Fatty acylation promotes fusion of transport vesicles with Golgi cisternae

Two different methods, stimulation of transport by fatty acyl-coenzyme A (CoA) and inhibition of transport by a nonhydrolyzable analogue of palmitoyl-CoA, reveal that fatty acylation is required to promote fusion of transport vesicles with Golgi cisternae. Specifically, fatty acyl-CoA is needed after the attachment of coated vesicles and subsequent uncoating of the vesicles, and after the binding of the NEM-sensitive fusion protein (NSF) to the membranes, but before the actual fusion event. We therefore suggest that an acylated transport component participates, directly or indirectly, in membrane fusion.     

A novel 115-kD peripheral membrane protein is required for intercisternal transport in the Golgi stack

We have used an in vitro Golgi protein transport assay dependent on high molecular weight (greater than 100 kD) cytosolic and/or peripheral membrane proteins to study the requirements for transport from the cis- to the medial-compartment. Fractionation of this system indicates that, besides the NEM-sensitive fusion protein (NSF) and the soluble NSF attachment protein (SNAP), at least three high molecular weight protein fractions from bovine liver cytosol are required. The activity from one of these fractions was purified using an assay that included the second and third fractions in a crude state. The result is a protein of 115-kD subunit molecular mass, which we term p115. Immunodepletion of the 115-kD protein from a purified preparation with mAbs removes activity. Peptide sequence analysis of tryptic peptides indicates that p115 is a "novel" protein that has not been described previously. Gel filtration and sedimentation analysis indicate that, in its native state, p115 is a nonglobular homo-oligomer. p115 is present on purified Golgi membranes and can be extracted with high salt concentration or alkaline pH, indicating that it is peripherally associated with the membrane. Indirect immunofluorescence indicates that p115 is associated with the Golgi apparatus in situ.     

Binding of an N-ethylmaleimide-sensitive fusion protein to Golgi membranes requires both a soluble protein(s) and an integral membrane receptor

An N-ethylmaleimide (NEM)-sensitive fusion protein (NSF) has recently been purified on the basis of its ability to restore transport to NEM-inactivated Golgi membranes in a cell-free transport system. NSF is a peripheral membrane protein required for the fusion of transport vesicles. We now report the existence of two novel components that together bind NSF to Golgi membranes in a saturable manner. These components were detected by examining the requirements for reassociation of purified NSF with Golgi membranes in vitro. One component is an integral membrane receptor that is heat sensitive, but resistant to Na2CO3 extraction and to all proteases tested. The second component is a cytosolic factor that is sensitive to both proteases and heat. This soluble NSF attachment protein (SNAP) is largely resistant to NEM and is further distinguished from NSF by chromatography. SNAP appears to act stoichiometrically in promoting a high-affinity interaction between NSF and the membrane receptor. Because NSF promotes vesicle fusion, it seems likely that these two new factors that allow NSF to bind to the membrane are also part of the fusion machinery.     

The activity of Golgi transport vesicles depends on the presence of the N-ethylmaleimide-sensitive factor (NSF) and a soluble NSF attachment protein (alpha SNAP) during vesicle formation

An assay designed to measure the formation of functional transport vesicles was constructed by modifying a cell-free assay for protein transport between compartments of the Golgi (Balch, W. E., W. G. Dunphy, W. A. Braell, and J. E. Rothman. 1984. Cell. 39:405-416). A 35-kD cytosolic protein that is immunologically and functionally indistinguishable from alpha SNAP (soluble NSF attachment protein) was found to be required during vesicle formation. SNAP, together with the N-ethylmaleimide-sensitive factor (NSF) have previously been implicated in the attachment and/or fusion of vesicles with their target membrane. We show that NSF is also required during the formation of functional vesicles. Strikingly, we found that after vesicle formation, the NEM-sensitive function of NSF was no longer required for transport to proceed through the ensuing steps of vesicle attachment and fusion. In contrast to these functional tests of vesicle formation, SNAP was not required for the morphological appearance of vesicular structures on the Golgi membranes. If SNAP and NSF have a direct role in transport vesicle attachment and/or fusion, as previously suggested, these results indicate that these proteins become incorporated into the vesicle membranes during vesicle formation and are brought to the fusion site on the transport vesicles.     

Retrograde vesicle transport in the Golgi

The Golgi apparatus is the central sorting and biosynthesis hub of the secretory pathway, and uses vesicle transport for the recycling of its resident enzymes. This system must operate with high fidelity and efficiency for the correct modification of secretory glycoconjugates. In this review, we discuss recent advances on how coats, tethers, Rabs and SNAREs cooperate at the Golgi to achieve vesicle transport. We cover the well understood vesicle formation process orchestrated by the COPI coat, and the comprehensively documented fusion process governed by a set of Golgi localised SNAREs. Much less clear are the steps in-between formation and fusion of vesicles, and we therefore provide a much needed update of the latest findings about vesicle tethering. The interplay between Rab GTPases, golgin family coiled-coil tethers and the conserved oligomeric Golgi (COG) complex at the Golgi are thoroughly evaluated.     

GATE-16, a membrane transport modulator, interacts with NSF and the Golgi v-SNARE GOS-28

Membrane proteins located on vesicles (v-SNAREs) and on the target membrane (t-SNAREs) mediate specific recognition and, possibly, fusion between a transport vesicle and its target membrane. The activity of SNARE molecules is regulated by several soluble cytosolic proteins. We have cloned a bovine brain cDNA encoding a conserved 117 amino acid polypeptide, denoted Golgi-associated ATPase Enhancer of 16 kDa (GATE-16), that functions as a soluble transport factor. GATE-16 interacts with N-ethylmaleimidesensitive factor (NSF) and significantly stimulates its ATPase activity. It also interacts with the Golgi v-SNARE GOS-28 in an NSF-dependent manner. We propose that GATE-16 modulates intra-Golgi transport through coupling between NSF activity and SNAREs activation.     

Sequential SNARE disassembly and GATE-16-GOS-28 complex assembly mediated by distinct NSF activities drives Golgi membrane fusion

Characterization of mammalian NSF (G274E) and Drosophila NSF (comatose) mutants revealed an evolutionarily conserved NSF activity distinct from ATPase-dependent SNARE disassembly that was essential for Golgi membrane fusion. Analysis of mammalian NSF function during cell-free assembly of Golgi cisternae from mitotic Golgi fragments revealed that NSF disassembles Golgi SNAREs during mitotic Golgi fragmentation. A subsequent ATPase-independent NSF activity restricted to the reassembly phase is essential for membrane fusion. NSF/alpha-SNAP catalyze the binding of GATE-16 to GOS-28, a Golgi v-SNARE, in a manner that requires ATP but not ATP hydrolysis. GATE-16 is essential for NSF-driven Golgi reassembly and precludes GOS-28 from binding to its cognate t-SNARE, syntaxin-5. We suggest that this occurs at the inception of Golgi reassembly to protect the v-SNARE and regulate SNARE function.     

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) bind to a multi-SNAP receptor complex in Golgi membranes.

Soluble N-ethylmaleimide-sensitive fusion attachment proteins (SNAPs) are required for the binding of N-ethylmaleimide-sensitive fusion protein (NSF) to Golgi membranes and are, therefore, required for intra-Golgi transport. We report the existence of distinct alpha/beta-SNAP and gamma-SNAP-binding sites in Golgi membranes that appear to be part of the same receptor complex. Cross-linking studies with alpha-SNAP demonstrate that an integral membrane protein of between 30-40 kDa is the alpha-SNAP binding component of the multi-SNAP receptor complex. These data suggest that SNAPs function by independently binding to a multi-SNAP membrane-receptor complex, thereby activating them to serve as adaptors for the targeting of NSF.     

Sequential involvement of p115, SNAREs, and Rab proteins in intra-Golgi protein transport

Delivery of transport vesicles to their receptor compartment involves tethering, priming, and fusion. Soluble NSF attachment protein-alpha (alphaSNAP) mediates the disruption of SNAREs by N-ethylmaleimide sensitive factor (NSF) and was employed to determine the hierarchy of proteins responsible for intra-Golgi protein transport. The N-terminal 23 amino acids of alphaSNAP are necessary for SNARE binding. The antibody 2F10 recognizes this SNARE interaction domain of alphaSNAP and inhibits intra-Golgi protein transport reversibly. This antibody was applied to modify the transport assay to determine the protein requirements relative to the action of alphaSNAP and NSF. We found that 1) p115 acts independently of alphaSNAP and NSF, 2) SNAREs are required after tethering and interact selectively after activation by alphaSNAP and NSF, and 3) Rab proteins act after SNARE activation and before fusion.     

Entamoeba histolytica: identification and characterization of an N-ethylmaleimide sensitive fusion protein homologue

Entamoeba histolytica is a phagocytic cell with numerous vesicles of different sizes and shapes but without a well-defined Golgi apparatus. Despite this, genes implied in membrane trafficking have been identified in the genome of this parasite. One of these genes is homologous to the N-ethylmaleimide sensitive fusion factor (NSF), whose protein has been shown to play an important role in vesicle fusion in other eukaryotic cells. In this report, we investigated the NSF homologue gene from a pathogenic E. histolytica, characterized its protein product and two of its activities, ATPase and in vitro intra-Golgi transport. The finding of an active NSF protein in E. histolytica indicates that a simple or primordial Golgi apparatus probably exists in this microorganism, as has been proposed by others.     

Purification of a novel class of coated vesicles mediating biosynthetic protein transport through the Golgi stack

We describe a scheme for the purification of the nonclathrin-coated vesicles that mediate transport of proteins between Golgi cisternae and probably from ER to Golgi. These "Golgi-derived coated vesicles" accumulate when Golgi membranes are incubated with ATP and cytosol in the presence of GTP gamma S, a compound that blocks vesicle fusion. The coated vesicles dissociate from the Golgi cisternae in high salt and can then be purified by employing differential and density gradient centrifugation. Golgi-derived coated vesicles have a putative polypeptide composition that is distinct from both cytosol and Golgi membranes, as well as from that of clathrin-coated vesicles.     

Identification of a novel, N-ethylmaleimide-sensitive cytosolic factor required for vesicular transport from endosomes to the trans-Golgi network in vitro

We have recently described a cell-free system that reconstitutes the vesicular transport of 300-kD mannose 6-phosphate receptors from late endosomes to the trans-Golgi network (TGN). We report here that the endosome----TGN transport reaction was significantly inhibited by low concentrations of the alkylating agent, N-ethylmaleimide (NEM). Addition of fresh cytosol to NEM-inactivated reaction mixtures restored transport to at least 80% of control levels. Restorative activity was only present in cytosol fractions, and was sensitive to trypsin treatment or incubation at 100 degrees C. A variety of criteria demonstrated that the restorative activity was distinct from NSF, an NEM-sensitive protein that facilitates the transport of proteins from the ER to the Golgi complex and between Golgi cisternae. Cytosol fractions immunodepleted of greater than or equal to 90% of NSF protein, or heated to 37 degrees C to inactivate greater than or equal to 93% of NSF activity, were fully able to restore transport to NEM-treated reaction mixtures. The majority of restorative activity sedimented as a uniform species of 50-100 kD upon glycerol gradient centrifugation. We have termed this activity ETF-1, for endosome----TGN transport factor-1. Kinetic experiments showed that ETF-1 acts at a very early stage in vesicular transport, which may reflect a role for this factor in the formation of nascent transport vesicles. GTP hydrolysis appears to be required throughout the transport reaction. The ability of GTP gamma S to inhibit endosome----TGN transport required the presence of donor, endosome membranes, and cytosol, which may reflect a role for guanine nucleotides in vesicle budding. Finally, ETF-1 appears to act before a step that is blocked by GTP gamma S, during the process by which proteins are transported from endosomes to the TGN in vitro.     

SNAPs, a family of NSF attachment proteins involved in intracellular membrane fusion in animals and yeast

Three new and likely related components of the cellular fusion machinery have been purified from bovine brain cytosol, termed alpha-SNAP (35 kd), beta-SNAP (36 kd), and gamma-SNAP (39 kd). Transport between cisternae of the Golgi complex measured in vitro requires SNAP activity during the membrane fusion stage, and each SNAP is capable of binding the general cellular fusion protein NSF to Golgi membranes. The SNAP-NSF-membrane complex may be an early stage in the assembly of a proposed multisubunit "fusion machine" on the target membrane. SNAP transport factor activity is also found in yeast. Yeast cytosol prepared from a secretion mutant defective in export from the endoplasmic reticulum (sec17) lacks SNAP activity, which can be restored in vitro by the addition of pure alpha-SNAP, but not beta- or gamma-SNAPs. These data suggest that the mechanism of action of SNAPs in membrane fusion is conserved in evolution.     

On vesicles and membrane compartments

Summary Two different mechanisms have been proposed to explain transport along the endocytic and biosynthetic transport routes in cells. The first involves stable compartments connected by vesicular traffic while the second argues that the key organelles (early endosomes or the cis Golgi) form de novo by fusion of vesicles and subsequently mature into later forms. In the first part of this article, I propose a classification that distinguishes between stable, preexisting membrane compartments and vesicles that are, by definition, transient organelles. In this scheme, compartments, but not vesicles, are capable of homotypic fusion while vesicles, but not compartments, are able to mature, a process defined as an irreversible set of biochemical events which lead to a physiologically distinct end-state of the vesicle prior to its vectorial fusion with a target compartment. In the second part, I summarize my current ideas about the ultrastructural organization of the ER-Golgi region. Finally, I review the cell biology of selected examples of different vesicle types in order to exemplify the fascinating diversity of functions that this class of membrane organelles has evolved.Abbreviations COP coatomer - ECV endosome carrier vesicle - ER endoplasmic reticulum - HRP horseradish peroxidase - IC intermediate compartment between ER and Golgi - MVB multivesicular body - NSF N-ethyl maleimide sensitive factor - SNAPS soluble NSF associated proteins - TGN trans Golgi network Dedicated to Professor Eldon H. Newcomb in recognition of his contributions to cell biology     

An NSF function distinct from ATPase-dependent SNARE disassembly is essential for Golgi membrane fusion

The precise biochemical role of N-ethylmaleimide-sensitive factor (NSF) in membrane fusion mediated by SNARE proteins is unclear. To provide further insight into the function of NSF, we have introduced a mutation into mammalian NSF that, in Drosophila dNSF-1, leads to temperature-sensitive neuroparalysis. This mutation is like the comatose mutation and renders the mammalian NSF temperature sensitive for fusion of postmitotic Golgi vesicles and tubules into intact cisternae. Unexpectedly, at the temperature that is permissive for membrane fusion, this mutant NSF binds to, but cannot disassemble, SNARE complexes and exhibits almost no ATPase activity. A well-charaterized NSF mutant containing an inactivating point mutation in the catalytic site of its ATPase domain is equally active in the Golgi-reassembly assay. These data indicate that the need for NSF during postmitotic Golgi membrane fusion may be distinct from its ATPase-dependent ability to break up SNARE pairs.     

Reconstitution of vesiculated Golgi membranes into stacks of cisternae: requirement of NSF in stack formation

We have developed an in vitro system to study the biochemical events in the fusion of ilimaquinone (IQ) induced vesiculated Golgi membranes (VGMs) into stacks of cisternae. The Golgi complex in intact normal rat kidney cells (NRK) is vesiculated by treatment with IQ. The cells are washed to remove the drug and then permeabilized by a rapid freeze-thaw procedure. VGMs of 60 nm average diameter assemble into stacks of Golgi cisternae by a process that is temperature dependent, requires ATP and a high speed supernatant from cell extract (cytosol), as revealed by immunofluorescence and electron microscopy. The newly assembled stacks are functionally active in vesicular protein transport and contain processing enzymes that carry out Golgi specific modifications of glycoproteins. The fusion of VGMs requires NSF, a protein known to promote fusion of transport vesicles with the target membrane in the exocytic and endocytic pathways. Immunoelectron microscopy using Golgi specific anti-mannosidase II antibody reveals that VGMs undergo sequential changes in their morphology, whereby they first fuse to form larger vesicles of 200-300-nm average diameter which subsequently extend into tubular elements and finally assemble into stacks of cisternae.     

Vesicle fusion in protein transport through the Golgi in vitro does not involve long-lived prefusion intermediates. A reassessment of the kinetics of transport as measured by glycosylation.

The well-characterized cell-free assay measuring protein transport between compartments of the Golgi [Balch, W. E., Dunphy, W. G., Braell, W. A., & Rothman, J. E. (1984) Cell 39, 405-416] utilizes glycosylation of a glycoprotein to mark movement of that protein from one Golgi compartment to the next. Glycosylation had been thought to occur immediately after vesicles carrying the glycoprotein fuse with their transport target. Therefore, the kinetics of glycosylation were taken to reflect the kinetics of vesicle fusion. We previously isolated and raised monoclonal antibodies against a protein (the prefusion operating protein, POP) which is required in this assay at a step after vesicles have apparently been formed and interacted with the target membranes, but long before glycosylation takes place. This was therefore presumed to be a reaction involving targeted but unfused vesicles. Here we report that POP is identical to uridine monophosphokinase, as revealed by molecular cloning. We show that POP is not active in transport per se but instead enhances the glycosylation used to mark transport. This indicated that, contrary to previous assumptions, glycosylation might lag significantly behind vesicle fusion. We directly show this to be true. This alters the interpretation of several earlier studies. In particular, the previously reported existence of a late, prefusion intermediate, the "NEM-resistant intermediate", can be seen to be due to effects on glycosylation and not indicative of true fusion events.     

Asymmetric requirements for a Rab GTPase and SNARE proteins in fusion of COPII vesicles with acceptor membranes

Soluble NSF attachment protein receptor (SNARE) proteins are essential for membrane fusion in transport between the yeast ER and Golgi compartments. Subcellular fractionation experiments demonstrate that the ER/Golgi SNAREs Bos1p, Sec22p, Bet1p, Sed5p, and the Rab protein, Ypt1p, are distributed similarly but localize primarily with Golgi membranes. All of these SNARE proteins are efficiently packaged into COPII vesicles and suggest a dynamic cycling of SNARE machinery between ER and Golgi compartments. Ypt1p is not efficiently packaged into vesicles under these conditions. To determine in which membranes protein function is required, temperature-sensitive alleles of BOS1, BET1, SED5, SLY1, and YPT1 that prevent ER/Golgi transport in vitro at restrictive temperatures were used to selectively inactivate these gene products on vesicles or on Golgi membranes. Vesicles bearing mutations in Bet1p or Bos1p inhibit fusion with wild-type acceptor membranes, but acceptor membranes containing these mutations are fully functional. In contrast, vesicles bearing mutations in Sed5p, Sly1p, or Ypt1p are functional, whereas acceptor membranes containing these mutations block fusion. Thus, this set of SNARE proteins is symmetrically distributed between vesicle and acceptor compartments, but they function asymmetrically such that Bet1p and Bos1p are required on vesicles and Sed5p activity is required on acceptor membranes. We propose the asymmetry in SNARE protein function is maintained by an asymmetric distribution and requirement for the Ypt1p GTPase in this fusion event. When a transmembrane-anchored form of Ypt1p is used to restrict this GTPase to the acceptor compartment, vesicles depleted of Ypt1p remain competent for fusion.     

Function of intracellular phospholipase A2 in vectorial transport of apoproteins from ER to Golgi.

1. The cytosolic fraction required in in vitro reconstituted intracellular transport of mucus glycoprotein apopeptide (apomucin) was isolated and its potential as transport supporting factor assessed by the quantitation of the gastric apomucin transferred to Golgi. 2. The experiments with the fraction promoting transport and delivery of apomucin to Golgi revealed that the active protein has the property of phospholipase A2 (PLA2) which assists ER vesicles fusion with Golgi. 3. The ability of the 76 kDa PLA2 to hydrolyze phospholipids and to support transport and fusion of ER vesicles with Golgi was abolished by phosphorylation and regained following dephosphorylation. 4. The data provide evidence that 76 kDa intracellular PLA2 is responsible for the fusion of ER-transport vesicles with Golgi. The process of fusion is accomplished by generation of lysophospholipids in fusing membranes.     

N-ethylmaleimide-sensitive fusion protein: a trimeric ATPase whose hydrolysis of ATP is required for membrane fusion

The NEM-sensitive fusion protein, NSF, together with SNAPs (soluble NSF attachment proteins) and the SNAREs (SNAP receptors), is thought to be generally used for the fusion of transport vesicles to their target membranes. NSF is a homotrimer whose polypeptide subunits are made up of three distinct domains: an amino-terminal domain (N) and two homologous ATP-binding domains (D1 and D2). Mutants of NSF were produced in which either the order or composition of the three domains were altered. These mutants could not support intra-Golgi transport, but they indicated that the D2 domain was required for trimerization of the NSF subunits. Mutations of the first ATP-binding site that affected either the binding (K266A) or hydrolysis (E329Q) of ATP completely eliminated NSF activity. The hydrolysis mutant was an effective, reversible inhibitor of Golgi transport with an IC50 of 125 ng/50 microliters assay. Mutants in the second ATP-binding site (binding, K549A; hydrolysis, D604Q) had either 14 or 42% the specific activity of the wild-type protein, respectively. Using coexpression of an inactive mutant with wild-type subunits, it was possible to produce a recombinant form of trimeric NSF that contained a mixture of subunits. The mixed NSF trimers were inactive, even when only one mutant subunit was present, suggesting that NSF action requires each of the three subunits in a concerted mechanism. These studies demonstrate that the ability of the D1 domain to hydrolyze ATP is required for NSF activity and, therefore is required for membrane fusion. The D2 domain is required for trimerization, but its ability to hydrolyze ATP is not absolutely required for NSF function.