Structural basis for the cooperative interplay between the two causative gene products of combined factor V and factor VIII deficiency.

Combined deficiency of coagulation factors V and VIII (F5F8D), an autosomal recessive disorder characterized by coordinate reduction in the plasma levels of factor V (FV) and factor VIII (FVIII), is genetically linked to mutations in the transmembrane lectin ERGIC-53 and the soluble calcium-binding protein MCFD2. Growing evidence indicates that these two proteins form a complex recycling between the endoplasmic reticulum (ER) and the ER-Golgi intermediate compartment and thereby function as a cargo receptor in the early secretory pathway of FV and FVIII. For better understanding of the mechanisms underlying the functional coordination of ERGIC-53 and MCFD2, we herein characterize their interaction by x-ray crystallographic analysis in conjunction with NMR and ultracentrifugation analyses. Inspection of the combined data reveals that ERGIC-53-CRD binds MCFD2 through its molecular surface remote from the sugar-binding site, giving rise to a 11 complex in solution. The interaction is independent of sugar-binding of ERGIC-53 and involves most of the missense mutation sites of MCFD2 so far reported in F5F8D. Comparison with the previously reported uncomplexed structure of each protein indicates that MCFD2 but not ERGIC-53-CRD undergoes significant conformational alterations upon complex formation. Our findings provide a structural basis for the cooperative interplay between ERGIC-53 and MCFD2 in capturing FV and FVIII.

Role of the lectin VIP36 in post-ER quality control of human alpha1-antitrypsin.

The leguminous-type (L-type) lectin VIP36 localizes to the Golgi apparatus and cycles early in the secretory pathway. In vitro, VIP36 binds high-mannose glycans with a pH optimum of 6.5, a value similar to the luminal pH of the Golgi apparatus. Although the sugar-binding properties of VIP36 in vitro have been characterized in detail, the function of VIP36 in the intact cell remains unclear as no convincing glycoprotein cargo has been identified. Here, we used yellow fluorescent protein (YFP) fragment complementation to identify luminal interaction partners of VIP36. By screening a human liver cDNA library, we identified the glycoprotein alpha1-antitrypsin (alpha1-AT) as a cargo of VIP36. The VIP36/alpha1-AT complex localized to Golgi and endoplasmic reticulum (ER). In the living cell, VIP36 bound exclusively to the high-mannose form of alpha1-AT. The binding was increased when complex glycosylation was prevented by kifunensine and abolished when the glycosylation sites of alpha1-AT were inactivated by mutagenesis. Silencing VIP36 accelerated alpha1-AT transport, arguing against a role of VIP36 in anterograde traffic. The complex formed by VIP36 and alpha1-AT in the Golgi recycled back to the ER. The combined data are most consistent with a function of VIP36 in post-ER quality control of alpha1-AT.

p28, a novel ERGIC/cis Golgi protein, required for Golgi ribbon formation.

The mammalian Golgi apparatus consists of individual cisternae that are stacked in a polarized manner to form the compact zones of the Golgi. Several stacks are linked to form a ribbon via dynamic lateral bridges. The determinants required for maintaining the characteristic Golgi structure are incompletely understood. Here, we have characterized p28, a new gamma-subfamily member of p24 membrane proteins. p28 localized to endoplasmic reticulum-Golgi intermediate compartment (ERGIC) and cis Golgi and accumulated in the ERGIC upon Brefeldin A treatment, typical for a protein cycling in the early secretory pathway. p28 interacted with a subset of p24 proteins. Its depletion by small interfering RNA (siRNA) led to fragmentation of the Golgi without affecting the overall organization of microtubules but considerably reducing the amount of acetylated tubulin. The distribution of COPI and tethers, including GM130, was not affected. At the ultrastructural level, the Golgi fragments appeared as mini-stacks with apparently unchanged cis-trans topology. Golgi fragmentation did not impair anterograde or retrograde traffic. Fluorescence recovery after photobleaching (FRAP) experiments revealed that silencing p28 prevents protein exchange between Golgi stacks during reassembly after Brefeldin A-induced Golgi breakdown. These results show that the formation of a Golgi ribbon requires the structural membrane protein p28 in addition to previously identified SNAREs, coat proteins and tethers.

Adaptation of endoplasmic reticulum exit sites to acute and chronic increases in cargo load.

The biogenesis of endoplasmic reticulum (ER) exit sites (ERES) involves the formation of phosphatidylinositol-4 phosphate (PI4) and Sec16, but it is entirely unknown how ERES adapt to variations in cargo load. Here, we studied acute and chronic adaptive responses of ERES to an increase in cargo load for ER export. The acute response (within minutes) to increased cargo load stimulated ERES fusion events, leading to larger but less ERES. Silencing either PI4-kinase IIIalpha (PI4K-IIIalpha) or Sec16 inhibited the acute response. Overexpression of secretory cargo for 24 h induced the unfolded protein response (UPR), upregulated COPII, and the cells formed more ERES. This chronic response was insensitive to silencing PI4K-IIIalpha, but was abrogated by silencing Sec16. The UPR was required as the chronic response was absent in cells lacking inositol-requiring protein 1. Mathematical model simulations further support the notion that increasing ERES number together with COPII levels is an efficient way to enhance the secretory flux. These results indicate that chronic and acute increases in cargo load are handled differentially by ERES and are regulated by different factors.

Selective export of human GPI-anchored proteins from the endoplasmic reticulum.

Selective export of transmembrane proteins from the endoplasmic reticulum (ER) relies on recognition of cytosolic-domain-localized transport signals by the Sec24 subunit of the COPII vesicle coat. Human cells express four Sec24 isoforms, termed Sec24A, Sec24B, Sec24C and Sec24D that are differentially required for selective, signal-mediated ER export of transmembrane proteins. By contrast, luminally exposed glycosylphosphatidylinositol (GPI)-anchored membrane proteins cannot bind directly to Sec24 and must either use membrane-spanning cargo receptors or alternative mechanisms for ER export. Little is known about the mechanism underlying export of GPI-anchored proteins from the ER in higher eukaryotes. Using siRNA-based silencing, we identified that ER-to-Golgi transport of the human GPI-anchored protein CD59 requires Sec24, with preference for the Sec24C and Sec24D isoforms, and the recycling transmembrane protein complex p24-p23 that exhibited the same Sec24C-Sec24D isoform preference for ER export. Co-immunoprecipitation indicated unprecedented physical interaction of CD59 as well as a GFP-folate-receptor-GPI-anchor hybrid with a p24-p23 complex. Density gradient centrifugation revealed co-partitioning of CD59 and p24-p23 into biosynthetically early lipid raft fractions, and CD59 transport to the Golgi was cholesterol dependent. The results suggest that the 24p-23p complex acts as a cargo receptor for GPI-anchored proteins by facilitating their export from the ER in a Sec24-isoform-selective manner involving lipid rafts as early sorting platforms.

Sec24- and ARFGAP1-dependent trafficking of GABA transporter-1 is a prerequisite for correct axonal targeting.

The GABA transporter-1 (GAT1) is a prototypical protein of the synaptic specialization. Export of GAT1 from the endoplasmic reticulum (ER) is contingent on its interaction with the COPII (coatomer protein-II) coat subunit Sec24D. Here we show that silencing all four Sec24 isoforms strongly inhibits transport of GAT1 to the cell surface. In contrast, transport of GAT1-RL/AS, a mutant that is deficient in Sec24D recruitment, was not inhibited, suggesting a nonconventional, COPII-independent pathway. However, ARFGAP1 bound directly to the C terminus of both GAT1-RL/AS and wild-type GAT1. Surface expression of GAT1-RL/AS involved ARFGAP1. GAT1-RL/AS appeared to bypass the ER-Golgi-intermediate compartment, but its pathway to the plasma membrane still involved passage through the Golgi. Thus, the GAT1-RL/AS mutant allowed to test whether COPII-dependent ER-export is required for correct sorting of GAT1 to the axon terminal in neuronal cells. In contrast to wild-type GAT1, GAT1-RL/AS failed to be specifically enriched at the tip of neurite extensions of CAD.a cells (a neuroblastoma cell line that can be differentiated into a neuron-like phenotype) and in the axon terminals of hippocampal neurons. These findings indicate that correct sorting to the axon is contingent on ER export via the COPII machinery and passage through the ER-Golgi-intermediate compartment.

Improved maturation of CFTR by an ER export signal.

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel in the plasma membrane of several epithelial cells. Maturation of CFTR is inefficient in most cells, with only a fraction of nascent chains being properly folded and transported to the cell surface. The most common mutation in CFTR, CFTR-deltaF508, leads to the genetic disease cystic fibrosis. CFTR-deltaF508 has a temperature-sensitive folding defect and is almost quantitatively degraded in the endoplasmic reticulum (ER). Here we tested whether a strong ER export signal appended to CFTR improves its transport and surface expression. We show that a single valine ER export signal at the C terminus of the cytoplasmic tail of CFTR improves maturation of wild-type CFTR by 2-fold. This conservative mutation interfered with neither plasma membrane localization nor stability of mature CFTR. In contrast, the valine signal was unable to rescue CFTR-deltaF508 from ER-associated degradation. Our finding of improved maturation of CFTR mediated by a valine signal may be of potential use in gene therapy of cystic fibrosis. Moreover, failure of the valine signal to rescue CFTR-deltaF508 from ER degradation indicates that the inability of CFTR-deltaF508 to leave the ER is unlikely to be due to a malfunctioning ER export signal.

Phosphorylation controls CLIMP-63-mediated anchoring of the endoplasmic reticulum to microtubules.

The microtubule-binding 63-kDa cytoskeleton-linking membrane protein (CLIMP-63) is an integral membrane protein that links the endoplasmic reticulum (ER) to microtubules. Here, we tested whether this interaction is regulated by phosphorylation. Metabolic labeling with (32)P showed that CLIMP-63 is a phosphoprotein with increased phosphorylation during mitosis. CLIMP-63 of mitotic cells is unable to bind to microtubules in vitro. Mitotic phosphorylation can be prevented by mutation of serines 3, 17, and 19 in the cytoplasmic domain of CLIMP-63. When these residues are mutated to glutamic acid, and hence mimic mitotic phosphorylation, CLIMP-63 does no longer bind to microtubules in vitro. Overexpression of the phospho-mimicking mitotic form of CLIMP-63 in interphase cells leads to a collapse of the ER around the nucleus, leaving the microtubular network intact. The results suggest that CLIMP-63-mediated stable anchoring of the ER to microtubules is required to maintain the spatial distribution of the ER during interphase and that this interaction is abolished by phosphorylation of CLIMP-63 during mitosis.

Carbohydrate- and conformation-dependent cargo capture for ER-exit.

Some secretory proteins leave the endoplasmic reticulum (ER) by a receptor-mediated cargo capture mechanism, but the signals required for the cargo-receptor interaction are largely unknown. Here, we describe a novel targeting motif that is composed of a high-mannose type oligosaccharide intimately associated with a surface-exposed peptide beta-hairpin loop. The motif accounts for lectin ERGIC-53-assisted ER-export of the lyososomal enzyme procathepsin Z. The second oligosaccharide chain of procathepsin Z exhibits no binding activity for ERGIC-53, illustrating the selective lectin properties of ERGIC-53. Our data suggest that the conformation-based motif is only present in fully folded procathepsin Z and that its recognition by ERGIC-53 reflects a quality control mechanism that acts complementary to the primary folding machinery in the ER. A similar oligosaccharide/beta-hairpin loop structure is present in cathepsin C, another cargo of ERGIC-53, suggesting the general nature of this ER-exit signal. To our knowledge this is the first documentation of an ER-exit signal in soluble cargo in conjunction with its decoding by a transport receptor.

ER export: call 14-3-3.

Forward transport of proteins from the ER to the plasma membrane requires escape from the ER's retention machinery. Recent studies suggest that 14-3-3 proteins may mediate ER export of potassium channels destined for the plasma membrane by interfering with dibasic-motif-mediated retention.

Lectins and protein traffic early in the secretory pathway.

Lectins of the early secretory pathway are involved in selective transport of newly synthesized glycoproteins from the endoplasmic reticulum (ER) to the ER-Golgi intermediate compartment (ERGIC). The most prominent cycling lectin is the mannose-binding type I membrane protein ERGIC-53 (ERGIC protein of 53 kDa), a marker for the ERGIC, which functions as a cargo receptor to facilitate export of an increasing number of glycoproteins with different characteristics from the ER. Two ERGIC-53-related proteins, VIP36 (vesicular integral membrane protein 36) and a novel ERGIC-53-like protein, ERGL, are also found in the early secretory pathway. ERGL may act as a regulator of ERGIC-53. Studies of ERGIC-53 continue to provide new insights into the organization and dynamics of the early secretory pathway. Analysis of the cycling of ERGIC-53 uncovered a complex interplay of trafficking signals and revealed novel cytoplasmic ER-export motifs that interact with COP-II coat proteins. These motifs are common to type I and polytopic membrane proteins including presenilin 1 and presenilin 2. The results support the notion that protein export from the ER is selective.

ADP ribosylation factors 1 and 4 and group VIA phospholipase A2 regulate morphology and intraorganellar traffic in the endoplasmic reticulum-Golgi intermediate compartment.

Organelle morphology of the endomembrane system is critical for optimal organelle function. ADP ribosylation factors (Arfs), a family of small GTPases, are required for maintaining the structure of the Golgi and endosomes. What determines the discontinuous nature of the endoplasmic reticulum (ER)-Golgi intermediate compartment (ERGIC) as tubulovesicular clusters is unknown. In search of morphological determinants for the ERGIC, we found that a double knockdown of Arf1+Arf4 induced dynamic ERGIC tubules that connect ERGIC clusters, indicating that the tubules mediated lateral intraERGIC traffic. Tubule formation was inhibited by an antagonist of group VI calcium-independent phospholipase A2 (PLA2G6) and by silencing the A isoform of PLA2G6 (PLA2G6-A). Arf1+Arf4 depletion altered the expression of PLA2G6-A splice variants and relocalized PLA2G6-A from the cytosol to ERGIC clusters and tubules, suggesting that the enzyme became locally active. We show that changes in Arf1 can modulate the activity of PLA2G6-A. We propose that a concerted action of Arf1, Arf4, and PLA2G6-A controls the architecture of the ERGIC in a way that is predicted to impact the rate and possibly the destination of cargos. Our findings have identified key components in the molecular mechanism underlying the regulation of tubules in the ERGIC and uncover tubular carriers as tightly controlled machinery.

MAPK signaling to the early secretory pathway revealed by kinase/phosphatase functional screening.

To what extent the secretory pathway is regulated by cellular signaling is unknown. In this study, we used RNA interference to explore the function of human kinases and phosphatases in controlling the organization of and trafficking within the secretory pathway. We identified 122 kinases/phosphatases that affect endoplasmic reticulum (ER) export, ER exit sites (ERESs), and/or the Golgi apparatus. Numerous kinases/phosphatases regulate the number of ERESs and ER to Golgi protein trafficking. Among the pathways identified, the Raf-MEK (MAPK/ERK [extracellular signal-regulated kinase] kinase)-ERK cascade, including its regulatory proteins CNK1 (connector enhancer of the kinase suppressor of Ras-1) and neurofibromin, controls the number of ERESs via ERK2, which targets Sec16, a key regulator of ERESs and COPII (coat protein II) vesicle biogenesis. Our analysis reveals an unanticipated complexity of kinase/phosphatase-mediated regulation of the secretory pathway, uncovering a link between growth factor signaling and ER export.

Membrane biogenesis: networking at the ER with atlastin.

The peripheral endoplasmic reticulum forms a dynamic network of interconnected membrane tubules. Although some determinants of this striking architecture are known, the mechanism underlying fusion of individual tubules has remained elusive. Two studies now identify atlastin proteins as key mediators of homotypic fusion of endoplasmic reticulum membranes.

Role of syntaxin 18 in the organization of endoplasmic reticulum subdomains.

The presence of subdomains in the endoplasmic reticulum (ER) enables this organelle to perform a variety of functions, yet the mechanisms underlying their organization are poorly understood. In the present study, we show that syntaxin 18, a SNAP (soluble NSF attachment protein) receptor localized in the ER, is important for the organization of two ER subdomains, smooth/rough ER membranes and ER exit sites. Knockdown of syntaxin 18 caused a global change in ER membrane architecture, leading to the segregation of the smooth and rough ER. Furthermore, the organization of ER exit sites was markedly changed concomitantly with dispersion of the ER-Golgi intermediate compartment and the Golgi complex. These morphological changes in the ER were substantially recovered by treatment of syntaxin-18-depleted cells with brefeldin A, a reagent that stimulates retrograde membrane flow to the ER. These results suggest that syntaxin 18 has an important role in ER subdomain organization by mediating the fusion of retrograde membrane carriers with the ER membrane.

Signal-dependent export of GABA transporter 1 from the ER-Golgi intermediate compartment is specified by a C-terminal motif.

The C-terminus of GABA transporter 1 (GAT1, SLC6A1) is required for trafficking of the protein through the secretory pathway to reach its final destination, i.e. the rim of the synaptic specialization. We identified a motif of three hydrophobic residues (569VMI571) that was required for export of GAT1 from the ER-Golgi intermediate compartment (ERGIC). This conclusion was based on the following observations: (i) GAT1-SSS, the mutant in which 569VMI571 was replaced by serine residues, was exported from the ER in a COPII-dependent manner but accumulated in punctate structures and failed to reach the Golgi; (ii) under appropriate conditions (imposing a block at 15 degrees C, disruption of COPI), these structures also contained ERGIC53; (iii) the punctae were part of a dynamic compartment, because it was accessible to a second anterograde cargo [the temperature-sensitive variant of vesicular stomatitis virus G protein (VSV-G)] and because GAT1-SSS could be retrieved from the punctate structures by addition of a KKxx-based retrieval motif, which supported retrograde transport to the ER. To the best of our knowledge, the VMI-motif of GAT1 provides the first example of a cargo-based motif that specifies export from the ERGIC.

Identification of ERGIC-53 as an intracellular transport receptor of alpha1-antitrypsin.

Secretory proteins are exported from the endoplasmic reticulum (ER) by bulk flow and/or receptor-mediated transport. Our understanding of this process is limited because of the low number of identified transport receptors and cognate cargo proteins. In mammalian cells, the lectin ER Golgi intermediate compartment 53-kD protein (ERGIC-53) represents the best characterized cargo receptor. It assists ER export of a subset of glycoproteins including coagulation factors V and VIII and cathepsin C and Z. Here, we report a novel screening strategy to identify protein interactions in the lumen of the secretory pathway using a yellow fluorescent protein-based protein fragment complementation assay. By screening a human liver complementary DNA library, we identify alpha1-antitrypsin (alpha1-AT) as previously unrecognized cargo of ERGIC-53 and show that cargo capture is carbohydrate- and conformation-dependent. ERGIC-53 knockdown and knockout cells display a specific secretion defect of alpha1-AT that is corrected by reintroducing ERGIC-53. The results reveal ERGIC-53 to be an intracellular transport receptor of alpha1-AT and provide direct evidence for active receptor-mediated ER export of a soluble secretory protein in higher eukaryotes.

The cargo receptors Surf4, endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53, and p25 are required to maintain the architecture of ERGIC and Golgi.

Rapidly cycling proteins of the early secretory pathway can operate as cargo receptors. Known cargo receptors are abundant proteins, but it remains mysterious why their inactivation leads to rather limited secretion phenotypes. Studies of Surf4, the human orthologue of the yeast cargo receptor Erv29p, now reveal a novel function of cargo receptors. Surf4 was found to interact with endoplasmic reticulum-Golgi intermediate compartment (ERGIC)-53 and p24 proteins. Silencing Surf4 together with ERGIC-53 or silencing the p24 family member p25 induced an identical phenotype characterized by a reduced number of ERGIC clusters and fragmentation of the Golgi apparatus without effect on anterograde transport. Live imaging showed decreased stability of ERGIC clusters after knockdown of p25. Silencing of Surf4/ERGIC-53 or p25 resulted in partial redistribution of coat protein (COP) I but not Golgi matrix proteins to the cytosol and partial resistance of the cis-Golgi to brefeldin A. These findings imply that cargo receptors are essential for maintaining the architecture of ERGIC and Golgi by controlling COP I recruitment.

Molecular basis of sugar recognition by the human L-type lectins ERGIC-53, VIPL, and VIP36.

ERGIC-53, VIPL, and VIP36 are related type 1 membrane proteins of the mammalian early secretory pathway. They are classified as L-type lectins because of their luminal carbohydrate recognition domain, which exhibits homology to leguminous lectins. These L-type lectins have different intracellular distributions and dynamics in the endoplasmic reticulum-Golgi system of the secretory pathway and interact with N-glycans of glycoproteins in a Ca(2+)-dependent manner, suggesting a role in glycoprotein sorting and trafficking. To understand the function of these lectins, knowledge of their carbohydrate specificity is crucial but only available for VIP36 (Kamiya, Y., Yamaguchi, Y., Takahashi, N., Arata, Y., Kasai, K. I., Ihara, Y., Matsuo, I., Ito, Y., Yamamoto, K., and Kato, K. (2005) J. Biol. Chem. 280, 37178-37182). Here we provide a comprehensive and quantitative analysis of sugar recognition of the carbohydrate recognition domains of ERGIC-53 and VIPL in comparison with VIP36 using a pyridylaminated sugar library in conjunction with frontal affinity chromatography. Frontal affinity chromatography revealed selective interaction of VIPL and VIP36 with the deglucosylated trimannose in the D1 branch of high-mannose-type oligosaccharides but with different pH dependence. ERGIC-53 bound high-mannose-type oligosaccharides with low affinity and broad specificity, not discriminating between monoglucosylated and deglucosylated high-mannosetype oligosaccharides. Based on the sugar-binding properties in conjunction with known features of these proteins, we propose a model for the action of the three lectins in glycoprotein guidance and trafficking. Moreover, structure-based mutagenesis revealed that the sugar-binding properties of these L-type lectins can be switched by single amino acid substitutions.

Bap31 is an itinerant protein that moves between the peripheral endoplasmic reticulum (ER) and a juxtanuclear compartment related to ER-associated Degradation.

Certain endoplasmic reticulum (ER)-associated degradation (ERAD) substrates with transmembrane domains are segregated from other ER proteins and sorted into a juxtanuclear subcompartment, known as the ER quality control compartment. Bap31 is an ER protein with three transmembrane domains, and it is assumed to be a cargo receptor for ER export of some transmembrane proteins, especially those prone to ERAD. Here, we show that Bap31 is a component of the ER quality control compartment and that it moves between the peripheral ER and a juxtanuclear ER or ER-related compartment distinct from the conventional ER-Golgi intermediate compartment. The third and second transmembrane domains of Bap31 are principally responsible for the movement to and recycling from the juxtanuclear region, respectively. This cycling was blocked by depolymerization of microtubules and disruption of dynein-dynactin function. Overexpression of Sar1p and Arf1 mutants affected Bap31 cycling, suggesting that this cycling pathway is related to the conventional vesicular transport pathways.