Steady-state regulation of COPII-dependent secretory cargo sorting by inositol trisphosphate receptors, calcium, and penta EF hand proteins.

Recently, we demonstrated that agonist-stimulated Ca2+ signaling involving IP3 receptors modulates ER export rates through activation of the penta-EF Hand proteins apoptosis-linked gene-2 (ALG-2) and peflin. It is unknown, however, whether IP3Rs and penta-EF proteins regulate ER export rates at steady state. Here we tested this idea in normal rat kidney epithelial cells by manipulation of IP3R isoform expression. Under standard growth conditions, spontaneous cytosolic Ca2+ oscillations occurred simultaneously in successive groups of contiguous cells, generating intercellular Ca2+ waves that moved across the monolayer periodically. Depletion of IP3R-3, typically the least promiscuous IP3R isoform, caused increased cell participation in intercellular Ca2+ waves in unstimulated cells. The increased spontaneous signaling was sufficient to cause increased ALG-2 and COPII coat subunit Sec31A and decreased peflin localization at ER exit sites, resulting in increased ER-to-Golgi transport of the COPII client cargo VSV-G. The elevated ER-to-Golgi transport caused greater concentration of VSV-G at ER exit sites and had reciprocal effects on transport of VSV-G and a bulk-flow cargo, though both cargos equally required Sec31A. Inactivation of client cargo sorting using 4-phenylbutyrate had opposing reciprocal effects on client and bulk-flow cargo and neutralized any effect of ALG-2 activation on transport. This work extends our knowledge of ALG-2 mechanisms and indicates that in normal rat kidney cells, IP3R isoforms regulate homeostatic Ca2+ signaling that helps determine the basal secretion rate and stringency of COPII-dependent cargo sorting.

Ca2+ regulation of constitutive vesicle trafficking.

Constitutive vesicle trafficking is the default pathway used by all cells for movement of intracellular cargoes between subcellular compartments and in and out of the cell. Classically, constitutive trafficking was thought to be continuous and unregulated, in contrast to regulated secretion, wherein vesicles are stored intracellularly until undergoing synchronous membrane fusion following a Ca2+ signal. However, as shown in the literature reviewed here, many continuous trafficking steps can be up- or down-regulated by Ca2+, including several steps associated with human pathologies. Notably, we describe a series of Ca2+ pumps, channels, Ca2+-binding effector proteins, and their trafficking machinery targets that together regulate the flux of cargo in response to genetic alterations as well as baseline and agonist-dependent Ca2+ signals. Here, we review the most recent advances, organized by organellar location, that establish the importance of these components in trafficking steps. Ultimately, we conclude that Ca2+ regulates an expanding series of distinct mechanistic steps. Furthermore, the involvement of Ca2+ in trafficking is complex. For example, in some cases, the same Ca2+ effectors regulate surprisingly distinct trafficking steps, or even the same trafficking step with opposing influences, through binding to different target proteins.

Genetically encoded fluorescent tools: Shining a little light on ER-to-Golgi transport.

Since the first fluorescent proteins (FPs) were identified and isolated over fifty years ago, FPs have become commonplace yet indispensable tools for studying the constitutive secretory pathway in live cells. At the same time, genetically encoded chemical tags have provided a new use for much older fluorescent dyes. Innovation has also produced several specialized methods to allow synchronous release of cargo proteins from the endoplasmic reticulum (ER), enabling precise characterization of sequential trafficking steps in the secretory pathway. Without the constant innovation of the researchers who design these tools to control, image, and quantitate protein secretion, major discoveries about ER-to-Golgi transport and later stages of the constitutive secretory pathway would not have been possible. We review many of the tools and tricks, some 25 years old and others brand new, that have been successfully implemented to study ER-to-Golgi transport in intact and living cells.

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.

Endoplasmic reticulum tubules limit the size of misfolded protein condensates.

The endoplasmic reticulum (ER) is composed of sheets and tubules. Here we report that the COPII coat subunit, SEC24C, works with the long form of the tubular ER-phagy receptor, RTN3, to target dominant-interfering mutant proinsulin Akita puncta to lysosomes. When the delivery of Akita puncta to lysosomes was disrupted, large puncta accumulated in the ER. Unexpectedly, photobleach analysis indicated that Akita puncta behaved as condensates and not aggregates, as previously suggested. Akita puncta enlarged when either RTN3 or SEC24C were depleted, or when ER sheets were proliferated by either knocking out Lunapark or overexpressing CLIMP63. Other ER-phagy substrates that are segregated into tubules behaved like Akita, while a substrate (type I procollagen) that is degraded by the ER-phagy sheets receptor, FAM134B, did not. Conversely, when ER tubules were augmented in Lunapark knock-out cells by overexpressing reticulons, ER-phagy increased and the number of large Akita puncta was reduced. Our findings imply that segregating cargoes into tubules has two beneficial roles. First, it localizes mutant misfolded proteins, the receptor, and SEC24C to the same ER domain. Second, physically restraining condensates within tubules, before they undergo ER-phagy, prevents them from enlarging and impacting cell health.

ER-to-Golgi Transport in HeLa Cells Displays High Resilience to Ca2+ and Energy Stresses.

One third of all human proteins are either transmembrane or soluble secretory proteins that first target the endoplasmic reticulum (ER). These proteins subsequently leave the ER and enter the Golgi apparatus via ER-Golgi intermediate vesicular structures. Live-cell imaging of cargos fused to fluorescent proteins (FPs) enables the high-resolution visualization and characterization of secretory transport processes. Here, we performed fluorescence time-lapse imaging to assess the Ca2+ and energy dependency of ER-to-Golgi transport in living HeLa cells, a cancer cell model which has been well investigated. Our data revealed that ER-to-Golgi transport remained highly efficient in the absence of ATP-generating substrates, despite clear reductions in cytosolic and mitochondrial ATP levels under these energy stress conditions. However, cell treatment with 2-deoxy-D-glucose (2-DG), which severely diminished subcellular ATP levels, abolished ER-to-Golgi transport. Interestingly, while 2-DG elevated cytosolic Ca2+ levels and reduced long-distance movements of glycosylphosphatidylinositol (GPI)-positive vesicles, robust short-term ER Ca2+ mobilizations, which strongly affected the motility of these vesicles, did not considerably impair ER-to-Golgi transport. In summary, we highlight that ER-to-Golgi transport in HeLa cells remains functional despite high energy and Ca2+ stress levels.

Live cell imaging of signaling and metabolic activities.

The interplay of metabolic and signaling processes is prerequisite for the functionality of cells. Any disturbances may have severe consequences, resulting in the development of diseases. However, the complex coordination of metabolism and signaling events makes it difficult to decipher the link between molecular irregularities and pathogenesis. An excellent way to provide more clarity is to see into the living cell and watch cellular processes in real-time, with the add-on of being able to manipulate certain processes. Live cell imaging enables us to do exactly that, with steadily improving spatial and temporal resolution. Modern genetically encoded fluorescent probes in combination with state-of-the-art high-resolution imaging devices have proven themselves as a valuable approach for monitoring, manipulating and ultimately understanding the interaction of cell metabolism and signaling. These probes also represent powerful tools for detecting biomarkers of disease, identifying new drug targets and elucidating drug actions at the cellular to the molecular level.

pH-Lemon, a Fluorescent Protein-Based pH Reporter for Acidic Compartments.

Distinct subcellular pH levels, especially in lysosomes and endosomes, are essential for the degradation, modification, sorting, accumulation, and secretion of macromolecules. Here, we engineered a novel genetically encoded pH probe by fusing the pH-stable cyan fluorescent protein (FP) variant, mTurquoise2, to the highly pH-sensitive enhanced yellow fluorescent protein, EYFP. This approach yielded a ratiometric biosensor-referred to as pH-Lemon-optimized for live imaging of distinct pH conditions within acidic cellular compartments. Protonation of pH-Lemon under acidic conditions significantly decreases the yellow fluorescence while the cyan fluorescence increases due to reduced Förster resonance energy transfer (FRET) efficiency. Because of its freely reversible and ratiometric responses, pH-Lemon represents a fluorescent biosensor for pH dynamics. pH-Lemon also shows a sizable pH-dependent fluorescence lifetime change that can be used in fluorescence lifetime imaging microscopy as an alternative observation method for the study of pH in acidic cellular compartments. Fusion of pH-Lemon to the protein microtubule-associated protein 1A/1B-light chain 3B (LC3B), a specific marker of autophagic membranes, resulted in its targeting within autolysosomes of HeLa cells. Moreover, fusion of pH-Lemon to a glycophosphatidylinositol (GPI) anchor allowed us to monitor the entire luminal space of the secretory pathway and the exoplasmic leaflet of the plasma membrane. Utilizing this new pH probe, we revealed neutral and acidic vesicles and substructures inside cells, highlighting compartments of distinct pH throughout the endomembrane system. These data demonstrate, that this novel pH sensor, pH-Lemon, is very suitable for the study of local pH dynamics of subcellular microstructures in living cells.

The enigmatic ATP supply of the endoplasmic reticulum.

The endoplasmic reticulum (ER) is a functionally and morphologically complex cellular organelle largely responsible for a variety of crucial functions, including protein folding, maturation and degradation. Furthermore, the ER plays an essential role in lipid biosynthesis, dynamic Ca2+ storage, and detoxification. Malfunctions in ER-related processes are responsible for the genesis and progression of many diseases, such as heart failure, cancer, neurodegeneration and metabolic disorders. To fulfill many of its vital functions, the ER relies on a sufficient energy supply in the form of adenosine-5'-triphosphate (ATP), the main cellular energy source. Despite landmark discoveries and clarification of the functional principles of ER-resident proteins and key ER-related processes, the mechanism underlying ER ATP transport remains somewhat enigmatic. Here we summarize ER-related ATP-consuming processes and outline our knowledge about the nature and function of the ER energy supply.

Penta-EF-Hand Protein Peflin Is a Negative Regulator of ER-To-Golgi Transport.

Luminal calcium regulates vesicle transport early in the secretory pathway. In ER-to-Golgi transport, depletion of luminal calcium leads to significantly reduced transport and a buildup of budding and newly budded COPII vesicles and vesicle proteins. Effects of luminal calcium on transport may be mediated by cytoplasmic calcium sensors near ER exits sites (ERES). The penta-EF-hand (PEF) protein apoptosis-linked gene 2 (ALG-2) stabilizes sec31A at ER exit sites (ERES) and promotes the assembly of inner and outer shell COPII components. However, in vitro and intact cell approaches have not determined whether ALG-2 is a negative or positive regulator, or a regulator at all, under basal physiological conditions. ALG-2 interacts with another PEF protein, peflin, to form cytosolic heterodimers that dissociate in response to calcium. However, a biological function for peflin has not been demonstrated and whether peflin and the ALG-2/peflin interaction modulates transport has not been investigated. Using an intact, single cell, morphological assay for ER-to-Golgi transport in normal rat kidney (NRK) cells, we found that depletion of peflin using siRNA resulted in significantly faster transport of the membrane cargo VSV-G. Double depletion of peflin and ALG-2 blocked the increased transport resulting from peflin depletion, demonstrating a role for ALG-2 in the increased transport. Furthermore, peflin depletion caused increased targeting of ALG-2 to ERES and increased ALG-2/sec31A interactions, suggesting that peflin may normally inhibit transport by preventing ALG-2/sec31A interactions. This work identifies for the first time a clear steady state role for a PEF protein in ER-to-Golgi transport-peflin is a negative regulator of transport.

Alpha-synuclein Toxicity in the Early Secretory Pathway: How It Drives Neurodegeneration in Parkinsons Disease.

Alpha-synuclein is a predominant player in the pathogenesis of Parkinson's Disease. However, despite extensive study for two decades, its physiological and pathological mechanisms remain poorly understood. Alpha-synuclein forms a perplexing web of interactions with lipids, trafficking machinery, and other regulatory factors. One emerging consensus is that synaptic vesicles are likely the functional site for alpha-synuclein, where it appears to facilitate vesicle docking and fusion. On the other hand, the dysfunctions of alpha-synuclein are more dispersed and numerous; when mutated or over-expressed, alpha-synuclein affects several membrane trafficking and stress pathways, including exocytosis, ER-to-Golgi transport, ER stress, Golgi homeostasis, endocytosis, autophagy, oxidative stress, and others. Here we examine recent developments in alpha-synuclein's toxicity in the early secretory pathway placed in the context of emerging themes from other affected pathways to help illuminate its underlying pathogenic mechanisms in neurodegeneration.

p115-SNARE interactions: a dynamic cycle of p115 binding monomeric SNARE motifs and releasing assembled bundles.

Tethering factors regulate the targeting of membrane-enclosed vesicles under the control of Rab GTPases. p115, a golgin family tether, has been shown to participate in multiple stages of ER/Golgi transport. Despite extensive study, the mechanism of action of p115 is poorly understood. SNARE proteins make up the machinery for membrane fusion, and strong evidence shows that function of p115 is directly linked to its interaction with SNAREs. Using a gel filtration binding assay, we have demonstrated that in solution p115 stably interacts with ER/Golgi SNAREs rbet1 and sec22b, but not membrin and syntaxin 5. These binding preferences stemmed from selectivity of p115 for monomeric SNARE motifs as opposed to SNARE oligomers. Soluble monomeric rbet1 can compete off p115 from coat protein II (COPII) vesicles. Furthermore, excess p115 inhibits p115 function in trafficking. We conclude that monomeric SNAREs are a major binding site for p115 on COPII vesicles, and that p115 dissociates from its SNARE partners upon SNAREpin assembly. Our results suggest a model in which p115 forms a mixed p115/SNARE helix bundle with a monomeric SNARE, facilitates the binding activity and/or concentration of the SNARE at prefusion sites and is subsequently ejected as SNARE complex formation and fusion proceed.

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.

Sit4p/PP6 regulates ER-to-Golgi traffic by controlling the dephosphorylation of COPII coat subunits.

Traffic from the endoplasmic reticulum (ER) to the Golgi complex is initiated when the activated form of the GTPase Sar1p recruits the Sec23p-Sec24p complex to ER membranes. The Sec23p-Sec24p complex, which forms the inner shell of the COPII coat, sorts cargo into ER-derived vesicles. The coat inner shell recruits the Sec13p-Sec31p complex, leading to coat polymerization and vesicle budding. Recent studies revealed that the Sec23p subunit sequentially interacts with three different binding partners to direct a COPII vesicle to the Golgi. One of these binding partners is the serine/threonine kinase Hrr25p. Hrr25p phosphorylates the COPII coat, driving the membrane-bound pool into the cytosol. The phosphorylated coat cannot rebind to the ER to initiate a new round of vesicle budding unless it is dephosphorylated. Here we screen all known protein phosphatases in yeast to identify one whose loss of function alters the cellular distribution of COPII coat subunits. This screen identifies the PP2A-like phosphatase Sit4p as a regulator of COPII coat dephosphorylation. Hyperphosphorylated coat subunits accumulate in the sit4Δ mutant in vivo. In vitro, Sit4p dephosphorylates COPII coat subunits. Consistent with a role in coat recycling, Sit4p and its mammalian orthologue, PP6, regulate traffic from the ER to the Golgi complex.

Poliovirus infection transiently increases COPII vesicle budding.

Poliovirus (PV) requires membranes of the host cell's secretory pathway to generate replication complexes (RCs) for viral RNA synthesis. Recent work identified the intermediate compartment and the Golgi apparatus as the precursors of the replication "organelles" of PV (N. Y. Hsu et al., Cell 141:799-811, 2010). In this study, we examined the effect of PV on COPII vesicles, the secretory cargo carriers that bud from the endoplasmic reticulum and homotypically fuse to form the intermediate compartment that matures into the Golgi apparatus. We found that infection by PV results in a biphasic change in functional COPII vesicle biogenesis in cells, with an early enhancement and subsequent inhibition. Concomitant with the early increase in COPII vesicle formation, we found an increase in the membrane fraction of Sec16A, a key regulator of COPII vesicle formation. We suggest that the early burst in COPII vesicle formation detected benefits PV by increasing the precursor pool required for the formation of its RCs.

R-SNARE ykt6 resides in membrane-associated protease-resistant protein particles and modulates cell cycle progression when over-expressed.

BACKGROUND INFORMATION: The arginine-type soluble N-ethylmaleimide-sensitive factor attachment protein receptor (R-SNARE) ykt6 possesses several atypical properties including selective high expression in neurons, a lipidated C-terminus, localization to punctae that do not correspond with known endomembrane markers, a potent ability to protect the secretory pathway from alpha-synuclein over-expression and specific up-regulation in tumors. We have followed up on several of these features that together suggest nontraditional SNARE structures and functions. RESULTS: A significant portion of ykt6 in PC12 cells was found in a protease-resistant state suggestive of a large complex or aggregate. Other endoplasmic reticulum/Golgi SNAREs were not protease resistant, demonstrating that SNARE complexes per se did not cause protease resistance. Mutagenesis indicated that lipidation of the ykt6 C-terminus was also not involved, implicating its longin domain in particle formation. Immunogold electron microscopy revealed ykt6 labeling of ∼100 nm electron densities associated with diverse membranes. Density gradient analysis of the protease-resistant structures confirmed their tight association with membranes. Since excess ykt6 has been correlated with tumorigenesis, we tested whether ykt6 over-expression in normal rat kidney cells that normally express little ykt6 affected the cell cycle. Ykt6 over-expression was found to result in altered cell division cycles as evidenced by significantly smaller cells, a higher mitotic index and increased DNA synthesis. Mutagenesis studies dis-correlated SNARE function with the cell cycle effects; instead, the cell cycle effects correlated better with ykt6 properties related to the longin domain or particle formation. CONCLUSIONS: The ykt6 particles/aggregates may represent ykt6 engaged in a non-SNARE function(s) or else nonfunctional, stored and/or excess ykt6. Whether the particulate ykt6 structures represent a means of buffering the apparent proliferative activity or are in fact mechanistically related to this activity will be of future interest in neuroscience and cancer biology.

Transport vesicle uncoating: it's later than you think.

Transport vesicle coat proteins play active roles in vesicle cargo sorting as well as membrane deformation and fission during vesicle biogenesis. For years, it was assumed that this was the extent of the coats' function and that the coats depolymerized immediately after vesicle budding, leaving the exposed fusion machinery free to find, dock, and fuse with the proper target membrane. Recently, however, it has become increasingly clear that the coat remains on transport vesicles during their post-budding life and in fact helps properly pair up the vesicle with its intended target membrane. These data have brought up urgent questions about exactly when vesicles do uncoat and how uncoating is regulated. Here, we summarize the latest round of evidence for post-budding roles for coats, including a few hints about how the uncoating process may be coupled to docking and fusion. We also speculate about the possibility of post-fusion functions for residual coats.

Alpha-synuclein delays endoplasmic reticulum (ER)-to-Golgi transport in mammalian cells by antagonizing ER/Golgi SNAREs.

Toxicity of human alpha-synuclein when expressed in simple organisms can be suppressed by overexpression of endoplasmic reticulum (ER)-to-Golgi transport machinery, suggesting that inhibition of constitutive secretion represents a fundamental cause of the toxicity. Whether similar inhibition in mammals represents a cause of familial Parkinson's disease has not been established. We tested elements of this hypothesis by expressing human alpha-synuclein in mammalian kidney and neuroendocrine cells and assessing ER-to-Golgi transport. Overexpression of wild type or the familial disease-associated A53T mutant alpha-synuclein delayed transport by up to 50%; however, A53T inhibited more potently. The secretory delay occurred at low expression levels and was not accompanied by insoluble alpha-synuclein aggregates or mistargeting of transport machinery, suggesting a direct action of soluble alpha-synuclein on trafficking proteins. Co-overexpression of ER/Golgi arginine soluble N-ethylmaleimide-sensitive factor attachment protein receptors (R-SNAREs) specifically rescued transport, indicating that alpha-synuclein antagonizes SNARE function. Ykt6 reversed alpha-synuclein inhibition much more effectively than sec22b, suggesting a possible neuroprotective role for the enigmatic high expression of ykt6 in neurons. In in vitro reconstitutions, purified alpha-synuclein A53T protein specifically inhibited COPII vesicle docking and fusion at a pre-Golgi step. Finally, soluble alpha-synuclein A53T directly bound ER/Golgi SNAREs and inhibited SNARE complex assembly, providing a potential mechanism for toxic effects in the early secretory pathway.

Vesicular calcium regulates coat retention, fusogenicity, and size of pre-Golgi intermediates.

The significance and extent of Ca(2+) regulation of the biosynthetic secretory pathway have been difficult to establish, and our knowledge of regulatory relationships integrating Ca(2+) with vesicle coats and function is rudimentary. Here, we investigated potential roles and mechanisms of luminal Ca(2+) in the early secretory pathway. Specific depletion of luminal Ca(2+) 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 Ca(2+) in COPII vesicle homotypic fusion was demonstrated in vitro using Ca(2+) 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 Ca(2+)-requiring manner, suggesting that ALG-2 may be a sensor for the effects of vesicular Ca(2+) on homotypic fusion. Immunoisolation established that Ca(2+) 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 Ca(2+), acting through ALG-2, favors the retention of residual coat molecules that seem to suppress membrane fusion. We propose that in cells, these Ca(2+)-dependent mechanisms temporally regulate COPII vesicle interactions, VTC biogenesis, cargo sorting, and VTC maturation.

Calcium: a fundamental regulator of intracellular membrane fusion?

For many years, it has been known that an increase in cytosolic calcium triggers the fusion of secretory granules and synaptic vesicles with the plasma membrane. However, the role of calcium in the intracellular membrane-fusion reactions that coordinate the secretory and endocytic pathways has been less clear. Initially, there was accumulating evidence to indicate that a focally localized and transient calcium signal is required to trigger even those fusion events formerly classified as 'constitutive'-that is, those that normally occur in the absence of global cytosolic calcium increases. Therefore, calcium seemed to be a required fundamental co-factor underlying all biological membrane-fusion steps, perhaps with a conserved mechanism of action. However, although such unification would be gratifying, new data indicate that several intracellular fusion events do not require calcium after all. In this review, the evidence for calcium requirements and its modes of action in constitutive trafficking are discussed. As a challenging perspective, I suggest that the specific absence of calcium requirements for some transport steps in fact expands the function of calcium in trafficking, because divergent luminal calcium concentrations and requirements for fusion might increase the specificity with which intracellular membrane-fusion partners are determined.