JOHN ARMSTRONG MEMBRANE FUSION

Latest episodes in the Golgi serial An assay for vesicle transport in the Colgi complex has

led to the isolation of two proteins, NSF and SNAP, which lmay mediate membrane fusions throughout the cell.

There-are two ways of ,getting your protein known: one is to give it a snappy name; the other is to do innovative work in identifying the protein and establishing its func- tional significance. A flurry of recent and forthcoming pa- pers from Rothman’s laboratory, on proteins required for membrane fusion during vesicle transport through the Golgi complex, neatly combine these two approaches. Meanwhile, the same combination by Wattenberg and colleagues has revealed that another putative hsion fac- tor is an interesting and instructive experimental artefact.

The source of all the proteins is an assay, using Golgi membranes, for attachment of N-acetyl glucosamine to vesicular stomatitis virus (VSV) G glycoprotein. As the VSV G protein and the glycosyl transferase start in sep- arate membrane compartments, the assay is thought to reconstitute vesicle-mediated transport within the Golgi complex to the compartment where the transferase acts. The assay is now thoroughly characterized, yet surpris- ingly there is still some (debate as to the precise cellular event that has been reconstituted [1,2]. By ingeniously combining fractionation with the use of inhibitors, the assay was differentiated into partial reactions: vesicle for- mation and budding; vesicle targetting and attachment; and fusion of the vesick? membrane with its ‘acceptor’ compartment. From this, last stage, a protein called N- ethyl-maleimide-Sensitive Fusion protein (NSF) was the first factor to be purified [3].

NSF is a tetramer of 761kD subunits; remarkably it acts not only within the Golgi, but also in transport between endoplasmic reticulum and the Golgi [4], and in fusion of endocytic vesicles IS]. Its yeast homologue, seclsp, is the product of a gene irwolved in secretion, which acts at multiple stages of vesicular transport [ 61.

Fractionation of salt-extractable membrane components identified a group of three small proteins also involved in the last stage of the assay 171. The three proteins were then isolated by searching for factors involved in attach- ing NSF to membranes [S]. Thus, it seemed that these Soluble NSF Attachment Proteins (SNAPS) assemble into a complex with NSF that is associated with membrane fusion. Each of the three SNAPS, CL, p and y, could ~LKK- tion alone in NSF attachment; c1 and p are similar in se- quence, as judged by mapping of peptide fragments, but y is quite different. In a further convergence, s&7, a gene previously shown to interact with secl8 [91, seemed to encode a yeast SNAP [ 81, The cloning of secZ7 and isolation of its encoded protein has now confirmed that it is the homologue of &NAP [lo].

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Why are there three SNAPS, each of which appears to function alone? Can ‘each one genuinely substitute for the others? What do the SNAP proteins attach to in the membrane? These points are addressed in two further papers [I lJ.2 J, which exploit several new tools: anti- bodies raised against peptides based on sequences from c1- and Y-SNAPS; labelled U-SNAP prepared in vitro from cDNA; and an epitope-tagged form of NSF purified from bacteria. The first new result is that NSF and SNAPS do not bind to each other in solution. The SNAP must first bind to a protein in the Golgi membrane, perhaps inducing in it a conformational change. Curiously, this change can also be induced by adsorbing SNAPS to a polypropylene surfze, for instance a microcentrifuge tube. The mem- brane binding of labelled a-SNAP was both saturable and compatible with unlabelled CI- or P-SNAP, but not with y-SNAP. The latter also bound to Go@ membranes, irn- plying the existence of separate binding sites, although technical reasons have, so far, prevented a demonstration of saturability, But the sites do not appear to be far apart; a column of immobilized cl-SNAP can bind ?-SNAP, but only in the presence of solubilized membrane extracts. This suggests that the two binding sites are part of the same protein complex, or even the same polypeptide chain. A candidate receptor was identified by chemical crosslinking of labelled a-SNAP to membranes: several products appeared, but only one showed the expected properties of an intrinsic membrane protein. Thus, it appears that the components of this putative -XI- sion particle’ assemble sequentially on the membrane to form a multi-subunit complex (Fig. 1). The assembly and disassembly of the particle are examined further in the second paper [ 121. The formation of the particle, requir- ing NSF, cl-SNAP and membrane extract, was monitored by velocity sedimentation. The particle had a sedimenta- tion coeficient of 20 S. When the gradient fractions were subsequently assayed for their ability to promote co-pre- cipitation of NSF and a-SNAP, the ‘assembIy factor’ also sedimented at 20 S. In contrast, if NSF was omitted from the gradient, the assembly factor sedimented at 10 S. Size exclusion chromatography of the fusion particle, com- bined with its sedimentation coefficient, suggest a molec- ular weight of approximately 700 kD. This is sufficient to include a tetramer of NSF along with SNAPS, assembly’ factors and perhaps other things. The assays for co-precipitation and sedimentation made it possible to discriminate further between CI- and y- SNAPS. As with the measurements of membrane binding,

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Membrane receptors

Fig. 1. A speculative scheme for the assembly of SNAPS (green) and NSF (red). The stoichiometry of SNAPS in the complex is unknown, and the presence of th’e receptor protein in the vesicle membrane has yet to be demonstrated. Other factors, as yet unidentified, may act after NSF in the fusion process.

co-precipitation of labelled cl-SNAP and NSF was inhib- ited by u&belled a-SNAP and less efficiently by P-SNAP. y-SNAP, however, promoted assembly and was itself as- sembled into the complex. Addition of Mg-ATP, but not non-hydrolysable ATP analogues, caused the particle to dissociate.

Both approaches suggest that the particle assembles pro- gressively on the membrane receptor, first with c1- (or j%) SW and y-SNAP, and then NSF. Membrane fusion may then be coupled to ATP hydrolysis, releasing NSF for an- other round of assembly (Fig. 1). As with every previous advance in the Golgi transport assay, a number of new questions have now emerged. The SNAP receptor (or assembly factor) has been identified in Golgi membranes; is it only there or is it, or another receptor, also present in the vesicle membrane, forming a bridge between the vesicle and Golgi membranes (as shown speculatively in figure I)? The tetrameric form of NSF seems to be an invitation to form such a symmetrical structure. At what stage of the reaction do the SNAPS attach to the Golgi (and vesicle) membrane? Are all the components of the particle as ubiquitous as NSF? Does a-SNAP/see 17 act at different stages of membrane traffic? Now that y- SNAP is functionally resolved from a-SNAP, does it too have a yeast homologue?

So do any factors act downstream of NSF? Previous work had suggested that the attachment of SNAPS and NSF was just the beginning of a long and relatively slow series of reactions before fusion occurs, involving at least two intermediates and several essential factors (summarized in IS]). A Pratein Operating in Pre-fusion (POP) was identified in yeast [ 131. However purification and cloning of this protein led to the startling discovery that POP is, in fact, uridine-monophosphate (UMP) kinase [ 141.

A neat piece of detective work explained this mystery. At later stages the assay is limited not by fusion but by availability of the sugar substrate, N-acetyl glucosamine, and raising the substrate concentration accelerates the glycosylation of VSV G glycoprotein. UMP kinase removes UMP, an inhibitor of the translocator that delivers UDP N- acetyl glucosamine into the Go@. Thus, it appears that all of the previously identified factors and intermediates after the NSF step relate to glycosylation, not fusion, and we are back to where we started-NSF and SNAP act at the last resolvable step in the Golgi assay. Clearly there are important lessons to be learned here by all practitioners of cell-free assays.

Finally, what exactly is in the NSF particle, and how many of each component are there? While the columns and centrifuges run, gifted post-doctoral minds will doubtless

If this particle is really involved in fusion, why is it so big? The fusion activities of enveloped viruses are generally provided by a single polypeptide, which sometimes does other things as well. The point has been made that pro- gressive assembly of a fision complex reduces the risk to the cell of potentially disastrous inappropriate fusions [ 11. But viral fusion proteins too can be tightly controlled, restraining their activity even when synthesized in massive quantities in the host cell.

The topology and function of the two processes are entirely different., but nevertheless it is noticeable that neither of the cloned components of the particle have hydrophobic sequences reminiscent of the ‘fusion pep-

’ tides’ of viral prsoteins. It should also be borne in mind that NSF acts at the last stage of the Golgi transport assay, but as yet there is no proof that it is a fusion protein.

turn to the search for further suitable acronyms. Surely there cannot be a Cytoplasmic Regulator of Attachment, Coalescence Kinetics and Lipid Exchange?

References

ROTHMAN JE, 0x1 L: Molecular dissection of the secretory pathway. Nature 1992, 355:409415.

BELLMAN I, SMONS K: The Golgi Complex: In Vitro Veritas? Cell 1992, 68:829-+340.

BLOCK m GUCK BS, WILCOX CA, WIEUND FT, ROTHMAN JE: Purification of an N-ethyhnaleimide-sensitive protein cat- alyzing vesicular transport. Proc Nat1 Acad Sci USA 1988, 85:7852-7856.

BECKER?! CJM, BIOCK MR, GLICK BS, ROTHMAN JE, BALCH WE: Vesicular transport between the endoplasmic reticulum and

336 @ 1992 Current Biology

the Golgi stack requires the NEM-sensitive fusion protein. Nature 1989, 3391:397-398.

5. DIAZ R, MAYORGA Ls, WEJDMAN PJ, ROTHMAN JE, STAHL PD: Vesi- cle fusion following receptor-mediated endocytosis requires a protein active in Goigi transport. Nature 1989, 339:398-400.

6. GRAHAM T, EMR !SD: Compartmental organization of Golgi- specitic protein modification and vacuolar protein sorting events defined in a yeast secl8 (NSF) mutant. J Cell Biol 1991, 114:207-218.

7. CLARY DO, ROTHMAN JE: Purification of three related periph- eral membrane proteins needed for vesicular transport. J Biol Cbem 1990, 265:1010+10117.

8. CIARY DO, GRIFF I:G, ROTHMAN JE: SNAPS, a family of NSF at- tachment proteins involved in intracelhtlar membrane fusion in animals and yeast. Cell 1990, 61:70~721.

9. KAISER C, SCHEKMAN R: Distinct sets of SEC genes govern trans- port vesicle formation and fusion early in the secretory path- way. Cell 1990, 61.:723-733.

10. GRIFF IC, SCKEJSMA,Y R, ROTHMAN JE, KAISER CA The yeast SEC1 7 gene product is functionally equivalent to mammalian u- SNAP protein. J Viol Chem 1992, in press.

11. WHITEHEART SW, BRUNNER M, WILSON DW, WIEDMANN M, ROTHMAIU JE; Soluble NSF Attachment Proteins (SNAPS) bind to a multi-SNAP receptor complex in Golgi membranes. J Biol Cbem 1992, in press.

12. WILSON DW, WHITEHEART SW, WIEDMANN M, BRUNNER M, ROTHMAN JE: A multisubunit particle implicated in membrane fusion. jI Cell Biol 1992, 117:531-538.

13. WATTENBERG BW, HIEBSCH RR, LECUREUX LW, WHITE MP: Iden- thication of a 25 kD protein from yeast cytosol that operates in a pretirsion step of vesicular transport between compart- ments of the Golgi. J Cell Biol 1990, 110:947-954.

14. HIEBSCH RR, WATIENBERG BW: Vesicle fusion in protein trans- port through the Golgi does not involve long lived pre-fusion intermediates. Biocbemishy 1992, in press.

John Armstrong, Membrane Molecular Biology Labora- tory, Imperial Cancer Research Fund, P.O.Box 123, Lin- coln’s Inn Fields, London WC2A 3PX, UK.

IN THE AUGUST 1992 ISSUE OF CURRENT OPINION IN CELL BIOLOGY

David Sabatini, Milton Adesnik and Daniel Louva will edit the following reviews on Membranes:

Functional interrelationships between the endoplasmic reticulum and golgi apparatus by H Haurl

The modification of proteins by lipid addition by M Chow Molecular trafficking across the nuclear pore complex by L Gerace

Mechanisms determining the transmembrane disposition of proteins by B Dobberstein Gen’etic and biochemical analysis of vesicular traffic in yeast by R Schekman

Adaptors, p-cops and non-clathrin and clathrin-coated vesicles by ‘I Kreis Biochemistry of vesicle transport and membrane fusion by J Gruenberg

The biogenesis of secretory vesicles and granules by M Rindler Transport of proteins into mitochondria by G Schatz Protein targetting to and translocation across the

membrane of the endoplasmic reticulum by P Walter Protein sorting, endocytosis and plasma membrane

domain dynamics in polarized cells by K Matlin

Ernest Wright will edit the following reviews on Membrane Permeability:

Pumps by N Nelson Cotransport systems by E Wright Cation exchangers by K Philipson

Bacterial transport systems by P Henderson Plant membrane transport by D Sanders

Ion channels by S Heinemann

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