Anna M. Castle

Intracellular Transport and Secretion of Salivary Proteins

Intracellular transport and secretion of salivary proteins are major activities of salivary acinar cells. While the major intracellular pathway followed by salivary proteins following their synthesis has been described previously, there is only limited understanding of how this process is regulated at the molecular level. Studies of salivary proteins, especially proline-rich proteins, expressed in an endocrine cell line have begun to provide insight regarding intermolecular interactions during transport and the role played by structural signals during intracellular sorting. Analysis of the secretion of newly synthesized salivary proteins in parotid tissue has shown that there are multiple pathways of discharge from acinar cells. While granule exocytosis is the major pathway, at least two other pathways that export salivary proteins have been found to originate from maturing secretion granules. These pathways may contribute to other acinar cell functions, including secretion of proteins in the absence of acute stimulation and support of the secretory process for fluid and electrolytes.

An intracellular role for ABCG1-mediated cholesterol transport in the regulated secretory pathway of mouse pancreatic β cells

Cholesterol is a critical component of cell membranes, and cellular cholesterol levels and distribution are tightly regulated in mammals. Recent evidence has revealed a critical role for pancreatic beta cell-specific cholesterol homeostasis in insulin secretion as well as in beta cell dysfunction in diabetes and the metabolic response to thiazolidinediones (TZDs), which are antidiabetic drugs. The ATP-binding cassette transporter G1 (ABCG1) has been shown to play a role in cholesterol efflux, but its role in beta cells is currently unknown. In other cell types, ABCG1 expression is downregulated in diabetes and upregulated by TZDs. Here we have demonstrated an intracellular role for ABCG1 in beta cells. Loss of ABCG1 expression impaired insulin secretion both in vivo and in vitro, but it had no effect on cellular cholesterol content or efflux. Subcellular localization studies showed the bulk of ABCG1 protein to be present in insulin granules. Loss of ABCG1 led to altered granule morphology and reduced granule cholesterol levels. Administration of exogenous cholesterol restored granule morphology and cholesterol content and rescued insulin secretion in ABCG1-deficient islets. These findings suggest that ABCG1 acts primarily to regulate subcellular cholesterol distribution in mouse beta cells. Furthermore, islet ABCG1 expression was reduced in diabetic mice and restored by TZDs, implicating a role for regulation of islet ABCG1 expression in diabetes pathogenesis and treatment.

Ubiquitously expressed secretory carrier membrane proteins (SCAMPs) 1-4 mark different pathways and exhibit limited constitutive trafficking to and from the cell surface.

Secretory carrier membrane proteins (SCAMPs) 1-4 are ubiquitously expressed and are major components of the eukaryotic cell surface recycling system. We investigated whether different SCAMPs function along distinct pathways and whether they behave like itinerant cargoes or less mobile trafficking machinery. In NRK cells, we show by immunofluorescence microscopy that different SCAMPs are concentrated mostly adjacent to one another in the trans-Golgi network and endosomal recycling compartment. By immunoelectron microscopy, they were shown to be close neighbors on individual transferrin-containing endosomal elements and on the plasma membrane. Within the internal endosomal network, SCAMPs are located distal to rab5-containing endosomes, and the individual isoforms appear to mark pathways that diverge from the constitutive recycling route and that may be distinguished by different adaptors, especially AP-1 and AP-3. Based on comparisons of SCAMP localization with endocytosed transferrin as well as live imaging of GFP-SCAMP1, we show that SCAMPs are concentrated within the motile population of early and recycling endosomes; however, they are not detected in newly formed transferrin-containing endocytic vesicles or in vesicles recycling transferrin to the surface. Also, they are not detected in constitutive secretory carriers marked by VSV-G. Their minimal recycling to the surface is reflected by their inability to relocate to the plasma membrane upon inhibition of endocytosis. Thus SCAMPs exhibit limited exchange between the cell surface and internal recycling systems, but within each of these sites, they form a mosaic with individual isoforms marking distinct pathways and potentially functioning as trafficking machinery at sites of vesicle formation and fusion. A corollary of these findings is that early endosomes exist as a distinct SCAMP-containing compartment and are not formed de novo by fusion of endocytic vesicles.

SCAMP3 Negatively Regulates Epidermal Growth Factor Receptor Degradation and Promotes Receptor Recycling

The epidermal growth factor receptor (EGFR) is targeted for lysosomal degradation by ubiquitin-mediated interactions with the ESCRTs (endosomal-sorting complexes required for transport) in multivesicular bodies (MVBs). We show that secretory carrier membrane protein, SCAMP3, localizes in part to early endosomes and negatively regulates EGFR degradation through processes that involve its ubiquitylation and interactions with ESCRTs. SCAMP3 is multimonoubiquitylated and is able to associate with Nedd4 HECT ubiquitin ligases and the ESCRT-I subunit Tsg101 via its PY and PSAP motifs, respectively. SCAMP3 also associates with the ESCRT-0 subunit Hrs. Depletion of SCAMP3 in HeLa cells by inhibitory RNA accelerated degradation of EGFR and EGF while inhibiting recycling. Conversely, overexpression enhanced EGFR recycling unless ubiquitylatable lysines, PY or PSAP motifs in SCAMP3 were mutated. Notably, dual depletions of SCAMP3 and ESCRT subunits suggest that SCAMP3 has a distinct function in parallel with the ESCRTs that regulates receptor degradation. This function may affect trafficking of receptors from prelysosomal compartments as SCAMP3 depletion appeared to sustain the incidence of EGFR-containing MVBs detected by immunoelectron microscopy. Together, our results suggest that SCAMP3, its modification with ubiquitin, and its interactions with ESCRTs coordinately regulate endosomal pathways and affect the efficiency of receptor down-regulation.

Nonredundant function of secretory carrier membrane protein isoforms in dense core vesicle exocytosis

Five secretory carrier membrane proteins (SCAMP-1, -2, -3, -4, and -5) have been characterized in mammalian cells. Previously, SCAMP-1 and -2 have been implicated to function in exocytosis. RNA inhibitor-mediated deficiency of one or both of these SCAMPs interferes with dense core vesicle (DCV) exocytosis in neuroendocrine PC12 cells as detected by amperometry. Knockdowns of these SCAMPs each decreased the number and frequency of depolarization-induced exocytotic events. SCAMP-2 but not SCAMP-1 depletion also delayed the onset of exocytosis. Both knockdowns, however, altered fusion pore dynamics, increasing rapid pore closure and decreasing pore dilation. In contrast, knockdowns of SCAMP-3 and -5 only interfered with the frequency of fusion pore opening and did not affect the dynamics of newly opened pores. None of the knockdowns noticeably affected upstream events, including the distribution of DCVs near the plasma membrane and calcium signaling kinetics, although norepinephrine uptake/storage was moderately decreased by deficiency of SCAMP-1 and -5. Thus, SCAMP-1 and -2 are most closely linked to the final events of exocytosis. Other SCAMPs collaborate in regulating fusion sites, but the roles of individual isoforms appear at least partially distinct.

Sorting and Secretion of Salivary Proteins

Most salivary proteins are stored in secretion granules prior to export from acinar cells in response to neural stimuli. A small subset of these proteins undergo unstimulated secretion without apparent storage. This pathway probably comprises vesicles that bud from maturing storage granules and carries proteins that do not aggregate efficiently at the storage site. Expression of a parotid proline-rich protein (and deletion mutants) in pituitary AtT-20 cells has shown that an N-terminal domain is necessary for storage in secretion granules. Evidence suggests that self-aggregation of proline-rich protein mediated by this domain may function in both efficient intracellular transport and storage. Thus selective aggregation may be an important secretory sorting mechanism.

Immunoglobulin‐derived polypeptides enter the regulated secretory pathway in AtT‐20 cells

Constitutively secreted proteins have traditionally been believed to be excluded from the regulated secretory pathway. In this work we show that kappa light chain and Fc fragment, two markers of the constitutive pathway, are present in the regulated pathway in AtT‐20 cells. They colocalize with the endogenous hormone ACTH and they exhibit stimulus‐dependent secretion. The Fc fragment, which undergoes intracellular transport at the same rate as the ACTH precursor POMC, enters the forming secretory granules, however, it is partially lost during granule maturation. These observations show that classic constitutive secretory markers are not excluded from the regulated secretory pathway and that efficient sorting for regulated secretion occurs above a background of proteins which enter the granules without sorting.

Reinterpretation of the localization of the ATP binding cassette transporter ABCG1 in insulin-secreting cells and insights regarding its trafficking and function

The ABC transporter ABCG1 contributes to the regulation of cholesterol efflux from cells and to the distribution of cholesterol within cells. We showed previously that ABCG1 deficiency inhibits insulin secretion by pancreatic beta cells and, based on its immunolocalization to insulin granules, proposed its essential role in forming granule membranes that are enriched in cholesterol. While we confirm elsewhere that ABCG1, alongside ABCA1 and oxysterol binding protein OSBP, supports insulin granule formation, the aim here is to clarify the localization of ABCG1 within insulin-secreting cells and to provide added insight regarding ABCG1’s trafficking and sites of function. We show that stably expressed GFP-tagged ABCG1 closely mimics the distribution of endogenous ABCG1 in pancreatic INS1 cells and accumulates in the trans-Golgi network (TGN), endosomal recycling compartment (ERC) and on the cell surface but not on insulin granules, early or late endosomes. Notably, ABCG1 is short-lived, and proteasomal and lysosomal inhibitors both decrease its degradation. Following blockade of protein synthesis, GFP-tagged ABCG1 first disappears from the ER and TGN and later from the ERC and plasma membrane. In addition to aiding granule formation, our findings raise the prospect that ABCG1 may act beyond the TGN to regulate activities involving the endocytic pathway, especially as the amount of transferrin receptor is increased in ABCG1-deficient cells. Thus, ABCG1 may function at multiple intracellular sites and the plasma membrane as a roving sensor and modulator of cholesterol distribution, membrane trafficking and cholesterol efflux.