Our overall goal is to understand the mechanism of protein secretion and biogenesis of the Golgi complex. The major ongoing research lines are listed in the section below.
- Mechanism of cargo sorting and export at the Trans Golgi Network (TGN). Our findings are beginning to unravel the mechanism of receptor independent sorting and export of secretory cargoes at the TGN. We discovered that a Ca2+ ATPase called SPCA1 was required for this process (Von Blume et al., 2011). SPCA1 interacted with Cab45, a soluble resident protein of the TGN, which bound secretory cargoes in Ca2+ dependent manner (Von Blume et al., 2012). Cab45 functions as a sponge to sequester secretory cargoes while excluding proteins that are targeted to the endosome/lysosome or to the endoplasmic reticulum (ER). This mode of secretory cargo sorting is conserved (Curwin et al., 2012) and our findings will help reveal the mechanism of this issue of fundamental importance. How are secretory cargoes, post sorting, exported from the TGN? We have reported before the involvement of Protein Kinase D (PKD) and diacylglycerol (DAG) in generation of specific carriers at the TGN (Campelo and Malhotra. 2012). We reported the identification of a new class of transport carriers (named them CARTS for CARriers of the TGN to cell Surface) that export specific secretory proteins from the TGN. Although we do not know the full complement of proteins and lipids contained in CARTS, they are found to contain a protein that is sorted by Cab45 dependent reaction (Wakana et al 2012). The trafficking of CARTS is mediated by a kinesin (Eg5) that prior to our findings was known only for its function in spindle dynamics in mitosis (Wakana et al., 2013). These findings have opened a new chapter for investigating the mechanism of vesicle-mediated transport from TGN. What is the role of specific lipids such as Sphingomyelin (SM) in events leading to cargo sorting and export? We found that lowering the synthesis of SM inhibited formation of export carriers from the Golgi membranes, but did not affect fusion of incoming transport carriers from ER and endosomes (Duran et al., 2012). We reasoned that SM concentration was crucial in clustering chemical components required to process secretory cargo and/or generate transport carriers (CARTS and COPI vesicles). As a first test of this hypothesis, we monitored glycosylation of a cargo by the TGN specific glycosyl transferase in cells depleted of SM. Our results revealed that lowering SM concentration segregated the glycosyl transferase from its substrate (Van Galen et al., 2014). This provides a good foundation for further investigating the role SM in creating platforms for chemical reactions involved in posttranslational modification, sorting, and export of membrane bound proteins at the TGN. Altogether our findings are revealing the principles by which divalent ions, lipids, and proteins function to sort proteins and help in their packaging for export from the TGN.
- Mechanism of mega cargo export from the ER. In 2009, we reported the requirement of TANGO1 in procollagen VII export from the ER (Saito et al., 2009). TANGO1 is one of the genes that we had identified in a genome wide screen for new components involved in Transport ANd Golgi Organization (Bard et al., 2006). Surprisingly, we discovered that membrane fusion proteins were required for collagen export from the ER (Nogueira et al., 2014). We found that a domain of TANGO1 recruited ERGIC53 containing membranes derived from the ER-Golgi intermediate compartment to collagen-enriched patches in the ER (Santos et al., 2015). Our findings reveal that ERGIC membrane is added to the ER at the site of collagen export so that the nascent carrier can accommodate collagen. In other words, membranes fuse with a collagen-enriched patch of the ER to promote growth of the export carrier. Altogether, we suggest that TANGO1 recruits collagen in the lumen of the ER and generates a collagen-enriched domain. The cytoplasmic domain of TANGO1 recruits inner COPII coat proteins Sec23/24 and ERGIC membranes. ERGIC membranes are used to generate a mega tubule, which is COPII coated and hence the need to increase the quantity/size of COPII coats. Dr. Schekman and colleagues (UC Berkeley) have suggested ubiquitin mediated increase in the outer COPII coat proteins Sec13/31, which fits well with the increased recruitment of inner COPII coat proteins by TANGO1 (Malhotra and Erlmann. 2015). We have more recently identified a TANGO1 like protein (TALI) that functions in the export of extremely bulky lipoprotein particles called chylomicrons from the ER. The basic mechanism of TALI function is the same as TANGO1except that the former binds ApoB coated lipid particles called chylomicrons and the later interacts with collagens (Santos et al., submitted). So, TANGO and TALI provide a means to understand the mechanism by which homeostasis of extracellular matrix (through collagen export and assembly) and cholesterol and dietary lipid (secreted chylomicrons traffic these components across tissues) is regulated in health and disease.
- Mechanism of unconventional protein secretion. We have known for 2 decades that eukaryotic cells secrete proteins that cannot enter the ER-Golgi pathway of conventional protein secretion. My lab is the first to identify a protein, called GRASP that is a component of the unconventional protein secretion pathway (Kinseth et al., 2007). In 2010, we reported the involvement of large number of new genes in this pathway (Duran et al., 2010). We identified a previously unknown organelle, called CUPS, that contains GRASP and forms under conditions that promote unconventional secretion (Bruns et al., 2011). Our new findings reveal that cells collect membranes from late Golgi and endosomes to generate CUPS during nutrient starvation in yeast. Interestingly, upon returning to normal growth conditions, CUPS fuse with the ER by a COPI dependent reaction. By this procedure, CUPS function during starvation and then, upon return to normal conditions, return components that had been barrowed from the late Golgi and endosomes by standard COPII mediated export from the ER (Cruz et al., 2014). More recently, we have reconstituted the unconventional protein secretion in yeast by a quantitative procedure. This procedure has confirmed the involvement of GRASP protein and revealed the requirement of ESCRT proteins of the multivesicular body pathway. Surprisingly, however, the key component of the MVB pathway, Vps4, is not involved in unconventional protein secretion. Altogether, our findings suggest a CUPS dependent, but MVB independent pathway of protein secretion (Curwin et al., in preparation for submission). We have also identified a number of new proteins that are secreted unconventionally, which should help in identifying a common signal that governs recruitment of cargoes into this pathway of secretion (Cruz et al., in preparation). These findings are thus beginning to unravel a novel pathway by which cells secrete essential cytokines, growth factors, adipokines, diazepam binding inhibitor, SOD1, and tissue transglutaminase in a signal dependent manner.
- Mechanism of mucous homeostasis. Dysregulation of mucin secretion is a major issue in human pathologies of chronic obstructive pulmonary disease (COPD: hyper secretion of mucins) and Crohn’s disease (hypo secretion of mucins). We performed a genome wide screen and identified 19 new genes required for mucin secretion. This pool included a gene called TRPM5, which upon stimulation by extracellular ATP promoted Na2+ entry into cells to trigger Ca2+ flux via the sodium/calcium exchanger (NCX). Inhibition of TRPM5 and NCX inhibited mucin (Muc5Ac) secretion (Mitrovic et al., 2013). Our new results reveal that NCX is also necessary for mucin secretion by cells of the respiratory lining of patients with cystic fibrosis (Cantero et al., to be submitted). Collectively, these genes are unravelling the mechanism of mucin secretion and our approaches also have the potential of generating valuable therapeutics.