regulator of the unfolded protein response that activates various endoplasmic reticulum (ER)-originating signaling pathways. In that report, we also showed that Golgi localization of NUCB1 was essential to regulate the unfolded protein response. However, the localization mechanism of NUCB1 is still unknown. Here, we report that the proline residue at the +2-position (Pro⁺²) from the signal peptide cleavage site is the determinant of NUCB1 protein export from the ER and subsequent transport to the Golgi. Fusion of the N-terminal amino acids 1–35 peptide region, including both signal peptide (amino acids 1–26) and Pro⁺², was sufficient for enhanced green fluorescent protein to localize in the Golgi, whereas single amino acid mutation of Pro⁺² resulted in defective export from the ER without affecting the protein maturation process. Furthermore, we demonstrated that Pro⁺² was important for the enhanced green fluorescent protein fusion protein to concentrate at a transport vesicle formation site within the ER, often termed the ER exit site. Interestingly, such a Pro⁺² has also been functionally conserved in other Golgi-localized soluble proteins, Cab45 (Ca²⁺-binding protein of 45 kDa), reticulocalbin 1, and calumenin. Our findings indicate that Pro⁺² can function as a novel ER export signal of some Golgi proteins.NUCB1 (nucleobindin 1), also known as calnuc, was first identified as a soluble secretory 55-kDa protein (461 amino acids) in lupus-prone mice with the lymphoproliferation (lpr) mutation (1). NUCB1 has also been shown to be secreted in culture supernatant of a murine B cell line established from the mice (2). Later studies also demonstrated that NUCB1 is expressed ubiquitously and localizes in the Golgi apparatus of intact cells (3, 4). NUCB1 contains multiple putative functional domains, including an N-terminal endoplasmic reticulum (ER)² signal peptide, a DNA binding site, a leucine zipper domain, two EF-hand Ca²⁺-binding sites, a nuclear localization signal, and G-protein-binding and cyclooxygenase-binding domains (1, 5, 6). Consistently, NUCB1 has been reported to function in various cellular processes, including osteogenesis, inflammation, autoimmunity, intracellular signaling, and cancer (6–10).Newly synthesized, premature NUCB1 protein is first targeted into the ER via its N-terminal ER signal peptide. After removal of the signal peptide in the ER, a mature NUCB1 protein is transported to the Golgi apparatus and then secreted to the extracellular matrix (11). NUCB1 in the Golgi pool is probably involved in establishing the agonist-mobilizable Golgi Ca²⁺ store (3). Furthermore, the Golgi-localized NUCB1 regulates the unfolded protein response, which is a cellular stress response that triggers various events, such as ER-resident molecular chaperone induction, translational repression, and apoptosis under ER stress conditions (12). On the other hand, extracellular NUCB1 has been suggested to serve as a modulator of matrix maturation in bone, based on the observations that NUCB1 is secreted by osteoblasts and osteocytes and can, indeed, be detected in the osteoid extracellular matrix (7, 13). Thus, Golgi transport and subsequent secretion of NUCB1 seem to be important to exert the protein''s activity, but little is known about its transport regulation mechanism.In eukaryotic cells, a tremendous variety of soluble and membrane cargo proteins are packaged into transport vesicles at the ER. Vesicle formation on the ER membrane begins with the assembly of a coat protein complex II (COPII) (14). This COPII coat consists of Sar1, Sec23-Sec24, and Sec13-Sec31 complexes that are sequentially recruited to the ER membrane. Sar1 is a small GTPase that regulates coat assembly and disassembly. To assemble the COPII coat, Sar1-GTP transiently associates with an export cargo protein and then binds to Sec23-Sec24, which in turn attracts Sec13-Sec31 (14). Polymerization of the formed COPII coat, which occurs at the so-called ER exit site (ERES), triggers transport vesicle budding on the ER membrane (14, 15). Then the vesicles fuse with the VTC compartment (vesiculo-tubular clusters, also called ERGIC) that mediates further protein transport to the Golgi apparatus. Cargo proteins are then carried to their final destinations, such as organelle, cell surface membrane, and extracellular matrix (14).Recent studies reveal that some transmembrane cargoes contain specific motifs to be selectively concentrated in the transport vesicle within the ER. This sorting motif is called the ER export signal. Representative ER export signals are the diacidic motif (DXE), dihydrophobic (LL) motif, and diaromatic motif (FF, YY) that have been found in vesicular stomatitis virus glycoprotein, ERGIC-53, and Emp46p, respectively (16–18). These export signals are present in the cytoplasmic region of the cargo proteins and mediate their interaction with the COPII complex at the outer side of the ER membrane, resulting in concentration in the newly formed budding vesicle. On the other hand, the soluble type of cargo proteins require their cargo receptor to be sorted into the COPII vesicle, because they cannot interact directly with COPII complex, since these proteins have no cytoplasmic region. Although recent studies have reported the existence of the cargo receptor and functional ER export signals found in some soluble cargo proteins, little is known about many other cargo receptors and the export signals of soluble cargo proteins (19, 20).Here, we report that Pro²⁸, which is located at the +2-position (Pro⁺²) from the signal peptide cleavage site of the NUCB1 protein, is a determinant of its export from the ER. In fact, single amino acid substitution (P28A) led to predominant ER distribution and reduced the secretion of NUCB1 without affecting its maturation process in the ER. We also demonstrated that Pro⁺² is required for concentration at the ERES. It is important to note that Pro⁺² was also conserved functionally in other proteins. Our results indicate that Pro⁺² can function as a new ER export signal.