Ca2+-independent phospholipase A2 participates in the vesicular transport of milk proteins

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.     

Inhibition of cellular protein secretion by poliovirus proteins 2B and 3A.

Poliovirus RNA replication occurs on the surface of membranous vesicles that proliferate throughout the cytoplasm of the infected cell. Since at least some of these vesicles are thought to originate within the secretory pathway of the host cell, we examined the effect of poliovirus infection on protein transport through the secretory pathway. We found that transport of both plasma membrane and secretory proteins was inhibited by poliovirus infection early in the infectious cycle. Transport inhibition did not require viral RNA replication or the inhibition of host cell translation by poliovirus. The viral proteins 2B and 3A were each sufficient to inhibit transport in the absence of viral infection. The intracellular localization of a secreted protein in the presence of 3A with the endoplasmic reticulum suggested that 3A directly blocks transport from the endoplasmic reticulum to the Golgi apparatus.     

Protein sorting upon exit from the endoplasmic reticulum

It is currently thought that all secretory proteins travel together to the Golgi apparatus where they are sorted to different destinations. However, the specific requirements for transport of GPI-anchored proteins from the endoplasmic reticulum to the Golgi apparatus in yeast could be explained if protein sorting occurs earlier in the pathway. Using an in vitro assay that reconstitutes a single round of budding from the endoplasmic reticulum, we found that GPI-anchored proteins and other secretory proteins exit the endoplasmic reticulum in distinct vesicles. Therefore, GPI-anchored proteins are sorted from other proteins, in particular other plasma membrane proteins, at an early stage of the secretory pathway. These results have wide implications for the mechanism of protein exit from the endoplasmic reticulum.     

Biosynthetic protein transport through the early secretory pathway

 Newly synthesized proteins destined for delivery to the cell surface are inserted cotranslationally into the endoplasmic reticulum (ER) and, after their correct folding, are transported out of the ER. During their transport to the cell surface, cargo proteins pass through the various cisternae of the Golgi apparatus and, in the trans-most cisternae of the stack, are sorted into constitutive secretory vesicles that fuse with the plasma membrane. Simultaneously with anterograde protein transport, retrograde protein transport occurs within the Golgi complex as well as from the Golgi back to the ER. Vesicular transport within the early secretory pathway is mediated by two types of non-clathrin coated vesicles: COPI- and COPII-coated vesicles. The formation of these carrier vesicles depends on the recruitment of cytosolic coat proteins that are thought to act as a mechanical device to shape a flattened donor membrane into a spherical vesicle. A general molecular machinery that mediates targeting and fusion of carrier vesicles has been identified as well. Beside a general overview of the various coat structures known today, we will discuss issues specifically related to the biogenesis of COPI-coated vesicles: (1) a possible role of phospholipase D in the formation of COPI-coated vesicles; (2) a functional role of a novel family of transmembrane proteins, the p24 family, in the initiation of COPI assembly; and (3) the direction COPI-coated vesicles may take within the early secretory pathway. Moreover, we will consider two alternative mechanisms of protein transport through the Golgi stack: vesicular transport versus cisternal maturation. Accepted: 24 October 1997     

The manganese cation disrupts membrane dynamics along the secretory pathway

The endoplasmic reticulum and Golgi apparatus play key roles in regulating the folding, assembly, and transport of newly synthesized proteins along the secretory pathway. We find that the divalent cation manganese disrupts the Golgi apparatus and endoplasmic reticulum (ER). The Golgi apparatus is fragmented into smaller dispersed structures upon manganese treatment. Golgi residents, such as TGN46, beta1,4-galactosyltransferase, giantin, and GM130, are still segregated and partitioned correctly into smaller stacked fragments in manganese-treated cells. The mesh-like ER network is substantially affected and peripheral ER elements are collapsed. These effects are consistent with manganese-mediated inhibition of motor proteins that link membrane organelles along the secretory pathway to the cytoskeleton. This divalent cation thus represents a new tool for studying protein secretion and membrane dynamics along the secretory pathway.     

Phospholipase D-dependent and -independent mechanisms are involved in milk protein secretion in rabbit mammary epithelial cells

Phospholipase D has been implicated in membrane traffic in the secretory pathway of yeast and of some mammalian cell lines. Here we investigated the involvement of phospholipase D in protein transport at various steps of the secretory pathway of mammary epithelial cells. Treatment of rabbit mammary explants with butanol, which blocks the formation of phosphatidic acid, decreased the secretion of caseins and to a lesser extent that of whey acidic protein. Butanol interfered with both the endoplasmic reticulum to Golgi complex transport of the caseins and secretory vesicle formation from the trans-Golgi network. In contrast, the transport of whey acidic protein to the Golgi was less affected. Activation of protein kinase C enhanced the overall secretion of both markers and interestingly, this stimulation of secretion was maintained for whey acidic protein in the presence of butanol. Transphosphatidylation assays demonstrated the existence of a constitutive phospholipase D activity which was stimulated by the activation of protein kinase C. We conclude that phospholipase D plays a role in casein transport from the endoplasmic reticulum to the Golgi and in the secretory vesicle formation from the trans-Golgi network. Moreover, our results suggest a differential requirement for phospholipase D in the secretion of caseins and that of whey acidic protein.     

Rab2 GTPase regulates vesicle trafficking between the endoplasmic reticulum and the Golgi bodies and is important to pollen tube growth

Pollen tube elongation depends on the secretion of large amounts of membrane and cell wall materials at the pollen tube tip to sustain rapid growth. A large family of RAS-related small GTPases, Rabs or Ypts, is known to regulate both anterograde and retrograde trafficking of transport vesicles between different endomembrane compartments and the plasma membrane in mammalian and yeast cells. Studies on the functional roles of analogous plant proteins are emerging. We report here that a tobacco pollen-predominant Rab2, NtRab2, functions in the secretory pathway between the endoplasmic reticulum and the Golgi in elongating pollen tubes. Green fluorescent protein-NtRab2 fusion protein localized to the Golgi bodies in elongating pollen tubes. Dominant-negative mutations in NtRab2 proteins inhibited their Golgi localization, blocked the delivery of Golgi-resident as well as plasmalemma and secreted proteins to their normal locations, and inhibited pollen tube growth. On the other hand, when green fluorescent protein-NtRab2 was over-expressed in transiently transformed leaf protoplasts and epidermal cells, in which NtRab2 mRNA have not been observed to accumulate to detectable levels, these proteins did not target efficiently to Golgi bodies. Together, these observations indicate that NtRab2 is important for trafficking between the endoplasmic reticulum and the Golgi bodies in pollen tubes and may be specialized to optimally support the high secretory demands in these tip growth cells.     

Heterogeneous distribution of phospholipids in membranes along the secretory pathway in pancreatic B-cells

The membrane content in phospholipids along the secretory pathway in rat pancreatic B-cells was studied in situ by high-resolution cytochemistry, applying the recently introduced phospholipase A2-gold technique. The gold particles were mostly associated with cell membranes, and the various types of membranes were labeled to a different extent. Quantitation of the labeling over these membranes revealed a heterogeneous distribution of the labeling across the secretory pathway. This heretogeneity occurred mainly as a progressive, decreasing gradient in the first half of this pathway, between the rough endoplasmic reticulum and the mi-cisternae of the Golgi apparatus. The labeling density remained at a lower level in the trans-most Golgi cisternae and immature secretory granule membranes, to increase in the mature secretory granule membrane, where it reached the value found in the plasma membrane. These results provide evidence that the functional heterogeneity existing across the membrane forming the secretory pathway is parallelled by substantial changes in their phospholipid content.     

A calcium-independent phospholipase activity insensitive to bromoenol lactone mediates arachidonic acid release by lindane in rat myometrial cells.

Arachidonic acid release is an important regulatory component of uterine contraction and parturition, and previous studies showed that lindane stimulates arachidonic acid release from myometrium. The present study partially characterized the enzyme activity responsible for lindane-induced arachidonic acid release in myometrial cells. Lindane released arachidonic acid from cultured rat myometrial cells in concentration- and time-dependent manners. This release was primarily from phosphatidylcholine and phosphatidylinositol, and was independent of intracellular and extracellular calcium. In cells prelabeled with [3H]arachidonic acid, 85% of radiolabel was recovered as free arachidonate and only 5% was recovered as eicosanoids. Pretreatment with the antioxidants Cu, Zn-superoxide dismutase, alpha-tocopherol or Trolox did not significantly modify lindane-induced arachidonic acid release. Pretreatment of cells with the phosphatidylcholine-specific phospholipase C inhibitor D609, phosphatidylinositol-specific phospholipase C inhibitor ET-18-OCH3, or an interrupter of the phospholipase D pathway (ethanol) did not suppress lindane-induced arachidonic acid release. Although these results are consistent with calcium-independent phospholipase A2 activation by lindane, the calcium-independent phospholipase A2 inhibitor bromoenol lactone failed to inhibit lindane-induced arachidonic acid release in myometrial cells, even though bromoenol lactone effectively blocked arachidonic acid release in neutrophils. These results suggest that myometrial cells express a novel, previously unidentified phospholipase that is arachidonate-specific, calcium-independent, insensitive to bromoenol lactone, insensitive to reactive oxygen species activation, shows substrate preference for phosphatidylcholine and phosphatidylinositol, and is stimulated by lindane. Moreover, the data show that the overwhelming majority of arachidonic acid released remains as arachidonate, but that lindane does not significantly inhibit metabolism of arachidonate to eicosanoids.     

Immunocytochemical localization of renin in juxtaglomerular cells

The involvement of various organelles in the synthesis, transport, and packaging of renin in the juxtaglomerular cells of newborn mice has been investigated by immunocytochemistry with the protein A-gold technique. Highly specific rabbit antibodies against mouse submandibular renin were used. Mild fixation and embedding in glycol methacrylate allowed enough sensitivity to identify a steep gradient of labeling from rough endoplasmic reticulum to Golgi complex to secretory granules. Routine fixation and embedding in Epon produced labeling differentials that allowed delineation of hitherto ill-defined types of secretory granules and vacuoles. The classical pattern of synthesis, transport, and packaging of secretory proteins involves the rough endoplasmic reticulum and Golgi complex and seems to apply to renin secretion. Immunoreactive renin is packaged as rhomboid crystals at the trans face of the Golgi complex. The limiting membrane of these rhomboids fuses to form coalescing protogranules where the crystals eventually yield their individuality maturing into secretory granules. Vacuoles containing a flocculent material, with or without a dense core, show significant immunocytochemical labeling. These vacuoles are not associated with the Golgi complex but occupy cytoplasmic areas well endowed with rough endoplasmic reticulum. As judged from their morphological features and their immunoreactivity, the vacuoles do not seem to follow the sequence of events typical of protogranules and coalescing protogranules. They possibly represent a parallel pathway of renin synthesis and transport, involving the nuclear envelope and bypassing the Golgi complex.     

The absence of Emp24p, a component of ER-derived COPII-coated vesicles, causes a defect in transport of selected proteins to the Golgi.

Emp24p is a type I transmembrane protein that is involved in secretory protein transport from the endoplasmic reticulum (ER) to the Golgi complex. A yeast mutant that lacks Emp24p (emp24 delta) is viable, but periplasmic invertase and the glycosylphosphatidyl-inositol-anchored plasma membrane protein Gas1p are delivered to the Golgi apparatus with reduced kinetics, whereas transport of alpha-factor, acid phosphatase and two vacuolar proteins is unaffected. Oligomerization and protease digestion studies of invertase suggest that the selective transport phenotype observed in the emp24 delta mutant is not due to a defect in protein folding or oligomerization. Consistent with a role in ER to Golgi transport, Emp24p is a component of COPII-coated, ER-derived transport vesicles that are isolated from a reconstituted in vitro budding reaction. We propose that Emp24p is involved in the sorting and/or concentration of a subset of secretory proteins into ER-derived transport vesicles.     

Interactions of 12-lipoxygenase with phospholipase A2 isoforms following platelet activation through the glycoprotein VI collagen receptor

Recent studies implicate the collagen receptor, glycoprotein VI (GPVI) in activation of platelet 12-lipoxygenase (p12-LOX). Herein, we show that GPVI-stimulated 12-hydro(peroxy)eicosatetraenoic acid (H(P)ETE) synthesis is inhibited by palmityl trifluromethyl ketone or oleyloxyethylphosphocholine , but not bromoenol lactone, implicating secretory and cytosolic, but not calcium-independent phospholipase A2 (PLA2) isoforms. Also, following GPVI activation, 12-LOX co-immunoprecipitates with both cytosolic and secretory PLA2 (sPLA2). Finally, venoms containing sPLA2 acutely activate p12-LOX in a dose-dependent manner. This study shows that platelet 12-H(P)ETE generation utilizes arachidonate substrate from both c- and sPLA2 and that 12-LOX functionally associates with both PLA2 isoforms.     

PAFAH Ib phospholipase A2 subunits have distinct roles in maintaining Golgi structure and function

Recent studies showed that the phospholipase subunits of Platelet Activating Factor Acetylhydrolase (PAFAH) Ib, α1 and α2 partially localize to the Golgi complex and regulate its structure and function. Using siRNA knockdown of individual subunits, we find that α1 and α2 perform overlapping and unique roles in regulating Golgi morphology, assembly, and secretory cargo trafficking. Knockdown of either α1 or α2 reduced secretion of soluble proteins, but neither single knockdown reduced secretion to the same degree as knockdown of both. Knockdown of α1 or α2 inhibited reassembly of an intact Golgi complex to the same extent as knockdown of both. Transport of VSV-G was slowed but at different steps in the secretory pathway: reduction of α1 slowed trans Golgi network to plasma membrane transport, whereas α2 loss reduced endoplasmic reticulum to Golgi trafficking. Similarly, knockdown of either subunit alone disrupted the Golgi complex but with markedly different morphologies. Finally, knockdown of α1, or double knockdown of α1 and α2, resulted in a significant redistribution of kinase dead protein kinase D from the Golgi to the plasma membrane, whereas loss of α2 alone had no such effect. These studies reveal an unexpected complexity in the regulation of Golgi structure and function by PAFAH Ib. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Drosophila arf72A acts as an essential regulator of endoplasmic reticulum quality control and suppresses autosomal-dominant retinopathy

The eukaryotic endoplasmic reticulum operates multiple quality control mechanisms to ensure that only properly folded proteins are exported to their final destinations via the secretory pathway and those that are not are destroyed via the degradation pathway. However, molecular mechanisms underlying such regulated exportation to these distinct routes are unknown. In this article, we report the role of Drosophila arf72A--the fly homologue of the mammalian Arl1 - in the quality checks of proteins and in the autosomal-dominant retinopathy. ARF72A localizes to the Golgi membranes of Drosophila photoreceptor cells, consistent with mammalian Arl1 localization in cell culture systems. A loss of arf72A function changes the membrane character of the endoplasmic reticulum and shifts the membrane balance between the endoplasmic reticulum and the Golgi complex toward the Golgi complex, resulting in over-proliferated Golgi complexes and accelerated protein secretion. Interestingly, our study indicated that more ARF72A localized on the endoplasmic reticulum in the ninaE(D1) photoreceptor cell, a Drosophila model of autosomal-dominant retinitis pigmentosa, compared to that in the wild-type. In addition, arf72A loss was shown to rescue the ninaE(D1)-related membrane accumulation and the rhodopsin maturation defect, and suppress ninaE(D1)-triggered retinal degeneration, indicating that rhodopsin accumulated in the endoplasmic reticulum bypasses the quality checks. While previous studies of ARF small GTPases have focused on their roles in vesicular budding and transport between the specific organelles, our findings establish an additional function of arf72A in the quality check machinery of the endoplasmic reticulum distinguishing the cargoes for secretion from those for degradation.     

The arrangement of the Golgi complex beads is not controlled by the cytoskeleton

Exposure of insect fat body to treatments which disrupt microtubules (colchicine, vinblastine sulfate and cold treatment) blocks intracellular transport between the Golgi complex and the plasma membrane but does not affect Golgi complex bead rings or transport from rough endoplasmic reticulum to the Golgi complex. Drugs which disrupt microfilaments (cytochalasins B and D) do not affect the bead rings or intracellular transport of secretory proteins at any level. Thus, intracellular transport between the rough endoplasmic reticulum and the Golgi complex and the arrangement of the beads in rings are both independent of the cytoskeleton. The ring arrangement is presumably maintained by interconnection(s) with rough endoplasmic reticulum membrane.     

The p24 family and selective transport processes at the ER—Golgi interface

The secretory pathway is of vital importance for eukaryotic cells and has a pivotal role in the synthesis, sorting, processing and secretion of a large variety of bioactive molecules involved in intercellular communication. One of the key processes in the secretory pathway concerns the transport of cargo proteins from the ER (endoplasmic reticulum) to the Golgi. Type‐I transmembrane proteins of ～24 kDa are abundantly present in the membranes of the early secretory pathway, and bind the COPI and COPII coat complexes that cover vesicles travelling between the membranes. These p24 proteins are thought to play an important role in the selective transport processes at the ER—Golgi interface, although their exact functioning is still obscure. One model proposes that p24 proteins couple cargo selection in the lumen with vesicle coat recruitment in the cytosol. Alternatively, p24 proteins may furnish subcompartments of the secretory pathway with the correct subsets of machinery proteins. Here we review the current knowledge of the p24 proteins and the various roles proposed for the p24 family members.     

Stimulation of Golgi membrane tubulation and retrograde trafficking to the ER by phospholipase A(2) activating protein (PLAP) peptide.

Recent pharmacological studies using specific antagonists of phospholipase A(2) (PLA(2)) activity have suggested that the formation of Golgi membrane tubules, 60-80 nm in diameter and up to several microns long, both in vivo and in a cell-free cytosol-dependent reconstitution system, requires the activity of a cytoplasmic Ca(2+)-independent PLA(2). We confirm and extend these studies by demonstrating that the stimulators of PLA(2), melittin and PLA(2) activating protein peptide (PLAPp), enhance cytosol-dependent Golgi membrane tubulation. Starting with preparations of bovine brain cytosol (BBC), or a fraction of BBC that is highly enriched in tubulation activity, called the gel filtration (GF) fraction, that are at subsaturating concentrations for inducing tubulation in vitro, we found that increasing concentrations of melittin or PLAPp produced a linear and saturable stimulation of Golgi membrane tubulation. This stimulation was inhibited by cytosolic PLA(2) antagonists, including the Ca(2+)-independent PLA(2)-specific antagonist, bromoenol lactone. The stimulatory effect of PLAPp, and its inhibition by PLA(2) antagonists, was reproduced using a permeabilized cell system, which reconstitutes both cytosol-dependent Golgi membrane tubulation and retrograde trafficking to the endoplasmic reticulum (ER). Taken together, these results are consistent with the idea that cytosolic PLA(2) activity is involved in the formation of Golgi membrane tubules, which can serve as trafficking intermediates in Golgi-to-ER retrograde movement.     

Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes

We have identified an important functional region of the yeast Arf1 activator Gea2p upstream of the catalytic Sec7 domain and characterized a set of temperature-sensitive (ts) mutants with amino acid substitutions in this region. These gea2-ts mutants block or slow transport of proteins traversing the secretory pathway at exit from the endoplasmic reticulum (ER) and the early Golgi, and accumulate both ER and early Golgi membranes. No defects in two types of retrograde trafficking/sorting assays were observed. We find that a substantial amount of COPI is associated with Golgi membranes in the gea2-ts mutants, even after prolonged incubation at the nonpermissive temperature. COPI in these mutants is released from Golgi membranes by brefeldin A, a drug that binds directly to Gea2p and blocks Arf1 activation. Our results demonstrate that COPI function in sorting of at least three retrograde cargo proteins within the Golgi is not perturbed in these mutants, but that forward transport is severely inhibited. Hence this region of Gea2p upstream of the Sec7 domain plays a role in anterograde transport that is independent of its role in recruiting COPI for retrograde transport, at least of a subset of Golgi-ER cargo.     

A membrane glycoprotein, Sec12p, required for protein transport from the endoplasmic reticulum to the Golgi apparatus in yeast

SEC12, a gene that is required for secretory, membrane, and vacuolar proteins to be transported from the endoplasmic reticulum to the Golgi apparatus, has been cloned from a genomic library by complementation of a sec12 ts mutation. Genetic analysis has shown that the cloned gene integrates at the SEC12 locus and that a null mutation at the locus is lethal. The DNA sequence predicts a protein of 471 amino acids containing a hydrophobic stretch of 19 amino acids near the COOH terminus. To characterize the gene product (Sec12p) in detail, a lacZ-SEC12 gene fusion has been constructed and a polyclonal antibody raised against the hybrid protein. The antibody recognizes Sec12p as a approximately 70-kD protein that sediments in a mixed membrane fraction that includes endoplasmic reticulum. Sec12p is not removed from the membrane fraction by treatment at high pH and high salt and is not degraded by exogenous protease unless detergent is present. Glycosylation of Sec12p during biogenesis is indicated by an electrophoretic mobility shift of the protein that is influenced by tunicamycin and by imposition of an independent secretory pathway block. We suggest that Sec12p is an integral membrane glycoprotein with a prominent domain that faces the cytoplasm where it functions to promote protein transport to the Golgi apparatus. In the process of transport, Sec12p itself may migrate to the Golgi apparatus and function in subsequent transport events.     

Actin microfilaments facilitate the retrograde transport from the Golgi complex to the endoplasmic reticulum in mammalian cells

The morphology and subcellular positioning of the Golgi complex depend on both microtubule and actin cytoskeletons. In contrast to microtubules, the role of actin cytoskeleton in the secretory pathway in mammalian cells has not been clearly established. Using cytochalasin D, we have previously shown that microfilaments are not involved in the endoplasmic reticulum–Golgi membrane dynamics. However, it has been reported that, unlike botulinum C2 toxin and latrunculins, cytochalasin D does not produce net depolymerization of actin filaments. Therefore, we have reassessed the functional role of actin microfilaments in the early steps of the biosynthetic pathway using C2 toxin and latrunculin B. The anterograde endoplasmic reticulum-to-Golgi transport monitored with the vesicular stomatitis virus-G protein remained unaltered in cells treated with cytochalasin D, latrunculin B or C2 toxin. Conversely, the brefeldin A-induced Golgi membrane fusion into the endoplasmic reticulum, the Golgi-to-endoplasmic reticulum transport of a Shiga toxin mutant form, and the subcellular distribution of the KDEL receptor were all impaired when actin microfilaments were depolymerized by latrunculin B or C2 toxin. These findings, together with the fact that COPI-coated and uncoated vesicles contain β/γ-actin isoforms, indicate that actin microfilaments are involved in the endoplasmic reticulum/Golgi interface, facilitating the retrograde Golgi-to-endoplasmic reticulum membrane transport, which could be mediated by the orchestrated movement of transport intermediates along microtubule and microfilament tracks.