Based on the model the newly synthesized protein is transported directly from the endoplasmic

What Kinds of Proteins Are Targeted to the Secretory Pathway?

The proteins that are targeted to the secretory pathway can be separated into two groups – those that function in the ER and Golgi to ensure proper protein folding and modification (i.e., resident proteins) and those that are processed in the ER and Golgi and are transported to later compartments such as the lysosome, plasma membrane, and extracellular space (Figure 1). Each of these proteins not only possesses a signal to enter the secretory pathway but may also have a secondary signal to localize it to a particular organelle within the pathway.

Abstract

The secretory pathway carries proteins to the cell surface membrane where they can be released. This process can be divided into two systems: the regulated secretory pathway, and the constitutive secretory pathway. For many proteins in the constitutive secretory pathway, this transport process occurs at a relatively constant rate that is determined by how quickly those proteins are synthesized. However, the regulated secretory pathway operates by creating a storage compartment within cells to store secretory cargo. Upon the appropriate stimulus, these storage compartments can fuse with the cell surface and allow a burst of contents to be rapidly secreted. Regulated secretion is used throughout multicellular organisms to accomplish a wide variety of tasks such as hormonal control and neuronal communication.

Production of Secretory Granules

The secretory pathway occurs in a vectorial manner and begins with uptake of amino acids that are used by the rough endoplasmic reticulum to produce newly synthesized proteins. These new proteins are transported to the Golgi complex for further processing and sorting. Proteins exit the Golgi complex in membrane-bound condensing vacuoles. As these vacuoles mature to secretory granules, they bud off from the membrane and the proteins stored inside are posttranslationally modified prior to exocytosis (Castle, 1993; Marino and Gorelick, 2009). As the proteins move through each of the stages of the secretory pathway toward the apical surface, they become more concentrated. Granules then dock and fuse with the apical membrane, which is mediated by SNARE proteins (Castle, 1993).

2.6 Sorting to the Secretory Vesicle

Secretory pathways may be either constitutive (unregulated) or regulated (requiring an extracellular stimulus). The two pathways diverge after secretory vesicles have formed from the trans-Golgi network112 by budding mechanisms that differ from ER vesicle formation and that are still incompletely understood.113–115 Much of the knowledge in this field is based on work with the dense-core secretory granules (DCSG) of neurones and neuroendocrine cells, and is yet to be confirmed for prohormones in enteroendocrine cells.

Chromogranin A plays a critical role in DCSG biogenesis. This conclusion was elegantly demonstrated by the observation that expression of chromogranin A, in a variant of the AtT20 pituitary cell line that lacks both chromogranin A and DCSG, induced granule biosynthesis and restored regulated secretion.116 The importance of chromogranin A117–120 and other members of the granin family (such as chromogranin B121 and secretogranin II122) was subsequently confirmed in antisense cell lines and in knockout (KO) mouse models. Recent studies indicate that the N-terminal 115 residues of chromogranin A are able to form vesicular structures even in COS-1 fibroblasts.123,124 One proposed mechanism envisages granin aggregation, stimulated by the weakly acidic, high Ca2+ conditions found in the trans-Golgi network, as the driving force for vesicle formation.115 An alternative mechanism proposes that protein kinase D is a key player based on the observations that excessive protein kinase D activation by the marine sponge metabolite ilimaquinone resulted in Golgi fragmentation, whereas expression of an inactive protein kinase D led to defective vesicle formation.125 Microtubule motors and the actin cytoskeleton are also involved in the elongation of the trans-Golgi network into tubules prior to vesicle formation.126

Alternative mechanisms for sorting of proteins to the secretory vesicle have been discussed in several recent reviews.1,114,127 In “aggregation-mediated sorting” the granin aggregates mentioned in the previous paragraph also contain prohormones, and binding of the granin N-terminal disulfide loop to the vesicular membrane is responsible for sorting. In “receptor-mediated sorting” carboxypeptidase E and/or secretogranin III bind prohormones, and a domain that recognizes membranes rich in cholesterol (the so-called “lipid raft”) targets the prohormone to the secretory vesicle.114 A third mechanism has been proposed for sorting of PC1/3, PC2, and PC5/6A to the vesicle.128 In particular, an α-helical region in the C-terminal tail of PC1/3 (residues 738–750), which aggregates in the presence of Ca2+ ions, has been shown to be necessary and sufficient to target a normally constitutively secreted protein to vesicles.129 A fourth mechanism130 involves the neuroendocrine secretory protein 7B2, which facilitates the transport of PC2 to secretory granules.131 Finally, the observation that trafficking of the enzyme peptidylglycine α-amidating monooxygenase (PAM), which contains two essential Cu atoms, is sensitive to intracellular Cu concentrations,132 suggests the existence of a Cu-dependent sorting mechanism.

The constitutive and regulated secretory pathways then diverge. Once formed, immature secretory vesicles fuse with each other in a process mediated by SNAREs.112 Membrane remodeling, achieved by the interaction of GTP-bound ADP-ribosylation factor (ARF), AP-1, and clathrin, results in the formation of clathrin-coated vesicles, which remove cargo and SNAREs that are not destined for the mature secretory vesicle and which enter the constitutive secretion pathway.112 Transport of the mature secretory vesicle to the site of release at the cell membrane is microtubule-dependent and mediated by carboxypeptidase E.133,134 Carboxypeptidase E is a transmembrane protein whose cytoplasmic tail binds to the adaptor protein dynactin, which tethers the vesicle to the motor proteins kinesin-2 or -3.134 Vesicle fusion with the cell membrane in response to a rise in intracellular Ca2+ is complex and involves tethering by a process dependent on GTPases of the Rab family and on the Ca2+-sensor synaptotagmin,135 followed by SNARE-dependent docking and exocytosis.135,136

3.2.1.2.1 Secretory pathway

In secretory pathway, E-cadherin transport out of the trans-Golgi network is mediated by the tethering protein Golgin 97 (Lock et al., 2005). It is dependent on association with ankyrin-G and β2 spectrin, two proteins stabilizing E-cadherin through binding to the actin cytoskeleton (Kizhatil et al., 2007). E-cadherin, synthesized as a precursor propeptide, is processed by a mechanism dependent of furin or other subtilisin-like convertases (Posthaus et al., 1998), with a tight coordination between cadherin and desmosome processings (Green et al., 2010).

16.1.10 The Secretory Pathway

The secretory pathway in T. cruzi involves the ER, the Golgi complex, and a system of vesicles that bud from the Golgi cisternae and migrate toward the flagellar pocket, where they fuse and discharge their contents into the flagellar pocket. ER cisternae are seen around the nucleus, and these radiate toward all regions of the cell, especially the peripheral microtubule-containing region. Both rough and smooth ER cisternae are present. The Golgi complex is always located close to the flagellar pocket and is essentially similar to that found in other cells. The Golgi complex can be labeled using gold-labeled lectins, which reveal sugar-containing molecules, indicating its involvement in protein glycosylation as reported for other eukaryotic cells. Rab7, a small GTPase involved in membrane trafficking, was also detected in the Golgi complex of trypanosomes (Araripe et al., 2005).

10.4.3 Role of the Secretory Pathway and Golgi Outposts in Dendritic Elaboration

The secretory pathway that delivers membrane to the cell surface consists of the endoplasmic reticulum (ER), and Golgi apparatus, and, in neurons, Golgi outposts that become apparent during periods of rapid dendritic growth. The Golgi outposts are a conserved component of the branching machinery of dendrites in vertebrates and invertebrates. Isolated outposts of Golgi were first identified and studied in mammalian neurons by Ehlers and colleagues (Horton and Ehlers, 2003; Horton et al., 2005). These studies provide context for genetic studies carried out in Drosophila. Somatic Golgi was found concentrated at the base of the largest most complex branch of vertebrate pyramidal neurons, the apical dendrite, and that isolated Golgi resided out along the arbor, primarily at branch points. Pharmacological disruption of forward Golgi trafficking with brefeldin A in dissociated cell culture resulted in decreased dendrite growth. Dispersion of the Golgi into multiple dendrites caused elaboration of a nonpolarized arbor with all branches of fairly equal length and branching complexity. These experiments reveal how polarized trafficking of membrane components can lead to specific patterning features of dendritic arbors.

Forward genetics likewise identified several components of the secretory pathway as important for normal dendritic growth in invertebrate neurons (Ye et al., 2007). Class IV neurons were screened for molecules that regulate dendritic growth and among those identified were several proteins that fit into a common forward secretory pathway, Sec23, Sar1, and Rab1 (Figure 10.3). Interestingly, in these mutant lines axon growth was disrupted to a lesser overall extent than dendrites suggesting a differential reliance of dendrite versus axon growth on ER-to-Golgi transport (Figure 10.3). The Sar1 gene was also studied in hippocampal cultures and a similar selective effect on dendrite but not axon growth was observed. Golgi outposts are likewise observed in the da neurons, and their morphology depends on intact Sar1. Golgi outposts are very dynamic in their movements along an arbor, and their forward or reverse movement (relative to the cell body) correlates with dendrite outgrowth or retraction, respectively (Ye et al., 2007). Just how this correlation relates to dendrite growth is still under investigation, but it has been shown that laser damaging of outposts halts branch dynamics, thus a role for Golgi outposts, and perhaps outpost movement, in arbor dynamic extension is strongly supported.

Studies of a C. elegans sensory neuron named PVD suggest that not only membrane deposition, but also membrane shaping, by the fusogen EFF-1 (epithelial fusion failure-1), is critical for dendritic branching and morphogenesis (Oren-Suissa et al., 2010). Fusogens are important for cell fusion events during development and control fusion by altering membrane curvature. The level of EFF-1 sets the proper branching complexity of PVD neurons with higher levels suppressing branching and lower levels leading to more branching and disorganization of the normal near 90° branching angles seen in PVD arbors (Figure 10.1(e)).

Summary

The secretory pathway needs a system to monitor its function; moreover, it should react to changes in secretion levels. The presence of a variety of signaling molecules, including G-proteins, kinases, phosphatases, and phospholipases, which are physically associated with secretory endomembranes, and in particular with the Golgi complex, might help to prevent structural and functional disruption (Cancino & Luini, 2013; Farhan & Rabouille, 2011). How these signaling pathways work in a coordinated fashion to keep the necessary precise balance is poorly understood. The amount of information collected over the last few decades is large, but it is mainly made up of a myriad of scattered data on the phosphorylation of molecular components of the transport machinery and sometimes on its effects on various trafficking steps. Thus, the precise functional “meaning” of this body of information remains for the most part mysterious (Cancino & Luini, 2013). To analyze the flow of information through the signaling pathways that control membrane trafficking, we need to be able to establish how and when Golgi-based signaling pathways are activated.

The experimental approaches herein described will help in the study of these Golgi-based signaling cascades.

3 Quality Control Mechanisms of the Secretory Pathway

The secretory pathway of eukaryotic cells is composed primarily of two organelles, the ER and Golgi, responsible for maintaining the fidelity of protein synthesis and maturation. The environment of the ER is specialized to properly fold secretory proteins due to an oxidizing redox potential, appropriate calcium levels, and dedicated enzymes for protein glycosylation and folding (i.e., chaperones and foldases; van Anken and Braakman, 2005). When abnormalities do occur, such as an overwhelming abundance of improperly folded protein retained in the ER, or a decrease in vesicle trafficking from the ER to Golgi, these phenomena, collectively termed “ER stress,” upregulate quality control mechanisms to ensure cellular homeostasis. Such stress can be caused by a variety of insults, including nutrient deprivation, pathogenic infection, chemical treatment, and the expression of heterologous protein.

ER-associated degradation (ERAD) and the UPR are two quality control mechanisms that are upregulated upon ER stress. They have several outcomes that occur at various timescales, depend on variations in the spatial organization of organelles, and alter selective protein concentrations and intracellular localization (Brodsky, 2007; McCracken and Brodsky, 2003; Midelfort and Wittrup, 2006; Ng et al., 2000). Removal of misfolded proteins through ERAD occurs by ubiquitination via ER-associated ubiquitin-conjugating enzymes, followed by retrotranslocation, and degradation in the cytoplasm by the proteasome (reviewed in Nakatsukasa and Brodsky, 2008). Local perturbations of unfolded protein levels in the ER activate the UPR, defined as a global cytoprotective signaling cascade that transcriptionally upregulates genes encoding ERAD components, chaperones, and oxidoreductases (Otte and Barlowe, 2004; Travers et al., 2000).

Once the ER's capacity to process nascent proteins is overwhelmed, the ER-resident chaperone BiP is sequestered from the transmembrane kinase protein Ire1p to counteract the stressful condition, which triggers the negative feedback loop known as the UPR. Unbound Ire1p dimerizes, autophosphorylates, and splices an intron from HAC1 mRNA. The resulting Hac1p transcription factor (TF) binds to the promoter regions of UPR targets, upregulating their expression. However, it must also be noted that unfolded protein may directly initiate the dimerization and activation of Ire1p (Kimata et al., 2007; Oikawa et al., 2007). Either mechanism results in the upregulation of diverse targets that include chaperones and foldases and many elements of the secretory pathway, as well as components of the ERAD machinery. In higher eukaryotes, several additional pathways are activated in response to ER stress, and a general attenuation of translation is observed.

1 Introduction

The secretory pathway, beginning from the initial targeting of secreted and membrane proteins to the endoplasmic reticulum (ER), transit through the Golgi, and finally on to organelles like the lysosome or the plasma membrane, has been well characterized in mammalian cells and in simple organisms like yeast (Schekman, 2010). Membrane proteins typically contain N-linked oligosaccharides (Landoltmarticorena & Reithmeier, 1994) that are involved in protein folding and quality control during the biosynthetic process (Benyair, Ron, & Lederkremer, 2011; Helenius, 1994). Some single-span membrane proteins, like red cell Glycophorin A or membrane-anchored enzymes, contain multiple short O-linked oligosaccharide chains in the external juxta-membrane region as well. Oligosaccharides can play a role in the mature protein by increasing the solubility of secreted proteins and protecting them from proteolysis (Elbein, 1991). The role of N-linked oligosaccharides on glycoproteins—including those in membranes—varies from essential to dispensable. In membrane glycoproteins, the oligosaccharides face the cell exterior or the lumen of organelles, “sugar coating” the membrane surface. This review focuses on studies of the effect of disease-causing mutations in erythroid and kidney anion exchanger 1 (AE1 and kAE1, respectively), other members of the SLC4 family of bicarbonate transports, and the SLC26 family of anion transporters, on the folding, trafficking, and functional expression of these membrane glycoproteins.