Anterograde transport through the Golgi complex: do Golgi tubules hold the key?

Biochemical studies have suggested that anterograde protein transport through the Golgi complex is mediated by coatomer-coated vesicles that bud from one compartment and then transfer to, and fuse with, the next. However, recent genetic studies have shown that coatomer mutations block retrograde, but not anterograde, transport in yeast, calling into question the role of coatomer vesicles in anterograde transport. Peggy Weidman proposes that these findings might be explained if anterograde transport occurs by transient fusion of Golgi tubules and if coatomers have related, but separable, functions in tubule and vesicle dynamics.

The G protein-activating peptide, mastoparan, and the synthetic NH2-terminal ARF peptide, ARFp13, inhibit in vitro Golgi transport by irreversibly damaging membranes.

Mastoparan is a cationic amphipathetic peptide that activates trimeric G proteins, and increases binding of the coat protein beta-COP to Golgi membranes. ARFp13 is a cationic amphipathic peptide that is a putative specific inhibitor of ARF function, and inhibits coat protein binding to Golgi membranes. Using a combination of high resolution, three-dimensional electron microscopy and cell-free Golgi transport assays, we show that both of these peptides inhibit in vitro Golgi transport, not by interfering in the normal functioning of GTP-binding proteins, but by damaging membranes. Inhibition of transport is correlated with inhibition of nucleotide sugar uptake and protein glycoslation, a decrease in the fraction of Golgi cisternae exhibiting normal morphology, and a decrease in the density of Golgi-coated buds and vesicles. At peptide concentrations near the IC50 for transport, those cisternae with apparently normal morphology had a higher steady state level of coated buds and vesicles. Kinetic analysis suggests that this increase in density was due to a decrease in the rate of vesicle fission. Pertussis toxin treatment of the membranes appeared to increase the rate of vesicle formation, but did not prevent the membrane damage induced by mastoparan. We conclude that ARFp13 is not a specific inhibitor of ARF function, as originally proposed, and that surface active peptides, such as mastoparan, have the potential for introducing artifacts that complicate the analysis of trimeric G protein involvement in regulation of Golgi vesicle dynamics.

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.

Vesicle fusion following receptor-mediated endocytosis requires a protein active in Golgi transport.

In reconstitution studies N-ethylmaleimide, a sulphydryl alkylating reagent, inhibits both fusion of endocytic vesicles and vesicular transport in the Golgi apparatus. We show here that the same N-ethylmaleimide-sensitive factor that catalyses the vesicle-mediated transport within Golgi stacks is also required for endocytic vesicle fusion. Thus, it is likely that a common mechanism for vesicle fusion exists for both the secretory and endocytic pathways of eukaryotic cells.

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.

Involvement of GTP-binding "G" proteins in transport through the Golgi stack.

GTP gamma S irreversibly inhibits protein transport between successive compartments of the Golgi stack in a cell-free system. Fluoride, potentiated by the addition of aluminum ion, also causes a strong inhibition. These are hallmarks of the involvement of a guanine nucleotide-binding or regulatory "G" protein. Inhibition by GTP gamma S requires a cytosolic inhibitory factor that binds to Golgi membranes during inhibition. Preincubation experiments reveal that GTP gamma S blocks the function of acceptor Golgi but not donor Golgi membranes. More specifically, a processing step in between vesicle attachment and the actual fusion event seems to be affected. Electron microscopy demonstrates a corresponding 5-fold accumulation of non-clathrin-coated buds and vesicles associated with the Golgi cisternae during inhibition by GTP gamma S.

Purification and characterization of proteoliaisin, a coordinating protein in fertilization envelope assembly.

We report the purification and characterization of proteoliaisin, a protein that participates in the assembly of the sea urchin fertilization envelope. Proteoliaisin was purified from egg cortical granule exudate to greater than 99% homogeneity using chromatography on DEAE-Sepharose and on phenyl-Sepharose. Native proteoliaisin is a highly asymmetric protein (f/fo = 2.0) composed of a single Mr approximately 230,000 peptide. Its asymmetry was demonstrated both by analytical ultracentrifugation and by nondenaturing polyacrylamide gel electrophoresis, a novel analysis that detects molecular asymmetry in heterogeneous protein mixtures. Proteoliaisin is enriched in six amino acids: aspartic acid/asparagine, glutamic acid/glutamine, glycine, and cysteine, which account for over 50% of its mass. Nearly all of the cysteine residues are disulfide bonded. The protein contains a small proportion of aromatic amino acids with phenylalanine greater than tyrosine greater than tryptophan. At neutral pH its absorbance maximum is at 274.5 nm, with an extinction coefficient of 0.43 ml mg-1 cm-1. Proteoliaisin forms a 1:1 Ca2+-stabilized complex with ovoperoxidase, another component of the fertilization envelope, with Kd = 1.1 X 10(-6) M. Proteoliaisin, a constituent of the specialized echinoderm extracellular matrix called the fertilization envelope, has certain structural similarities to mammalian extracellular matrix proteins.

Regulation of extracellular matrix assembly: in vitro reconstitution of a partial fertilization envelope from isolated components.

At fertilization, the glycocalyx (vitelline layer) of the sea urchin egg is transformed into an elevated fertilization envelope by the association of secreted peptides and the formation of intermolecular dityrosine bonds. Dityrosine cross-links are formed by a secreted ovoperoxidase that exists in a Ca2+-stabilized complex with proteoliaisin in the fertilization envelope. By using purified proteins, we now show that proteoliaisin is necessary and sufficient to link ovoperoxidase to the egg glycocalyx. Specifically, we have found that ovoperoxidase can associate with the vitelline layer only when complexed with proteoliaisin; proteoliaisin binds to the vitelline layer independently of its association with ovoperoxidase; proteolytic modification of the vitelline layer is not required for this interaction to occur; the binding of proteoliaisin to the vitelline layer is mediated by the synergistic action of the two major seawater divalent cations, Ca2+ and Mg2+; the number of proteoliaisin-binding sites on the vitelline layer of unfertilized eggs is equivalent to the amount of proteoliaisin secreted at fertilization; and the binding of ovoperoxidase to the vitelline layer, via proteoliaisin, permits the in vitro cross-linking of these two in vivo substrates. The association of purified ovoperoxidase and proteoliaisin with the vitelline layer of unfertilized eggs reconstitutes part of the morphogenesis of the fertilization envelope.

Assembly of the sea urchin fertilization membrane: isolation of proteoliaisin, a calcium-dependent ovoperoxidase binding protein.

Fertilization of the sea urchin egg is accompanied by the assembly of an extracellular glycoprotein coat, the fertilization membrane. Assembly of the fertilization membrane involves exocytosis of egg cortical granules, divalent cation-mediated association of exudate proteins with the egg glycocalyx (the vitelline layer), and cross-linking of the assembled structure by ovoperoxidase, a fertilization membrane component derived from the cortical granules. We have identified and isolated a new protein, which we call proteoliaisin, that appears to be responsible for inserting ovoperoxidase into the fertilization membrane. Proteoliaisin is a 250,000-Mr protein that binds ovoperoxidase in a Ca2+-dependent manner, with half-maximal binding at 50 microM Ca2+. Other divalent cations are less effective (Ba2+, Mn2+, and Sr2+) or ineffective (Mg2+ and Cd2+) in mediating the binding interaction. Binding is optimal over the physiological pH range of fertilization membrane assembly (pH 5.5-7.5). Both proteoliaisin and ovoperoxidase are found in isolated, uncross-linked fertilization membranes. We have identified several macromolecular aggregates that are released from uncross-linked fertilization membranes after dilution into divalent cation-free buffer. One of these is an ovoperoxidase-proteoliaisin complex that is further disrupted only upon the addition of EGTA. These results suggest that a Ca2+-stabilized complex of ovoperoxidase and proteoliaisin forms one structural subunit of the fertilization membrane.

Purification and properties of ovoperoxidase, the enzyme responsible for hardening the fertilization membrane of the sea urchin egg.

The ovoperoxidase from the egg of the sea urchin, Strongylocentrotus purpuratus, has been purified to apparent homogeneity. Ovoperoxidase is secreted from the egg at fertilization and is responsible, in vivo, for hardening of the fertilization membrane by forming cross-links between protein tyrosyl residues. Purification was accomplished by activation of cortical granule exocytosis with acetic acid, followed by NH4SO4 precipitation, DEAE-Sephacel chromatography in the absence of divalent cations, and CM-Sephadex chromatography. The purified enzyme is a glycoprotein of Mr 70,000, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibits a UV-visible spectrum typical of heme peroxidases (epsilon 412 = 1.19 X 10(5) M-1 cm-1). Ovoperoxidase catalyzes the oxidation of tyrosine, guaiacol, iodide, and bromide, but not chloride, and can employ either H2O2 or, with 8% relative efficiency, ethyl peroxide as an oxidative substrate. Phenylhydrazine, 3-amino-1,2,4-triazole, azide, and sulfite all inhibit purified ovoperoxidase at concentrations similar to those that inhibit hardening in vivo. Inhibition by 3-amino-1,2,4-triazole is reversible, requires H2O2, and is slow relative to substrate turnover. The purified enzyme is sensitive to protease cleavage in the native state, yielding an active product of Mr approximately 50,000 which varies slightly depending upon the protease employed. Ovoperoxidase should provide a useful tool for the study of fertilization membrane formation as a paradigm of macromolecular assembly and modification.