Effect of Rho and ADP-ribosylation factor GTPases on phospholipase D activity in intact human adenocarcinoma A549 cells. (25/1373)

Phospholipase D (PLD) has been implicated as a crucial signaling enzyme in secretory pathways. Two 20-kDa guanine nucleotide-binding proteins, Rho and ADP-ribosylation factor (ARF), are involved in the regulation of secretion and can activate PLD in vitro. We investigated in intact (human adenocarcinoma A549 cells) the role of RhoA and ARF in activation of PLD by phorbol 12-myristate 13-acetate, bradykinin, and/or sphingosine 1-phosphate. To express recombinant Clostridium botulinum C3 exoenzyme (using double subgenomic recombinant Sindbis virus C3), an ADP-ribosyltransferase that inactivates Rho, or dominant-negative Rho containing asparagine at position 19 (using double subgenomic recombinant Sindbis virus Rho19N), cells were infected with Sindbis virus, a novel vector that allows rapid, high level expression of heterologous proteins. Expression of C3 toxin or Rho19N increased basal and decreased phorbol 12-myristate 13-acetate-stimulated PLD activity. Bradykinin or sphingosine 1-phosphate increased PLD activity with additive effects that were abolished in cells expressing C3 exoenzyme or Rho19N. In cells expressing C3, modification of Rho appeared to be incomplete, suggesting the existence of pools that differed in their accessibility to the enzyme. Similar results were obtained with cells scrape-loaded in the presence of C3; however, results with virus infection were more reproducible. To assess the role of ARF, cells were incubated with brefeldin A (BFA), a fungal metabolite that disrupts Golgi structure and inhibits enzymes that catalyze ARF activation by accelerating guanine nucleotide exchange. BFA disrupted Golgi structure, but did not affect basal or agonist-stimulated PLD activity, i.e. it did not alter a rate-limiting step in PLD activation. It also had no effect on Rho-stimulated PLD activity, indicating that RhoA action did not involve a BFA-sensitive pathway. A novel PLD activation mechanism, not sensitive to BFA and involving RhoA, was identified in human airway epithelial cells by use of a viral infection technique that preserves cell responsiveness.  (+info)

Gbetagamma and palmitate target newly synthesized Galphaz to the plasma membrane. (26/1373)

The subcellular location of a signaling protein determines its ability to transmit messages accurately and efficiently. Three different lipid modifications tether heterotrimeric G proteins to membranes: alpha subunits are myristoylated and/or palmitoylated, and gamma subunits are prenylated. In a previous study, we examined the role of lipid modifications in maintaining the membrane attachment of a G protein alpha subunit, alphaz, which is myristoylated and palmitoylated (Morales, J., Fishburn, C. S., Wilson, P. T., and Bourne, H. R. (1998) Mol. Biol. Cell 9, 1-14). Now we extend this analysis by characterizing the mechanisms that target newly synthesized alphaz to the plasma membrane (PM) and analyze the role of lipid modifications in this process. In comparison with newly synthesized alphas, which is palmitoylated but not myristoylated, alphaz moves more rapidly to the membrane fraction following synthesis in the cytosol. Newly synthesized alphaz associates randomly with cellular membranes, but with time accumulates at the PM. Palmitoylated alphaz is present only in PM-enriched fractions, whereas a nonpalmitoylated mutant of alphaz (alphazC3A) associates less stably with the PM than does wild-type alphaz. Expression of a C-terminal fragment of the beta-adrenoreceptor kinase, which sequesters free betagamma, impairs association of both alphaz and alphazC3A with the PM, suggesting that the alpha subunit must bind betagamma in order to localize at the PM. Based on these findings, we propose a model in which, following synthesis on soluble ribosomes, myristoylated alphaz associates randomly and reversibly with membranes; upon association with the PM, alphaz binds betagamma, which promotes its palmitoylation, thus securing it in the proper place for transmitting the hormonal signal.  (+info)

Cell surface membrane homeostasis and intracellular membrane traffic balance in mouse L929 cells. (27/1373)

We have developed a simple method for synchronizing L929 mouse fibroblasts. Cultured as monolayers, these cells stop growing at confluency and arrest at the end of the G1 phase. Upon seeding at low density, they enter the S phase simultaneously. Using these cells we then looked at the evolution of the surface membrane area during the cell cycle using the fluorescence membrane probe TMA-DPH. In contact with cells, this probe partitions between the membrane (probe fluorescent) and the external medium (non-fluorescent), delivering a signal proportional to the membrane area. This area was constant until just before mitosis, when it increased at once. With the same probe as an endocytic marker, we examined how this membrane homeostasis could be consistent with intracellular membrane trafficking. The study was limited to one selected period of the cell cycle (6-9 hours). We observed that 14% of the membrane endocytosed was not recycled, but was replaced at the cell surface by newly formed membrane from biosynthetic pathways. Brefeldin A modified the membrane traffic, but not the overall membrane homeostasis. The results are discussed in the framework of a maturation model.  (+info)

Brefeldin A increases the quantal size and alters the kinetics of catecholamine release from rat adrenal chromaffin cells. (28/1373)

The fungal metabolite, brefeldin A (BFA), is known to inhibit guanine nucleotide exchange on the ADP-ribosylating factors that are involved in vesicle membrane trafficking. Here, we investigated the action of BFA on Ca2+-regulated exocytosis in single rat adrenal chromaffin cells. Incubation of chromaffin cells with BFA (1 or 10 microM) for 2 h effectively disrupted the Golgi membranes but did not affect the pattern of catecholamine release triggered by high extracellular K+, which was monitored with carbon fiber amperometry along with cytosolic Ca2+ measurement. The BFA treatment, however, increased the mean quantal size of catecholamine-containing vesicles and the occurrence of amperometric events with a "foot" or "stand alone" signal (which reflects sluggish or incomplete dilation of the fusion pore). To examine whether BFA altered the Ca2+-dependence of exocytosis, we employed the whole-cell recording technique in conjunction with the capacitance measurement to measure exocytosis evoked from the entire cell during voltage-gated Ca2+ entry. Our results suggested that BFA treatment did not alter either the initial rate of capacitance increase or the total amount of capacitance increase. Therefore, in chromaffin cells, BFA treatment affects Ca2+-regulated exocytosis predominantly by increasing the quantal size and by slowing the fusion kinetics of some vesicles.  (+info)

A complex web of signal-dependent trafficking underlies the triorganellar distribution of P-selectin in neuroendocrine PC12 cells. (29/1373)

By analyzing the trafficking of HRP-P-selectin chimeras in which the lumenal domain of P-selectin was replaced with horseradish peroxidase, we determined the sequences needed for targeting to synaptic-like microvesicles (SLMV), dense core granules (DCG), and lysosomes in neuroendocrine PC12 cells. Within the cytoplasmic domain of P-selectin, Tyr777 is needed for the appearance of P-selectin in immature and mature DCG, as well as for targeting to SLMV. The latter destination also requires additional sequences (Leu768 and 786DPSP789) which are responsible for movement through endosomes en route to the SLMV. Leu768 also mediates transfer from early transferrin (Trn)-positive endosomes to the lysosomes; i.e., operates as a lysosomal targeting signal. Furthermore, SLMV targeting of HRP-P-selectin chimeras, but not the endogenous SLMV protein synaptophysin/p38, previously shown to be delivered to SLMV directly from the plasma membrane, is a Brefeldin A-sensitive process. Together, these data are consistent with a model of SLMV biogenesis which involves an endosomal intermediate in PC12 cells. In addition, we have discovered that impairment of SLMV or DCG targeting results in a concomitant increase in lysosomal delivery, illustrating the entwined relationships between routes leading to regulated secretory organelles (RSO) and to lysosomes.  (+info)

p200 ARF-GEP1: a Golgi-localized guanine nucleotide exchange protein whose Sec7 domain is targeted by the drug brefeldin A. (30/1373)

The drug brefeldin A (BFA) disrupts protein traffic and Golgi morphology by blocking activation of ADP ribosylation factors (ARFs) through an unknown mechanism. Here, we investigated the cellular localization and BFA sensitivity of human p200 ARF-GEP1 (p200), a ubiquitously expressed guanine nucleotide exchange factor of the Sec7 domain family. Multiple tagged forms of the full-length polypeptide localized to tight ribbon-like perinuclear structures that overlapped with the Golgi marker mannosidase II and were distinct from the pattern observed with ERGIC53/58. Analysis of several truncated forms mapped the Golgi-localization signal to the N-terminal third of p200. BFA treatment of transiently or stably transfected cells resulted in the redistribution of Golgi markers and in loss of cell viability, thereby indicating that overproduction of p200 may not be sufficient to overcome the toxic effect. A 39-kDa fragment spanning the Sec7 domain catalyzed loading of guanosine 5'-[gamma-thio]triphosphate onto class I ARFs and displayed clear sensitivity to BFA. Kinetic analysis established that BFA did not compete with ARF for interaction with p200 but, rather, acted as an uncompetitive inhibitor that only targeted the p200-ARF complex with an inhibition constant of 7 microM. On the basis of these results, we propose that accumulation of an abortive p200-ARF complex in the presence of BFA likely leads to disruption of Golgi morphology. p200 mapped to chromosome 8q13, 3.56 centirays from WI-6151, and database searches revealed the presence of putative isoforms whose inhibition may account for the effects of BFA on various organelles.  (+info)

Phospholipid transfer protein enhances removal of cellular cholesterol and phospholipids by high-density lipoprotein apolipoproteins. (31/1373)

High-density lipoprotein (HDL) apolipoproteins remove excess cholesterol from cells by an active transport pathway that may protect against atherosclerosis. Here we show that treatment of cholesterol-loaded human skin fibroblasts with phospholipid transfer protein (PLTP) increased HDL binding to cells and enhanced cholesterol and phospholipid efflux by this pathway. PLTP did not stimulate lipid efflux in the presence of albumin, purified apolipoprotein A-I, and phospholipid vesicles, suggesting specificity for HDL particles. PLTP restored the lipid efflux activity of mildly trypsinized HDL, presumably by regenerating active apolipoproteins. PLTP-stimulated lipid efflux was absent in Tangier disease fibroblasts, induced by cholesterol loading, and inhibited by brefeldin A treatment, indicating selectivity for the apolipoprotein-mediated lipid removal pathway. The lipid efflux-stimulating effect of PLTP was not attributable to generation of prebeta HDL particles in solution but instead required cellular interactions. These interactions increased cholesterol efflux to minor HDL particles with electrophoretic mobility between alpha and prebeta. These findings suggest that PLTP promotes cell-surface binding and remodeling of HDL so as to improve its ability to remove cholesterol and phospholipids by the apolipoprotein-mediated pathway, a process that may play an important role in enhancing flux of excess cholesterol from tissues and retarding atherogenesis.  (+info)

GBF1: A novel Golgi-associated BFA-resistant guanine nucleotide exchange factor that displays specificity for ADP-ribosylation factor 5. (32/1373)

Expression cloning from a cDNA library prepared from a mutant CHO cell line with Golgi-specific resistance to Brefeldin A (BFA) identified a novel 206-kD protein with a Sec7 domain termed GBF1 for Golgi BFA resistance factor 1. Overexpression of GBF1 allowed transfected cells to maintain normal Golgi morphology and grow in the presence of BFA. Golgi- enriched membrane fractions from such transfected cells displayed normal levels of ADP ribosylation factors (ARFs) activation and coat protein recruitment that were, however, BFA resistant. Hexahistidine-tagged-GBF1 exhibited BFA-resistant guanine nucleotide exchange activity that appears specific towards ARF5 at physiological Mg2+concentration. Characterization of cDNAs recovered from the mutant and wild-type parental lines established that transcripts in these cells had identical sequence and, therefore, that GBF1 was naturally BFA resistant. GBF1 was primarily cytosolic but a significant pool colocalized to a perinuclear structure with the beta-subunit of COPI. Immunogold labeling showed highest density of GBF1 over Golgi cisternae and significant labeling over pleiomorphic smooth vesiculotubular structures. The BFA-resistant nature of GBF1 suggests involvement in retrograde traffic.  (+info)