Endoplasmic reticulum-Golgi Pathway Research Articles

An Annexin 2 Phosphorylation Switch Mediates p11-dependent Translocation of Annexin 2 to the Cell Surface

Annexin 2 is a profibrinolytic co-receptor for plasminogen and tissue plasminogen activator that stimulates activation of the major fibrinolysin, plasmin, at cell surfaces. In human subjects, overexpression of annexin 2 in acute promyelocytic leukemia leads to a bleeding diathesis reflective of excessive cell surface annexin 2-dependent generation of plasmin (Menell, J. S., Cesarman, G. M., Jacovina, A. T., McLaughlin, M. A., Lev, E. A., and Hajjar, K. A. (1999) N. Engl. J. Med. 340, 994-1004). In addition, mice completely deficient in annexin 2 display fibrin accumulation within blood vessels and impaired clearance of injury-induced thrombi (Ling Q., Jacovina, A.T., Deora, A.B., Febbraio, M., Simantov, R., Silverstein, R. L., Hempstead, B. L., Mark, W., and Hajjar, K. A. (2004) J. Clin. Investig. 113, 38-48). Here, we show that endothelial cell annexin 2, a protein that lacks a typical signal peptide, translocates from the cytoplasm to the extracytoplasmic plasma membrane in response to brief temperature stress both in vitro and in vivo in the absence of cell death or cell lysis. This regulated response is independent of new protein or mRNA synthesis and does not require the classical endoplasmic reticulum-Golgi pathway. Temperature stress-induced annexin 2 translocation is dependent on both expression of protein p11 (S100A10) and tyrosine phosphorylation of annexin 2 because annexin 2 release is completely eliminated on depletion of p11, inactivation of tyrosine kinase, or mutation of tyrosine 23. Translocation of annexin 2 to the cell surface dramatically increases tissue plasminogen activator-dependent plasminogen activation potential and may represent a novel stress-induced protein secretion pathway.

Cloning and characterization of a novel neurotoxin from the sea anemone Anthopleura sp.

A full-length cDNA of neurotoxin (Hk2a) was isolated by RT-PCR of total RNA isolated from tentacles of Anthopleura sp. using degenerate oligonucleotide primers and 3′,5′-RACE. The cDNA sequence of Hk2a encoded a polypeptide of 47 amino acids, which lacks a typical N-terminal signal sequences commonly found in proteins that are secreted via endoplasmic reticulum–Golgi pathway, indicating the possibility of secretion via a non-classical pathway. The neurotoxin has a predicted molecular mass of 4.8 kDa and a pI value of 7.62. The amino acid sequence of Hk2a is very similar to Anthopleurin C (Ap-C) and Neurotoxin I (Af I), and shares 95% amino acid sequence similarity to Ap-C. The coding region for the matured Hk2a toxin was cloned into the thioredoxin (TRX) fusion expression vector (pTRX) for the fusion expression in Escherichia coli. The recombinant polypeptide of Hk2a (rHk2a) was purified by the affinity chromatography, 15 mg/l of rHk2a was obtained after the digestion with protease 3C and further purification. The molecular weight of rHk2a (5.078 kDa) obtained by MALDI-TOF was very close to that (5 Da) calculated from the sequence. The results of the UV-circular dichroism spectra of rHk2a indicates that its secondary structure is similar to that of Ap-B ( Benzinger et al., 1998), having 61.7% β-sheet and no α-helix. Investigation on pharmacological effects of rHk2a in vitro was undertaken, and it was found that LD 50 of rHk2a was 1.4 mg/kg on NIH mice (i.p.). The rHk2a was demonstrated to increase contracting activity on isolated SD rat atria with the enhancing degree reaching 343.5±160.5%. The increase in contractile amplitude reached a plateau value within 3–5 min after addition of this toxin.

Lysosomal and Cytosolic Sialic Acid 9-O-Acetylesterase Activities Can Be Encoded by One Gene via Differential Usage of a Signal Peptide-encoding Exon at the N Terminus

9-O-Acetylation is one of the most common modifications of sialic acids, and it can affect several sialic acid-mediated recognition phenomena. We previously reported a cDNA encoding a lysosomal sialic acid-specific 9-O-acetylesterase, which traverses the endoplasmic reticulum-Golgi pathway and localizes primarily to lysosomes and endosomes. In this study, we report a variant cDNA derived from the same gene that contains a different 5' region. This cDNA has a putative open reading frame lacking a signal peptide-encoding sequence and is thus a candidate for the previously described cytosolic sialic acid 9-O-acetylesterase activity. Epitope-tagged constructs confirm that the new sequence causes the protein product to be targeted to the cytosol and has esterase activity. Using reverse transcription-polymerase chain reaction to distinguish the two forms of message, we show that although the lysosomal sialic acid-specific 9-O-acetylesterase message has a widespread pattern of expression in adult mouse tissues, this cytosolic sialic acid 9-O-acetylesterase form has a rather restricted distribution, with the strongest expression in the liver, ovary, and brain. Using a polyclonal antibody directed against the 69-amino acid region common to both proteins, we confirmed that the expression of glycosylated and nonglycosylated polypeptides occurred in appropriate subcellular fractions of normal mouse tissues. Rodent liver polypeptides reacting to the antibody also co-purify with previously described lysosomal sialic acid esterase activity and at least a portion of the cytosolic activity. Thus, two sialic acid 9-O-acetylesterases found in very different subcellular compartments can be encoded by a single gene by differential usage of a signal peptide-encoding exon at the N terminus. The 5'-rapid amplification of cDNA ends results and the differences in tissue-specific expression suggest that expression of these two products may be differentially regulated by independent promoters.

Processing, Secretion, and Anti-HIV-1 Activity of IL-16 With or Without a Signal Peptide in CD4+ T Cells

CD4+ T cells transfected with the C-terminal 130 aa of human IL-16 are rendered resistant to HIV infection. Whether the constitutively expressed IL-16 acts intracellularly, extracellularly, or both is not clear. To address this question and to further study the processing of IL-16, new constructs containing either the C-terminal 130 aa or the C-terminal 100 aa (PDZ-like motif) were constructed with and without a signal peptide. Pulse-chase experiments and treatment of cells with brefeldin A and/or tunicamycin showed that IL-16 is secreted despite the absence of a signal peptide, but with a signal peptide IL-16 is processed through the endoplasmic reticulum-golgi pathway and is glycosylated. Cells expressing IL-16 linked to a signal peptide secrete considerably more IL-16 into the supernatant than cells expressing IL-16 without a signal peptide and are considerably more resistant to HIV replication. Resistance extends to almost 25 days for cells expressing IL-16 with signal peptide as compared with only 15 days for cells without signal peptide. Cells expressing the C-terminal 100 aa not linked to a signal peptide are poor secretors of IL-16 and show little if any resistance to HIV. In contrast, cells expressing the C-terminal 100 aa linked to a signal peptide secrete IL-16 and are resistant to HIV replication. It is concluded that the secretion of IL-16 is required for HIV inhibition.

Blockade of the classical pathway of protein secretion does not affect the cellular exportation of lipocortin 1

The mechanism by which lipocortin 1 (LC1) is extruded from cells in the brain and periphery in response to a glucocorticoid challenge is unknown. This study examined the influence of three inhibitors of the classical endoplasmic reticulum–Golgi pathway of protein secretion on the dexamethasone-induced (0.1 μM, 2–3 h) cellular exportation of LC1 in vitro in brain (cortex, hippocampus, hypothalamus), anterior pituitary tissue and peritoneal macrophages. In all instances, the steroid-induced exportation of LC1 was unaffected by brefeldin A (1.4 μM), monensin (10 μM) and nocodazole (3.3 μM); however, these drugs readily blocked the release of corticotrophin from pituitary tissue. These data suggest that LC1 is exported by a mechanism distinct from the classical pathway of protein secretion.

Cholinergic agonists stimulate secretion of soluble full-length amyloid precursor protein in neuroendocrine cells.

The Abeta peptide of Alzheimer disease is derived from the proteolytic processing of the amyloid precursor proteins (APP), which are considered type I transmembrane glycoproteins. Recently, however, soluble forms of full-length APP were also detected in several systems including chromaffin granules. In this report we used antisera specific for the cytoplasmic sequence of APP to show that primary bovine chromaffin cells secrete a soluble APP, termed solAPPcyt, of an apparent molecular mass of 130 kDa. This APP was oversecreted from Chinese hamster ovary cells transfected with a full-length APP cDNA indicating that solAPPcyt contained both the transmembrane and Abeta sequence. Deglycosylation of solAPPcyt showed that it contained both N- and O-linked sugars, suggesting that this APP was transported through the endoplasmic reticulum-Golgi pathway. Secretion of solAPPcyt from primary chromatin cells was temperature-, time-, and energy-dependent and was stimulated by cell depolarization in a Ca2+-dependent manner. Cholinergic receptor agonists, including acetylcholine, nicotine, or carbachol, stimulated the rapid secretion of solAPPcyt, a process that was inhibited by cholinergic antagonists. Stimulation of solAPPcyt secretion was paralleled by a stimulation of secretion in catecholamines and chromogranin A, indicating that secretion of solAPPcyt was mediated by chromaffin granule vesicles. Taken together, our results show that release of the potentially amyloidogenic solAPPcyt is an active cellular process mediated by both the constitutive and regulated pathways. solAPPcyt was also detected in human cerebrospinal fluid. Combined with the neuronal physiology of chromaffin cells, our data suggest that cholinergic agonists may stimulate the release of this APP in neuronal synapses where it may exert its biological functions. Moreover, vesicular or secreted solAPPcyt may serve as a soluble precursor of Abeta.

Activation of ADP-ribosylation factor by Golgi membranes. Evidence for a brefeldin A- and protease-sensitive activating factor on Golgi membranes

Recent evidence has implicated ADP-ribosylation factor (ARF) proteins as critical regulators of the protein secretory pathway, particularly in the endoplasmic reticulum-Golgi pathway. We have examined whether Golgi membranes contain activators of ARF and the consequences of ARF activation and acylation on its membrane association. Two means were used to assess ARF activation. First, guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) binding to protein was found to be greater when ARF and Golgi were incubated together than when either was incubated alone. These data suggested that ARF GTP gamma S was formed. This was confirmed by showing that the GTP gamma S-bound protein functioned as a cofactor for cholera toxin-stimulated ADP-ribosylation of Gs alpha, a reaction for which activated ARF is a necessary cofactor. Trypsin treatment of Golgi, an inhibitory ARF peptide, and brefeldin A each inhibited Golgi-mediated activation by approximately 70%, demonstrating that a specific protein interaction is required for the majority of the ARF activation. This ARF-activating protein is a strong candidate for the molecular target for brefeldin A. The ubiquitous nature of ARF proteins and their importance in both the exocytic and endocytic pathways may explain the effects of brefeldin A on both exocytic and endocytic membrane traffic in animal cells. A protease-insensitive activation of ARF by Golgi could also be demonstrated and was the dominant activity observed in submicromolar concentrations of magnesium. We believe this to be the lipid-mediated process described previously for purified ARF proteins. ARF activation resulted in tight association of ARF with phospholipid vesicles. Vesicle association required amino-terminal myristoylation of ARF whereas activation did not. These studies indicate that the brefeldin A-sensitive ARF-activating protein and other factors that determine the level of activation of ARF in animal cells are fundamental regulators of membrane traffic in animal cells.

The endoplasmic reticulum—Golgi intermediate compartment

The recent identification of an endoplasmic reticulum-Golgi intermediate compartment has added to the complexity of the structural and functional organization of the early secretory pathway. Protein sorting along the endoplasmic reticulum-Golgi pathway depends on different signals and mechanisms, some of which guarantee recycling from various levels of the Golgi apparatus to biosynthetically earlier compartments.

Radioautographic characterization of successive compartments along the rough endoplasmic reticulum-Golgi pathway of collagen precursors in foot pad fibroblasts of [3H]proline-injected rats.

Young rats given an intravenous injection of [3H]proline were killed at successive times from 4 to 80 min later. Fibroblasts from the front foot pad were radioautographed ; silver grains were counted over several of the organelles and the results were expressed as percent radiolabel per unit volume. These percentages reached a peak over rough endoplasmic reticulum cisternae at 4 min, intermediate vesicles and tubules at 10 min, spherical distensions of cis-side Golgi saccules at 20 min, cylindrical distensions of trans-side saccules between 40 and 60 min, and secretory granules at 60 min. It is proposed that the succession of peaks corresponds to the migration pathway of collagen precursor proteins within fibroblasts; that is, the proteins synthesized in rough endoplasmic reticulum are delivered by intermediate vesicles and/or tubules to the spherical distensions of cis-side saccules, somehow pass from there to the cylindrical distensions of trans-side saccules and, finally, are carried by secretory granules to the extracellular space.