**Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo**

Douglas S. Darling, Srirangapatnam G. Venkatesh, Dipti Goyal and Anne L. Carenbauer *University of Louisville, Louisville, Kentucky USA* 

#### **1. Introduction**

38 Crosstalk and Integration of Membrane Trafficking Pathways

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Every cell must sort and transport proteins. This is true for soluble proteins as well as proteins that are in membranes, each of which need to be directed to appropriate subcellular or extracellular destinations in order to perform their essential functions. In eukaryotes, selective trafficking contributes to maintaining the different compositions of different membranes such as apical and basolateral plasma membranes, as well as directing appropriate proteins to lysosomes, endosomes, multivesicular bodies, or other intracellular compartments. The normal physiology of the cell is critically dependent on selective trafficking of proteins and membranes between different transport pathways within the cell. Other chapters in this book focus on the mechanics of transporting cargo membranes, including the molecular aspects of vesicle fusion to specific target membranes. This chapter will focus on the importance and mechanisms of sorting luminal cargo into different pathways, i.e., the "selective" aspect of selective trafficking, particularly with respect to exocrine secretion.

Selective trafficking of new proteins is largely achieved by budding of vesicles from the trans-Golgi network (TGN) for transport to specific organelles or to specific regions of the plasmalemma. Different terminology is used for these vesicles depending on their size, histological appearance, contents, or cell type. Granules (including dense-core secretory granules, DCSG) are secretory vesicles present in endocrine, exocrine, immune, and neuroendocrine cells, responsible for both storage and secretion of proteins. Lymphocytes, dendritic cells, and natural killer cells also contain secretory lysosomes for the release of lysosomal enzymes (Stanley and Lacy 2010), and neurons contain peptidergic synaptic vesicles (Park and Loh 2008; Park et al. 2011). However, all of these types of vesicles serve the same broad purpose of transporting specific cargo to specific destinations by an appropriately regulated pathway.

The lipid membranes of these vesicles carry tightly associated cytoplasmic proteins (termed coat proteins) which not only help form the vesicle, but also direct the vesicle to the correct destination (De Matteis and Luini 2008; Santiago-Tirado and Bretscher 2011; Wilson et al. 2011). The matrix of coat proteins on the cytosolic face of the membrane contributes to the bending of the TGN membrane during budding of the vesicle. This matrix is formed by multiple interactions, including binding of coat proteins to phosphatidylinositol phosphates

Phosphatidylinositol Bisphosphate Mediated Sorting of Secretory Granule Cargo 41

trafficking not only affect the physiology of the cell, but also embryonic development (Shilo and Schejter 2011) and disease. This chapter will focus on sorting (selective trafficking) for secretion of luminal cargo proteins. We will review some of the general aspects of selective trafficking, and then build on that background by focusing on our recent work suggesting a

Secreted proteins are translated at the rough endoplasmic reticulum and transit from the ER, through the ER-Golgi Intermediate Compartment (ERGIC) to the Golgi and subsequently the trans-Golgi network (TGN) (Shilo and Schejter 2011). Post-translational modifications such as glycosylation occur in the ER and Golgi. Membranes on the trans side of the Golgi apparatus form dynamic tubular reticular structures having a large surface area (De Matteis and Luini 2008). This network of saccules and tubes is continuously remodeled such that both the structure and size of the TGN varies depending on the secretory activity and the cell type (Trucco et al. 2004). Selective trafficking at the TGN will sort cargo into vesicles or carrier tubules (De Matteis and Luini 2008), and this sorting requires the genesis of carrier vesicles targeted to specific membranes within the cell. As noted above, these vesicles are distinguished by the combination of coat proteins on the cytosolic face of the membrane which determine the target membrane for that vesicle. In some pathways additional sorting occurs at the recycling endosome (reviewed in (De Matteis and Luini 2008; Santiago-Tirado

The initiating event in vesicle biogenesis may be driven by the local membrane lipid composition where asymmetry in the types of lipid in the two faces of the membrane bilayer can induce bending (van Meer and Sprong 2004). Initiation may also involve membrane rafts, which are reported to be present in the TGN of all cells (Park and Loh 2008), and on membranes of secretory granules (Hosaka et al. 2004; Lang 2007; Guerriero et al. 2008). While membrane rafts are well characterized to play important roles in endocytosis at the plasmalemma, their role in vesicle formation at the TGN is not as well understood. Many vesicle coat proteins have been localized to lipid rafts on vesicles (Puri and Roche 2006). This has been suggested to be important for the formation of the coat protein complexes on the TGN for the initial creation of vesicle buds and selective trafficking (Simons and Sampaio 2011). For example, SNARE proteins are enriched in cholesterol-dependent rafts in beta-cells and PC-12 cells (Lang 2007). However, technical issues have called into question the validity of some methods for isolation of 'lipid rafts', leading to a more stringent definition and the term 'membrane rafts' (Lang 2007). Nonetheless, even with the more stringent approach, SNAREs such as VAMP2 and VAMP3 are enriched in membrane rafts of vesicles. Membrane rafts can contain different complements of proteins due to specific protein interactions. One model for the formation of vesicle buds is that membrane rafts on the TGN (with associated transmembrane cargo proteins) coalesce creating a lipid domain that is favorable to bending the membrane, and containing transmembrane proteins which can facilitate the decoration of the cytosolic face of the membrane with adaptor and other coat proteins (De Matteis and Luini 2008; Simons and Sampaio 2011). Testing the relevance of this model to selective sorting for secretion in living cells is important, and requires determining whether trafficking vesicles in different pathways (e.g., regulated secretion versus constitutive secretion pathways) contain different types of membrane rafts, or that

novel mechanism for sorting in the parotid salivary gland.

and Bretscher 2011)).

**2. Biogenesis during trans-Golgi network vesicle trafficking** 

(PtdInsPs) in the TGN membrane, interactions between the coat proteins, and binding to integral proteins of the TGN membrane. Importantly, populations of vesicles are distinguished by the presence of specific combinations of coat proteins, such as clathrin, Adaptor Proteins (AP1-4), FAPP1/2, GGAs, ARF, v-snares, and synaptotagmin. In the parotid gland, VAMP2, VAMP8, syntaxin4/6, and synaptotagmin decorate the cytoplasmic side of secretory granules (Fujita-Yoshigaki et al. 2006; Wang et al. 2007). These different coat proteins on different vesicles direct the vesicles to the correct target membranes. For example, FAPP2 is critical for constitutive apical trafficking, whereas FAPP1 directs basolateral trafficking (Vieira et al. 2005). The coat proteins also mediate interactions with other proteins, including actins, to mediate transportation of that vesicle.

Having vesicles destined for different targets raises the central question of how does the correct cargo get put into just the correct type of vesicle? These post-TGN vesicles carry integral membrane proteins, which are one type of cargo delivered by this process. Transmembrane cargo proteins (such as MPR300) are localized by direct interactions with coat proteins (such as GGAs) on the outside of the forming vesicle as it buds from the TGN (Ghosh et al. 2003). Sorting sequences which mediate these interactions have been identified on the cytosolic tails of many transmembrane cargo proteins (Folsch et al. 2009). Hence, the problem of sorting membrane cargo proteins to the correct vesicle has an elegant solution based on direct interactions of transmembrane cargo with the coat protein complex which identifies that vesicle and targets it to the correct destination (recently reviewed in (De Matteis and Luini 2011)).

Importantly, the lumen of the vesicle contains a different type of cargo composed of specific soluble proteins. Luminal cargo proteins include lysosomal enzymes, hormones, cytokines, neurotransmitters, digestive enzymes, and salivary proteins. As can be seen from this list, soluble cargo proteins are present in a variety of different secretory cell types. These luminal cargo proteins cannot interact directly with the coat proteins on the cytoplasmic (outer) surface of the vesicle membrane; therefore, other mechanisms must be involved to localize the correct soluble cargo protein into the vesicle destined for the correct target, and not into the incorrect vesicles. This is an information transfer problem, i.e., how to get the information encoded by the cytoplasmic coat proteins (which determine the destination of the vesicle) to select the appropriate luminal cargo proteins.

Information transfer for sorting is a typical problem in any distribution network and must be solved by companies involved with distribution, such as UPS or FedEx. The need for solutions to such problems is reflected by the growing number of Logistics and Distribution programs at universities. Notably, eukaryotic cells developed solutions to these logistics problems many eons ago. For an exocrine or endocrine cell, the problem is how to get the lysosomal enzymes (soluble cargo proteins) into a forming TGN vesicle destined for the lysosome, and secretory cargo proteins into a different TGN vesicle destined for the plasmalemma. This requires the transfer of information from the cytosolic side of the forming vesicle membrane to the luminal side of the membrane.

While there are some good model systems, we do not have a clear understanding of the molecular mechanisms that direct the sorting of soluble cargo proteins between different vesicles. Nonetheless, this is an important issue since all eukaryotic cells produce several different types of vesicles at the TGN (Folsch et al. 2009), and many cell types secrete proteins by specific pathways such as apical versus basolateral pathways. Changes in

(PtdInsPs) in the TGN membrane, interactions between the coat proteins, and binding to integral proteins of the TGN membrane. Importantly, populations of vesicles are distinguished by the presence of specific combinations of coat proteins, such as clathrin, Adaptor Proteins (AP1-4), FAPP1/2, GGAs, ARF, v-snares, and synaptotagmin. In the parotid gland, VAMP2, VAMP8, syntaxin4/6, and synaptotagmin decorate the cytoplasmic side of secretory granules (Fujita-Yoshigaki et al. 2006; Wang et al. 2007). These different coat proteins on different vesicles direct the vesicles to the correct target membranes. For example, FAPP2 is critical for constitutive apical trafficking, whereas FAPP1 directs basolateral trafficking (Vieira et al. 2005). The coat proteins also mediate interactions with

Having vesicles destined for different targets raises the central question of how does the correct cargo get put into just the correct type of vesicle? These post-TGN vesicles carry integral membrane proteins, which are one type of cargo delivered by this process. Transmembrane cargo proteins (such as MPR300) are localized by direct interactions with coat proteins (such as GGAs) on the outside of the forming vesicle as it buds from the TGN (Ghosh et al. 2003). Sorting sequences which mediate these interactions have been identified on the cytosolic tails of many transmembrane cargo proteins (Folsch et al. 2009). Hence, the problem of sorting membrane cargo proteins to the correct vesicle has an elegant solution based on direct interactions of transmembrane cargo with the coat protein complex which identifies that vesicle and targets it to the correct destination (recently reviewed in (De

Importantly, the lumen of the vesicle contains a different type of cargo composed of specific soluble proteins. Luminal cargo proteins include lysosomal enzymes, hormones, cytokines, neurotransmitters, digestive enzymes, and salivary proteins. As can be seen from this list, soluble cargo proteins are present in a variety of different secretory cell types. These luminal cargo proteins cannot interact directly with the coat proteins on the cytoplasmic (outer) surface of the vesicle membrane; therefore, other mechanisms must be involved to localize the correct soluble cargo protein into the vesicle destined for the correct target, and not into the incorrect vesicles. This is an information transfer problem, i.e., how to get the information encoded by the cytoplasmic coat proteins (which determine the destination of

Information transfer for sorting is a typical problem in any distribution network and must be solved by companies involved with distribution, such as UPS or FedEx. The need for solutions to such problems is reflected by the growing number of Logistics and Distribution programs at universities. Notably, eukaryotic cells developed solutions to these logistics problems many eons ago. For an exocrine or endocrine cell, the problem is how to get the lysosomal enzymes (soluble cargo proteins) into a forming TGN vesicle destined for the lysosome, and secretory cargo proteins into a different TGN vesicle destined for the plasmalemma. This requires the transfer of information from the cytosolic side of the

While there are some good model systems, we do not have a clear understanding of the molecular mechanisms that direct the sorting of soluble cargo proteins between different vesicles. Nonetheless, this is an important issue since all eukaryotic cells produce several different types of vesicles at the TGN (Folsch et al. 2009), and many cell types secrete proteins by specific pathways such as apical versus basolateral pathways. Changes in

other proteins, including actins, to mediate transportation of that vesicle.

the vesicle) to select the appropriate luminal cargo proteins.

forming vesicle membrane to the luminal side of the membrane.

Matteis and Luini 2011)).

trafficking not only affect the physiology of the cell, but also embryonic development (Shilo and Schejter 2011) and disease. This chapter will focus on sorting (selective trafficking) for secretion of luminal cargo proteins. We will review some of the general aspects of selective trafficking, and then build on that background by focusing on our recent work suggesting a novel mechanism for sorting in the parotid salivary gland.
