ATF6 is an endoplasmic reticulum (ER) stress-regulated transmembrane transcription factor that activates the transcription of ER molecular chaperones. Upon ER stress, ATF6 translocates from the ER to the Golgi where it is processed to its active form. We have found that the ER chaperone BiP/GRP78 binds ATF6 and dissociates in response to ER stress. Loss of BiP binding correlates with the translocation of ATF6 to the Golgi, which was slowed in cells overexpressing BiP. Two Golgi localization signals (GLSs) were identified in ATF6. Removal of BiP binding sites from ATF6, while retaining a GLS, resulted in its constitutive translocation to the Golgi. These results suggest that BiP retains ATF6 in the ER by inhibiting its GLSs and that dissociation of BiP during ER stress allows ATF6 to be transported to the Golgi.

Sec1/Munc18 (SM) proteins are required for every step of intracellular membrane fusion, but their molecular mechanism of action has been unclear. In this work, we demonstrate a fundamental role of the SM protein: to act as a stimulatory subunit of its cognate SNARE fusion machinery. In a reconstituted system, mammalian SNARE pairs assemble between bilayers to drive a basal fusion reaction. Munc18-1/nSec1, a synaptic SM protein required for neurotransmitter release, strongly accelerates this reaction through direct contact with both t- and v-SNAREs. Munc18-1 accelerates fusion only for the cognate SNAREs for exocytosis, therefore enhancing fusion specificity.

ATF6 is an endoplasmic reticulum (ER) transmembrane transcription factor that is activated by the ER stress/unfolded protein response by cleavage of its Nterminal half from the membrane. We find that ER stress induces ATF6 to move from the ER to the Golgi, where it is cut in its luminal domain by site 1 protease. ATF6 contains a single transmembrane domain with 272 amino acids oriented in the lumen of the ER. We found that this luminal domain is required for the translocation of ATF6 to the Golgi and its subsequent cleavage, and we have mapped regions required for these properties. These results suggest that the conserved CD1 region is required for translocation, whereas the CD2 region is required for site 1 protease cleavage. We also find that ATF6's luminal domain is sufficient to sense ER stress and cause translocation to the Golgi when fused to LZIP, another ER transmembrane protein. These results show that ATF6 has a mechanism to sense ER stress and respond by translocation to the Golgi.

Endoplasmic reticulum (ER) stress-induced activation of ATF6, an ER membrane-bound transcription factor, requires a dissociation step from its inhibitory regulator, BiP. It has been generally postulated that dissociation of the BiP-ATF6 complex is a result of the competitive binding of misfolded proteins generated during ER stress. Here we present evidence against this model and for an active regulatory mechanism for dissociation of the complex. Contradictory to the competition model that is based on dynamic binding of BiP to ATF6, our data reveal relatively stable binding. First, the complex was easily isolated, in contrast to many chaperone complexes that require chemical cross-linking. Second, ATF6 bound at similar levels to wild-type BiP and a BiP mutant form that binds substrates stably because of a defect in its ATPase activity. Third, ER stress specifically induced the dissociation of BiP from ER stress transducers while the competition model would predict dissociation from any specific substrate. Fourth, the ATF6-BiP complex was resistant to ATP-induced dissociation in vitro when isolated without detergents, suggesting that cofactors stabilize the complex. In favor of an active dissociation model, one specific region within the ATF6 lumenal domain was identified as a specific ER stress-responsive sequence required for ER stress-triggered BiP release. Together, our results do not support a model in which competitive binding of misfolded proteins causes dissociation of the BiP-ATF6 complex in stressed cells. We propose that stable BiP binding is essential for ATF6 regulation and that ER stress dissociates BiP from ATF6 by actively restarting the BiP ATPase cycle.

Organelles are in constant communication with each other through exchange of proteins (mediated by trafficking vesicles) and lipids [mediated by both trafficking vesicles and lipid transfer proteins (LTPs)]. It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca 2+ , allowing membrane protein transport to be adjusted according to physiological demands. However, it remains unclear whether LTP-mediated lipid transport can also be regulated by Ca 2+ . In this work, we show that extended synaptotagmins (E-Syts), poorly understood membrane proteins at endoplasmic reticulum-plasma membrane contact sites, are Ca 2+ -dependent LTPs. Using both recombinant and endogenous mammalian proteins, we discovered that E-Syts transfer glycerophospholipids between membrane bilayers in the presence of Ca 2+ . E-Syts use their lipid-accommodating synaptotagmin-like mitochondrial lipid binding protein (SMP) domains to transfer lipids. However, the SMP domains themselves cannot transport lipids unless the two membranes are tightly tethered by Ca 2+ -bound C2 domains. Strikingly, the Ca 2+ -regulated lipid transfer activity of E-Syts was fully recapitulated when the SMP domain was fused to the cytosolic domain of synaptotagmin-1, the Ca 2+ sensor in synaptic vesicle fusion, indicating that a common mechanism of membrane tethering governs the Ca 2+ regulation of lipid transfer and vesicle fusion. Finally, we showed that microsomal vesicles isolated from mammalian cells contained robust Ca 2+ -dependent lipid transfer activities, which were mediated by E-Syts. These findings established E-Syts as a novel class of LTPs and showed that LTP-mediated lipid trafficking, like vesicular transport, can be subject to tight Ca 2+ regulation.lipid transfer | organelle | synaptotagmin | membrane contact sites T he endoplasmic reticulum (ER) is the primary site for the synthesis of proteins and lipids needed to maintain and propagate membrane-bound organelles (1). Proteins are transported from the ER to other organelles by small, sac-like trafficking vesicles, which shuttle between organelles through cycles of budding and fusion reactions (1, 2). Vesicles also carry lipids, but a substantial portion of intracellular lipid trafficking is mediated by lipid transfer proteins (LTPs) independent of vesicular transport (3). Operating at narrow membrane contact sites (MCSs) between organelles, LTPs extract lipids from one membrane and subsequently deliver them to another membrane (4, 5).It has long been known that vesicle trafficking can be tightly regulated by the second messenger Ca 2+ in certain pathways, allowing membrane protein flow to be adjusted according to physiological demands (6-8). One prominent example of Ca 2+ -regulated vesicle trafficking is the release of neurotransmitters at the chemical synapses of neurons, which serves as the brain's major form of cell-to-cell communication (2). Neurotransmitters are released when synaptic vesicles fuse with the plasma membrane (PM), a process driven by the v...