Dennis Shields

Secretory Vesicle Budding from the Trans-Golgi Network Is Mediated by Phosphatidic Acid Levels

Phospholipid metabolism plays a central role in regulating vesicular traffic in the secretory pathway. In mammalian cells, activation of a Golgi-associated phospholipase D activity by ADP-ribosylation factor results in hydrolysis of phosphatidylcholine to phosphatidic acid (PA). This reaction has been proposed to stimulate nascent secretory vesicle budding from the trans-Golgi network. It is unclear whether PA itself or diacylglycerol (DAG), a metabolite implicated in yeast secretory vesicle formation, regulates budding. To distinguish between these possibilities we have used a permeabilized cell system supplemented with phospholipid-modifying enzymes that generate either DAG or PA. The data demonstrate that in mammalian cells accumulation of PA rather than DAG is a key step in regulating budding of secretory vesicles from the trans-Golgi network.

A caspase cleavage fragment of p115 induces fragmentation of the Golgi apparatus and apoptosis

In mammalian cells, the Golgi apparatus undergoes extensive fragmentation during apoptosis. p115 is a key vesicle tethering protein required for maintaining the structural organization of the Golgi apparatus. Here, we demonstrate that p115 was cleaved during apoptosis by caspases 3 and 8. Compared with control cells expressing native p115, those expressing a cleavage-resistant form of p115 delayed Golgi fragmentation during apoptosis. Expression of cDNAs encoding full-length or an NH2-terminal caspase cleavage fragment of p115 had no effect on Golgi morphology. In contrast, expression of the COOH-terminal caspase cleavage product of p115 itself caused Golgi fragmentation. Furthermore, this fragment translocated to the nucleus and its expression was sufficient to induce apoptosis. Most significantly, in vivo expression of the COOH-terminal fragment in the presence of caspase inhibitors, or upon coexpression with a cleavage-resistant mutant of p115, showed that p115 degradation plays a key role in amplifying the apoptotic response independently of Golgi fragmentation.

Somatostatin-like immunoreactivity within neuritic plaques

Alzheimers disease or senile dementia of the Alzheimer type (SDAT) is a progressive neurodegenerative disease that is characterized pathologically by two types of microscopic lesions in the neocortex: the neurofibrillary tangle and neuritic plaque. The concentration of neuritic plaques is correlated with significant reductions in the level of specific neurotransmitter and neuropeptide systems in autopsied brains of patients with SDAT, including decreased amounts of the tetradecapeptide, somatostatin. The clinical effects of reduced cortical somatostatin activity in patients with SDAT is unclear, nor is it known whether somatostatinergic neurons participate in either lesion. In the present study we employed light microscopic immunocytochemistry to determine whether somatostatin-containing neurons participate in the formation of neuritic plaques. Examination of selected cortical regions from autopsied brains revealed 20-50% of all neuritic plaques contained somatostatin-positive profiles indicating that processes of somatostatinergic neurons are associated with neuritic plaque formation.

ADP-ribosylation Factor-1 Stimulates Formation of Nascent Secretory Vesicles from the trans-Golgi Network of Endocrine Cells

ADP-ribosylation factor (ARF) is a small GTP-binding protein that has been implicated in intracellular vesicular transport. ARF regulates the budding of vesicles that mediate endoplasmic reticulum to Golgi and intra-Golgi transport. It also plays an important role in maintaining the function and morphology of the Golgi apparatus. Using a permeabilized cell system derived from GH cells, we provide evidence that ARF-1 regulates the formation of nascent secretory vesicles from the trans-Golgi network. Both myristoylated and non-myristoylated forms of recombinant human ARF-1 enhanced secretory vesicle budding about 2-fold. A mutant lacking the first 17 N-terminal residues, as well as one that preferentially binds GDP (T31N) did not stimulate vesicle formation. In contrast, a mutant defective in GTP hydrolysis (Q71L) promoted vesicle budding. Strikingly, a peptide corresponding to the N terminus of human ARF-1 (amino acids 2-17) also stimulated vesicle budding from the trans-Golgi network, in marked contrast to its inhibitory effect on vesicular transport from the endoplasmic reticulum to Golgi. These data demonstrate that in endocrine cells, ARF-1 and in particular its N terminus play an essential role in the formation of secretory vesicles.

Fragmentation of the Golgi Apparatus: An Early Apoptotic Event Independent of the Cytoskeleton

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase‐mediated cleavage of several Golgi‐associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor‐activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine‐treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of β‐actin or α‐tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin‐160, underwent caspase‐mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.

Expression of preprosomatostatin in heterologous cells: Biosynthesis, posttranslational processing, and secretion of mature somatostatin

Somatostatin is a 14 amino acid peptide hormone that is synthesized as part of a larger precursor, preprosomatostatin, which comprises about 120 amino acids. The overall organization of the precursor is conserved in many species in that it consists of a signal peptide followed by a proregion of 90-100 amino acids and the mature hormone is located at the carboxyl terminus of the molecule. To understand the role of the propeptide in generating the mature hormone, we have used gene-transfer experiments to introduce angler fish preprosomatostatin into mammalian cells. Here we report the results of transfection of COS-7 cells with an SV40 expression vector containing preprosomatostatin cDNA cloned into the VP-1 late gene. Analysis of the parameters of somatostatin gene expression showed that COS cells synthesized prosomatostatin, which was detected intracellularly; the prosomatostatin, was proteolytically processed to mature somatostatin; and the mature hormone was secreted by the COS cells into the tissue culture medium. Our results suggest that COS cells, which do not normally secrete polypeptide hormones, contain the necessary proteolytic processing enzymes to convert preprosomatostatin to the mature hormone and the cellular apparatus necessary for its secretion.

Fragmentation of the Golgi Apparatus A ROLE FOR βIII SPECTRIN AND SYNTHESIS OF PHOSPHATIDYLINOSITOL 4,5-BISPHOSPHATE

Phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) synthesis has been implicated in maintaining the function of the Golgi apparatus. Here we demonstrate that the inhibition of PtdIns(4,5)P2 synthesis in vitro in response to primary alcohol treatment and the kinetics of Golgi fragmentation in vivo were very rapid and tightly coupled. Preloading Golgi membranes with short chain phosphatidic acid abrogated the alcohol-mediated inhibition of PtdIns(4,5)P2synthesis in vitro. We also show that fragmentation of the Golgi apparatus in response to diminished PtdIns(4,5)P2synthesis correlated with both the phosphorylation of a Golgi form of βIII spectrin, a PtdIns(4,5)P2-interacting protein, and changes in its intracellular redistribution. The data are consistent with a model suggesting that the decreased PtdIns(4,5)P2synthesis and the phosphorylation state of βIII spectrin modulate the structural integrity of the Golgi apparatus.

Prosomatostatin Processing in Permeabilized Cells CALCIUM IS REQUIRED FOR PROHORMONE CLEAVAGE BUT NOT FORMATION OF NASCENT SECRETORY VESICLES

Our laboratory has been using a permeabilized cell system derived from rat anterior pituitary GH cells expressing prosomatostatin (pro-SRIF) to study prohormone processing and nascent secretory vesicle formation in vitro. Because calcium is necessary for prohormone processing enzyme activity, secretory granule fusion with the plasma membrane, and possibly sorting to the regulated pathway, we treated permeabilized cells with the calcium ionophore A23187 to determine the role of calcium in pro-SRIF cleavage and nascent vesicle formation from the trans-Golgi network (TGN). Here we demonstrate that pro-SRIF cleavage was markedly inhibited when lumenal free calcium was chelated with EGTA in the presence of A23187. Surprisingly, submillimolar free calcium (15 μM) was sufficient to maintain prohormone cleavage efficiency, a value far lower than that estimated for total calcium levels in the TGN and secretory granules. Experiments using both A23187 and the protonophore CCCP revealed that free calcium is absolutely required for efficient pro-SRIF cleavage, even at the optimal pH of 6.1. Secretory vesicle formation by contrast was not inhibited by calcium chelation but rather by millimolar extralumenal free calcium. Together, these observations demonstrate that pro-SRIF processing and budding of nascent secretory vesicles from the TGN can be uncoupled and therefore have distinct biochemical requirements. Interestingly, our data using intact GH cells demonstrate that basal secretion of SRIF-related material is largely calcium-dependent and therefore cannot be equated with constitutive pathway secretion. These results underscore the importance of determining calcium requirements before assigning a secretion event to either the constitutive or regulated secretory pathway.

Opposing Action of Casein Kinase 1 and Calcineurin in Nucleo-cytoplasmic Shuttling of Mammalian Translation Initiation Factor eIF6

Eukaryotic initiation factor 6 (eIF6), a highly conserved protein from yeast to mammals, is essential for 60 S ribosome biogenesis and assembly. Both yeast and mammalian eIF6 are phosphorylated at Ser-174 and Ser-175 by the nuclear isoform of casein kinase 1 (CK1). The molecular basis of eIF6 phosphorylation, however, remains elusive. In the present work, we show that subcellular distribution of eIF6 in the nuclei and the cytoplasm of mammalian cells is mediated by dephosphorylation and phosphorylation, respectively. This nucleo-cytoplasmic shuttling is dependent on the phosphorylation status at Ser-174 and Ser-175 of eIF6. We demonstrate that Ca2+-activated calcineurin phosphatase binds to and promotes nuclear localization of eIF6. Increase in intracellular concentration of Ca2+ leads to rapid translocation of eIF6 from the cytoplasm to the nucleus, an event that is blocked by specific calcineurin inhibitors cyclosporin A or FK520. Nuclear export of eIF6 is regulated by phosphorylation at Ser-174 and Ser-175 by the nuclear isoform of CK1. Mutation of eIF6 at the phos-phorylatable Ser-174 and Ser-175 to alanine or treatment of cells with the CK1 inhibitor, D4476 inhibits nuclear export of eIF6 and results in nuclear accumulation of eIF6. Together, these results establish eIF6 as a substrate for calcineurin and suggest a novel paradigm for calcineurin function in 60 S ribosome biogenesis via regulating the nuclear accumulation of eIF6.

Biosynthesis and Packaging of Carboxypeptidase D into Nascent Secretory Vesicles in Pituitary Cell Lines

Metallocarboxypeptidase D (CPD) is a membrane-bound trans-Golgi network (TGN) protein. In AtT-20 cells, CPD is initially produced as a 170-kDa endoglycosidase H-sensitive glycoprotein. Within 30 min of chase, the CPD increases to 180 kDa and is resistant to endoglycosidase H as a result of carbohydrate maturation. CPD also undergoes an activation step required for binding to a substrate affinity resin. Blocking the protein exit from the endoplasmic reticulum inhibits the increase in molecular mass but not the step required for affinity column binding, suggesting that enzyme activation precedes carbohydrate maturation and that these reactions occur in distinct intracellular compartments. Only the higher molecular weight mature CPD enters nascent secretory vesicles, which bud from the TGN of permeabilized AtT-20 and GH3 cells. The budding efficiency of CPD into vesicles is 2–3-fold lower than that of endogenous proopiomelanocortin in AtT-20 cells or prolactin in GH3 cells. In contrast, the packaging of a truncated form of CPD, which lacks the cytoplasmic tail and transmembrane domain, was similar to that of proopiomelanocortin. Taken together, the results support the proposal that CPD functions in the TGN in the processing of proteins that transit the secretory pathway and that the C-terminal region plays a major role in TGN retention.