Recent evidence suggests that endoplasmic reticulum (ER) tubules mark the sites where the GTPase Drp1 promotes mitochondrial fission via a largely unknown mechanism. Here, we show that the SNARE protein syntaxin 17 (Syn17) is present on raft-like structures of ER-mitochondria contact sites and promotes mitochondrial fission by determining Drp1 localization and activity. The hairpin-like C-terminal hydrophobic domain, including Lys-254, but not the SNARE domain, is important for this regulation. Syn17 also regulates ER Ca(2+) homeostasis and interferes with Rab32-mediated regulation of mitochondrial dynamics. Starvation disrupts the Syn17-Drp1 interaction, thus favoring mitochondrial elongation during autophagy. Because we also demonstrate that Syn17 is an ancient SNARE, our findings suggest that Syn17 is one of the original key regulators for ER-mitochondria contact sites present in the last eukaryotic common ancestor. As such, Syn17 acts as a switch that responds to nutrient conditions and integrates functions for the ER and autophagosomes with mitochondrial dynamics.

We have isolated a membrane fraction enriched in a class of transport carriers that form at the trans Golgi network (TGN) and are destined for the cell surface in HeLa cells. Protein kinase D (PKD) is required for the biogenesis of these carriers that contain myosin II, Rab6a, Rab8a, and synaptotagmin II, as well as a number of secretory and plasma membrane-specific cargoes. Our findings reveal a requirement for myosin II in the migration of these transport carriers but not in their biogenesis per se. Based on the cargo secreted by these carriers we have named them CARTS for CARriers of the TGN to the cell Surface. Surprisingly, CARTS are distinct from the carriers that transport vesicular stomatitis virus (VSV)-G protein and collagen I from the TGN to the cell surface. Altogether, the identification of CARTS provides a valuable means to understand TGN to cell surface traffic.

Actin-severing proteins ADF/cofilin are required for the sorting of secretory cargo at the trans-Golgi network (TGN) in mammalian cells. How do these cytoplasmic proteins interact with the cargoes in the lumen of the TGN? Put simply, how are these two sets of proteins connected across the TGN membrane? Mass spectrometry of cofilin1 immunoprecipitated from HeLa cells revealed the presence of actin and the Ca(2+) ATPase SPCA1. Moreover, cofilin1 was localized to the TGN and bound to SPCA1 via dynamic actin. SPCA1 knockdown, like ADF/cofilin1 knockdown, inhibited Ca(2+) uptake into the TGN and caused missorting of secretory cargo. These defects were rescued by the overexpression of the TGN-localized SPCA1. We propose that ADF/cofilin-dependent severing of actin filaments exposes and promotes the activation of SPCA1, which pumps Ca(2+) into the lumen of the TGN for the sorting of the class of secretory cargo that binds Ca(2+).

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. INTRODUCTIONThe endoplasmic reticulum (ER) exhibits a reticular tubular network that extends from the nucleus to the cell periphery along microtubule tracks. It performs a variety of functions, including the synthesis, posttranslational modifications, quality control, and export of secretory and membrane proteins; the synthesis of lipids; stress response; Ca 2ϩ storage; and apoptosis. Most, if not all, of these functions are managed by subdomains, such as the rough and smooth ER and transitional ER sites. Each subdomain contains a unique set of proteins that are responsible for its function and organization. ER subdomains are relatively stable, but they can be transformed into alternative structures in response to cellular conditions (for review, see Voeltz et al., 2002;Levine and Rabouille, 2005;Borgese et al., 2006;Vedrenne and Hauri, 2006).Several studies showed the presence of a specialized ER subdomain that is related to ER-associated degradation (ERAD). ERAD is part of a quality control system that ensures the delivery of only correctly folded or assembled secretory and membrane proteins to their final destinations. Newly synthesized proteins that fail to fold or assemble correctly are retained in the ER, retrotranslocated to the cytosol, and degraded by the ubiquitin-proteasome system (for review, see Ellgaard and Helenius, 2003;Meusser et al., 2005;Rö misch, 2005). Studies using mammalian cells demonstrated that transmembrane ERAD substrates are segregated into "ER quality control compartments," which become discernible at the juxtanuclear region upon inhibition of ERAD by proteasome inhibitors (Kamhi-Nesher et al., 2001;Spiliotis et al., 2002). In Saccharomyces cerevisiae, ectopically expressed cystic fibrosis transmembrane conductance regulator (CFTR) is segregated from other ER proteins and accumulates in ER-associated compartments (Kiser et al., 2001;Zhang et al., 2001;Huyer et al., 2004). Degradation of CFTR in yeast is independent of ER-to-Golgi tr...

Biogenesis of the TGN-derived transport carriers CARTS requires the ER protein VAP and Golgi lipid transfer proteins, ceramide transfer protein and OSBP. Sac1 lipid phosphatase is recruited to a VAP–OSBP complex formed at an ER subdomain closely apposed to the trans-Golgi/TGN. Association–dissociation dynamics of ER–Golgi contacts are important for CARTS formation.