Retrograde Membrane Research Articles

Phospholipase A2 antagonists inhibit constitutive retrograde membrane traffic to the endoplasmic reticulum.

Eukaryotic cells contain a variety of cytoplasmic Ca(2+)-dependent and Ca(2+)-independent phospholipase A2s (PLA2s; EC 2.3.1.2.3). However, the physiological roles for many of these ubiquitously-expressed enzymes is unclear or not known. Recently, pharmacological studies have suggested a role for Ca(2+)-independent PLA2 (iPLA2) enzymes in governing intracellular membrane trafficking events in general and regulating brefeldin A (BFA)-stimulated membrane tubulation and Golgi-to-endoplasmic reticulum (ER) retrograde membrane trafficking, in particular. Here, we extend these studies to show that membrane-permeant iPLA2 antagonists potently inhibit the normal, constitutive retrograde membrane trafficking from the trans-Golgi network (TGN), Golgi complex, and the ERGIC-53-positive ER-Golgi-intermediate compartment (ERGIC), which occurs in the absence of BFA. Taken together, these results suggest that iPLA2 enzymes play a general role in regulating, or directly mediating, multiple mammalian membrane trafficking events.

COPI vesicles accumulating in the presence of a GTP restricted arf1 mutant are depleted of anterograde and retrograde cargo.

Microinjection of the slowly hydrolyzable GTP analogue GTP(gamma)S or the ectopic expression of a GTP restricted mutant of the small GTPase arf1 (arf1[Q71L]) leads to the rapid accumulation of COPI coated vesicles and buds in living cells. This effect is blocked at 15 degrees C and by microinjection of antibodies against (beta)-COP. Anterograde and retrograde membrane protein transport markers, which have been previously shown to be incorporated into COPI vesicles between the endoplasmic reticulum and Golgi complex, are depleted from the GTP(gamma)S or arf1[Q71L] induced COPI coated vesicles and buds. In contrast, in control cells 30 to 60% of the COPI carriers co-localize with these markers. These in vivo data corroborate recent in vitro work, suggesting that GTP(gamma)S and arf1[Q71L] interfere with the sorting of membrane proteins into Golgi derived COPI vesicles, and provide the first in vivo evidence for a role of GTP hydrolysis by arf1 in the sorting of cargo into COPI coated vesicles and buds.

Axonal Neurofilaments Are Nonessential Elements of Toxicant-Induced Reductions in Fast Axonal Transport: Video-Enhanced Differential Interference Microscopy in Peripheral Nervous System Axons

Neurofilament modification and accumulation, occurring in toxicant-induced neuropathies, has been proposed to compromise fast axonal transport and contribute to neurological symptoms or pathology. The current study compares the effects of the neurotoxicants acrylamide (ACR) and 2,5-hexanedione (2,5-HD) on the quantity of fast, bidirectional vesicular traffic within isolated mouse sciatic nerve axons from transgenic mice lacking axonal neurofilaments (Eyer and Peterson, Neuron 12, 1–20, 1994) and nontransgenic littermates possessing neurofilaments. Fast anterograde and retrograde membrane bound organelle (MBO) traffic was quantitated within axons, before and after toxicant exposure, using video-enhanced differential interference contrast (AVEC–DIC) microscopy. Addition of 0.7 mM ACR to the buffer bathing the nerve produced a time-dependent reduction in bidirectional transport with a similar time to onset and magnitude in both transgenic and nontransgenic mice. 2,5-HD (4 mM) exposure reduced bidirectional vesicle traffic by a similar amount in both transgenic and nontransgenic animals. The time to onset of the transport reduction was less and the magnitude of the reduction was greater with 2,5-HD compared to ACR. A single 10-min exposure to ACR or 2,5-HD produced a similar reduction in transport to that produced by prolonged (1 h) exposure. Nonneurotoxic propionamide or 3,4-hexanedione (3,4-HD) produced no changes in bidirectional transport in either transgenic or nontransgenic animals. We conclude that ACR or 2,5-HD produces a rapid, saturable, nonreversible, neurotoxicant-specific reduction in fast bidirectional transport within isolated peripheral nerve axons. These actions are mediated through direct modification of axonal component(s), which are independent of toxicant-induced modifications of, or accumulations of, neurofilaments.

PDMP blocks the BFA-induced ADP-ribosylation of BARS-50 in isolated Golgi membranes

We reported that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (PDMP), blocks brefeldin A (BFA)-induced retrograde membrane transport from the Golgi complex to the endoplasmic reticulum (ER) (Kok et al., 1998, J. Cell Biol. 142, 25–38). We now show that PDMP partially blocks the BFA-induced ADP-ribosylation of the cytosolic protein BARS-50. Moreover, PDMP does not interfere with the BFA-induced inhibition of the binding of ADP-ribosylation factor (ARF) and the coatomer component β-coat protein to Golgi membranes. These results are consistent with a role of ADP-ribosylation in the action of BFA and with the involvement of BARS-50 in the regulation of membrane trafficking.

Clofibrate inhibits membrane trafficking to the Golgi complex and induces its retrograde movement to the endoplasmic reticulum.

Insights into the function of the Golgi complex have been provided by experiments performed with various inhibitors of membrane trafficking, such as the macrocyclic lactone brefeldin A (BFA), a compound that inhibits constitutive secretion, prevents the formation of coatomer-coated transport vesicles, and stimulates the retrograde movement of Golgi resident enzymes back to the ER. We show here that the structurally unrelated compound clofibrate, a peroxisome proliferator (PP) and hypolipidemic agent, also reversibly disrupts the morphological and functional integrity of the Golgi complex in a manner similar to BFA. In the presence of clofibrate, the forward transport of newly synthesized secretory proteins from the ER to the Golgi is dramatically inhibited. Moreover, clofibrate causes Golgi membranes to travel rapidly in a microtubule-dependent manner back to the ER, forming a hybrid ER-Golgi tubulovesicular membrane network. These affects appear to be independent of clofibrate's ability to stimulate the PP-activated receptor (PPAR) alpha pathway because other PPAR stimulators (DEHP, WY-14643) did not alter the Golgi complex or induce retrograde trafficking. These data suggest that PPAR alpha-independent, clofibrate-sensitive proteins participate in regulating Golgi-to-ER retrograde membrane transport, and, equally importantly, that clofibrate may be used as a pharmacological tool for investigating Golgi membrane dynamics.

PDMP blocks brefeldin A-induced retrograde membrane transport from golgi to ER: evidence for involvement of calcium homeostasis and dissociation from sphingolipid metabolism.

In this study, we show that an inhibitor of sphingolipid biosynthesis, D,L-threo-1-phenyl-2- decanoylamino-3-morpholino-1-propanol (PDMP), inhibits brefeldin A (BFA)-induced retrograde membrane transport from Golgi to endoplasmic reticulum (ER). If BFA treatment was combined with or preceded by PDMP administration to cells, disappearance of discrete Golgi structures did not occur. However, when BFA was allowed to exert its effect before PDMP addition, PDMP could not "rescue" the Golgi compartment. Evidence is presented showing that this action of PDMP is indirect, which means that the direct target is not sphingolipid metabolism at the Golgi apparatus. A fluorescent analogue of PDMP, 6-(N-[7-nitro-2,1, 3-benzoxadiazol-4-yl]amino)hexanoyl-PDMP (C6-NBD-PDMP), did not localize in the Golgi apparatus. Moreover, the effect of PDMP on membrane flow did not correlate with impaired C6-NBD-sphingomyelin biosynthesis and was not mimicked by exogenous C6-ceramide addition or counteracted by exogenous C6-glucosylceramide addition. On the other hand, the PDMP effect was mimicked by the multidrug resistance protein inhibitor MK571. The effect of PDMP on membrane transport correlated with modulation of calcium homeostasis, which occurred in a similar concentration range. PDMP released calcium from at least two independent calcium stores and blocked calcium influx induced by either extracellular ATP or thapsigargin. Thus, the biological effects of PDMP revealed a relation between three important physiological processes of multidrug resistance, calcium homeostasis, and membrane flow in the ER/ Golgi system.

Retrograde trafficking of both Golgi complex and TGN markers to the ER induced by nordihydroguaiaretic acid and cyclofenil diphenol.

Previous studies have shown that the Golgi stack and the trans-Golgi network (TGN) may play a role in capturing escaped resident endoplasmic reticulum (ER) proteins, and directing their retrograde transport back to that organelle. Whether this retrograde movement represents a highly specific or more generalized membrane trafficking pathway is unclear. To better understand both the retrograde and anterograde trafficking pathways of the secretory apparatus, we examined more closely the in vivo effects of two structurally unrelated compounds, the potent lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA), and the non-steroidal estrogen cyclofenil diphenol (CFD), both of which are known to inhibit secretion. In the presence of these compounds, transport of vesicular stomatitis virus G membrane glycoprotein from the ER to the Golgi complex, and from the TGN to the cell surface, was inhibited potently and rapidly. Surprisingly, we found that NDGA and CFD stimulated the rapid, but not concomitant, retrograde movement of both Golgi stack and TGN membrane proteins back to the ER until both organelles were morphologically absent from cells. Both NDGA- and CFD-stimulated TGN and Golgi retrograde membrane trafficking were inhibited by microtubule depolymerizing agents and energy poisons. Removal of NDGA and CFD resulted in the complete, but not concomitant, reformation of both Golgi stacks and their closely associated TGN compartments. These studies suggest that NDGA and CFD unmask a generalized bulk recycling pathway to the ER for both Golgi and TGN membranes and, further, that NDGA and CFD are useful for investigating the molecular mechanisms that control the formation and maintenance of both the Golgi stack proper and the TGN.

Golgi tubule traffic and the effects of brefeldin A visualized in living cells.

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein-Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5-10 min until rapidly emptying Golgi contents into the ER within 15-30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.

Functional Protein Prenylation Is Required for the Brefeldin A-dependent Retrograde Transport from the Golgi Apparatus to the Endoplasmic Reticulum

In cells exposed to brefeldin A (BFA), enzymes of the Golgi apparatus are redistributed to the endoplasmic reticulum (ER) by retrograde membrane flow, where they may cause modifications on resident ER proteins. We have used a truncated form of the rough ER-specific type I transmembrane glycoprotein ribophorin I as a probe to detect Golgi glycosyltransferases relocated to the ER in a BFA-dependent fashion. This polypeptide (RI332) comprises the 332 amino-terminal amino acids of ribophorin I and behaves like a luminal ER protein when expressed in HeLa cells. Upon treatment of the cells with BFA, RI332 becomes quantitatively O-glycosylated by Golgi glycosyltransferases that are transported back to the ER. Here we demonstrate that pretreatment of the cells with lovastatin, an inhibitor of HMG-CoA reductase, abrogates this modification and that mevalonate, the product formed in the step inhibited by the drug, is able to counteract the effect of lovastatin. We also show by immunofluorescence using mannosidase II as a Golgi marker that the BFA-induced retrograde transport of Golgi enzymes is blocked by lovastatin, although electron microscopy indicates that BFA causes disassembly of the Golgi apparatus into swollen vesicles and tubules. Our observations support the role of a prenylated protein, such as the geranylgeranylated small G protein Rab6, in the retrograde transport from the Golgi apparatus to the ER, since lovastatin acts by inhibiting its prenylation.

Fast transport and retrograde movement of huntingtin and HAP 1 in axons.

Huntingtin, the protein product of the Huntington's disease gene, associates with vesicle membranes and microtubules in neurons. Analysis of axonal transport with a stop-flow, double crush ligation approach in rat sciatic nerve showed that full length huntingtin (350 kDa) and an N-terminal cleavage product (50 kD) were increased within 6-12 h on both the proximal and distal sides of the crush site when compared with normal unligated nerve. The huntingtin associated protein HAP 1 and the retrograde motor protein dynein also accumulated on both sides of the crush, whereas the vesicle docking protein SNAP-25 was elevated only proximally. The cytoskeletal protein alpha-tubulin was unaffected. The rapid anterograde accumulation of huntingtin and HAP 1 is compatible with their axonal transport on vesicular membranes. Retrograde movement of both proteins, as seen by accumulation distal to the nerve crush, may be necessary for their degradation at the soma or for a function in retrograde membrane trafficking.

Dissociation of coatomer from membranes is required for brefeldin A-induced transfer of Golgi enzymes to the endoplasmic reticulum.

Addition of brefeldin A (BFA) to mammalian cells rapidly results in the removal of coatomer from membranes and subsequent delivery of Golgi enzymes to the endoplasmic reticulum (ER). Microinjected anti-EAGE (intact IgG or Fab-fragments), antibodies against the "EAGE"-peptide of beta-COP, inhibit BFA-induced redistribution of beta-COP in vivo and block transfer of resident proteins of the Golgi complex to the ER; tubulo-vesicular clusters accumulate and Golgi membrane proteins concentrate in cytoplasmic patches containing beta-COP. These patches are devoid of marker proteins of the ER, the intermediate compartment (IC), and do not contain KDEL receptor. Interestingly, relocation of KDEL receptor to the IC, where it colocalizes with ERGIC53 and ts-O45-G, is not inhibited under these conditions. While no stacked Golgi cisternae remain in these injected cells, reassembly of stacks of Golgi cisternae following BFA wash-out is inhibited to only approximately 50%. Mono- or divalent anti-EAGE stabilize binding of coatomer to membranes in vitro, at least as efficiently as GTP(gamma)S. Taken together these results suggest that enhanced binding of coatomer to membranes completely inhibits the BFA-induced retrograde transport of Golgi resident proteins to the ER, probably by inhibiting fusion of Golgi with ER membranes, but does not interfere with the disassembly of the stacked Golgi cisternae and recycling of KDEL receptor to the IC. These results confirm our previous results suggesting that COPI is involved in anterograde membrane transport from the ER/IC to the Golgi complex (Pepperkok et al., 1993), and corroborate that COPI regulates retrograde membrane transport between the Golgi complex and ER in mammalian cells.

AP-2-containing clathrin coats assemble on mature lysosomes.

Coat proteins appear to play a general role in intracellular protein trafficking by coordinating a membrane budding event with cargo selection. Here we show that the AP-2 adaptor, a clathrin-associated coat-protein complex that nucleates clathrin-coated vesicle formation at the cell surface, can also initiate the assembly of normal polyhedral clathrin coats on dense lysosomes under physiological conditions in vitro. Clathrin coat formation on lysosomes is temperature dependent, displays an absolute requirement for ATP, and occurs in both semi-intact cells and on purified lysosomes, suggesting that clathrin-coated vesicles might regulate retrograde membrane traffic out of the lysosomal compartment.

Comparative behavior of membrane protein-antibody complexes on motile fibroblasts: implications for a mechanism of capping.

A characteristic feature of fibroblast locomotory activity is the rearward transport across the leading lamella of various materials used to mark the cell surface. The two processes most frequently invoked as explanations for this transport phenomenon, called capping, are (a) retrograde membrane flow arising from directed membrane insertion and (b) rearward cortical cytoskeletal flow arising from cytoskeletal assembly and contraction. The retrograde lipid flow hypothesis, the most current form of the membrane flow scheme, makes explicit predictions about the movement of membrane proteins subjected to the postulated rearward lipid flow. Several of these predictions were tested by comparing the behavior of four membrane proteins, Pgp-1, Thy-1, H-2, and influenza HA0, identified by fluorescent antibodies. With the exception of Pgp-1, these proteins were uniformly distributed under nonaggregated conditions but were capped when aggregated into patches. In contrast, Pgp-1 was capped in similar time frames in both nonaggregated and aggregated states where the lateral diffusion coefficients were very different. Furthermore, the capping behavior of two tagged membrane proteins was markedly different yet both had similar diffusion coefficients. The results from these tests disprove the bulk membrane flow hypothesis and are at odds with explicit predictions of the retrograde lipid flow hypothesis for the mechanism of capping. This work, therefore, supports the alternative cytoskeletal-based mechanism for driving capping. Requirements for coupling cytoskeletal movement to membrane components are discussed.