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Reconstitution of CUPS in vitro and assessing the mechanism of their cargo packing during unconventional protein secretion

Final Report Summary - CUPS (Reconstitution of CUPS in vitro and assessing the mechanism of their cargo packing during unconventional protein secretion)

Human cells express 28 different collagens that constitute roughly 25% of our dry body weight. Most of these collagens are secreted (Kadler et al., 2007; Malhotra et al., 2015). Given the abundance and importance of collagens, an understanding of the mechanism by which these proteins are exported from their site of synthesis in the endoplasmic reticulum (ER) is of invaluable importance. The challenge arises from the fact that collagens contain rigid triple helical domains that can reach 450 nm in length (Burgeson et al., 1985) and therefore are too big to be exported by conventional COPII (Coat Protein complex II)-coated vesicles of 60-90 nm diameter from the ER (Glick and Malhotra, 1998; Malhotra et al., 2015; Miller and Schekman, 2013; Saito and Katada, 2015). COPII-coated vesicles are formed with membranes moulded exclusively from the ER. These vesicles have been characterised extensively and it is clear they cannot accommodate bulky cargoes such as the collagens. An important step in understanding the mechanism by which collagens are exported from the ER was the identification of a resident protein at ER exit sites called TANGO1 (Bard et al., 2006; Saito et al., 2009). The ER-lumenal domain of TANGO1 is required for binding to cargos and on the cytoplasmic side, a proline rich domain interacts with the COPII coat proteins (Saito et al., 2009; Saito et al., 2011). It has been shown that TANGO1 mediates the export of the only collagen expressed in Drosophila, and TANGO1-knockout mice are defective in the secretion of many collagens (Wilson et al., 2011). The Malhotra lab showed previously that a fusion event of membranes from another compartment with the ER was required for collagen VII export (Nogueira et al., 2014). When I joined the lab, the source of these membranes and how were they recruited specifically to the sites of export of collagen VII was unknown. Also to be discovered were the other components of this SNARE-mediated fusion reaction. As a first project during my Post-doc I decided to address these issues. Together with others, I uncovered the entire complement of t-SNAREs required for collagen VII export from the ER - BNIP1, USE1 (in addition to the previously described Syntaxin 18); showed that ERGIC membranes containing the v-SNARE YKT6 are required for fusion with the ER; and that the first coiled coil domain of TANGO1 is sufficient to recruit these membranes (a domain we named TEER). The results of this were published at the end of 2015 in the highly prestigious journal eLife (Santos et al., 2015).

Besides collagens, other secreted cargoes, including chylomicrons and VLDLs (around 150-500 nm and 100-120 nm diameter, respectively), are too large to fit into COPII-coated vesicles (Fromme and Schekman, 2005; Malhotra and Erlmann, 2011; Malhotra et al., 2015). These lipid particles, decorated with apolipoprotein B (ApoB), are secreted by cells of the liver and the small intestine and a defect in their export impairs the homeostasis of cholesterol and triglycerides. A chimeric protein resulting from fusion of cTAGE5 and MIA2 genes, which shares extensive structural homology with TANGO1, was identified in mice (Pitman et al., 2011). A mutation in cTAGE5/MIA2 has been found to correlate with a systemic reduction in the plasma levels of cholesterol and triglycerides in mice (Pitman et al., 2011). In addition, many GWAS (Genome-Wide Association Studies) showed that a SNP in TANGO1 confers higher risk of coronary artery disease and atherosclerosis (Garcia-Bermudez et al., 2012; Li et al., 2013; Roder et al., 2011). Since there was no known receptor for the exit of bulky ApoB-containing particles, like chylomicrons and VLDLs, I tested whether their export from the ER required TANGO1 and cTAGE5/MIA2, which we named TALI (for TANGO1-LIke). This was my second project during my Post-Doc studies in the Malhotra lab. My data showed that loss of TANGO1 or TALI in intestinal and liver cells, that produce and secrete chylomicrons and VLDLs, respectively (Jammart et al., 2013; Meex et al., 2011; Nauli et al., 2014; van Greevenbroek et al., 2000), reduces ApoB secretion. During this work, which is going to publish soon as an article in the Journal of Cell Biology (JCB), I further showed that TANGO1 interacts with TALI, and both interact with ApoB, to recruit bulky lipid particles to ER exit sites. Loss of either TANGO1 or TALI leads to an accumulation of chylomicrons/VLDLs in the ER. Several studies have identified circulating chylomicrons and VLDLs as a major contributor to the accumulation of the atherosclerotic plaque and therefore an association between their secretion and clearance and the development of atherosclerosis is currently well accepted (Nakano et al., 2008; Pal et al., 2003; Proctor and Mamo, 2003; Tomkin and Owens, 2012). My work further helps in the mechanistic understanding of this disease.