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Q &A

SNARE complexes is there sufficient complexity for vesicle targeting specificity?

What are SNAREs and why are they important?

SNAREs (soluble N-ethylmaleimidesensitive factor attachment protein receptors) are a family of cytoplasmically orientated membrane-associated proteins that are found throughout the endomembrane system of eukaryotic cells. In addition to probably being core components of the membrane fusion apparatus itself, the diverse intracellular distributions of SNAREs make them attractive candidates for the determinants of transport vesicleorganelle targeting specificity, which is crucial for maintaining the organization of the secretory pathway and for the correct modification and sorting of proteins.
How do SNAREs work?

easily placed into family groups on the basis of amino acid sequence similarities than other SNAREs (such as the syntaxins, SNAP-25 family members and the synaptobrevins). More recently, SNAREs have been categorized as either Q- or RSNAREs depending on whether they contain a conserved glutamine or arginine, respectively, in their SNARE-binding domain.
How is SNARE function studied?

drives membrane fusion, in part by bringing the two lipid bilayers into close proximity. Following membrane fusion the SNARE complex now a resident of the target organelle membrane (termed a cis-SNARE complex) is dissociated (through the action of NSF and -SNAP) and recycled.
What do SNARE complexes look like?

A variety of approaches have been used including the characterization of yeast strains with defects in SNARE genes, the development of in vitro transport assays that reconstitute membrane targeting and fusion, SNARE protein structure and function studies, as well as through the study of exocytosis in neuronal cells. More recently, an in vitro assay that measures SNARE-mediated fusion between liposomes has been used to test the fusion activity of various combinations of a subset of yeast SNAREs.
How and where are SNARE complexes formed?

As originally proposed in the SNARE hypothesis (circa 1993), SNARESNARE pairing was sufficient to ensure proper vesicle targeting and fusion. A SNARE on the surface of a secretory organelle (or target compartment; called a tSNARE) paired specifically with a SNARE on a transport vesicle (a vSNARE), and this v/tSNARE pairing provided a means by which cells could control the specificity of intracellular membrane fusion events. However, if the tenants of the original SNARE hypothesis are strictly followed, there appears to be an insufficient number of SNAREs to account for the diversity of vesicle trafficking pathways. This is particularly apparent for traffic through the yeast Golgi where only one tSNARE is required. Furthermore, there is an increasing number of examples in which a single SNARE can participate in more than one trafficking step or has numerous SNARE-binding partners, an observation that has led to the suggestion that SNARESNARE interactions in vivo are promiscuous.
Are all SNAREs categorized as either v- or tSNAREs?

Complexes form between SNAREs in opposing vesicle and target organelle membranes. SNARE pairing is mediated by the membrane proximal heptad-repeatcontaining region of these proteins (usually referred to as the core domain or SNARE domain, see Fig. 1). Such trans-SNARE complexes are reportedly resistant to dissociation by NSF (an ATPase, the product of the SEC18 gene in yeast) and -SNAP (a SNARE-binding protein, the product of the SEC17 gene in yeast). One view is that the formation of the trans-SNARE complex
N

SNARE complexes form stable parallel -helical bundles. In the case of the neuronal exocytic SNARE complex, one helix is contributed by syntaxin, one by synaptobrevin and two helices are contributed by SNAP-25. A similar composition has also been proposed for the yeast exocytic SNARE complex. The scenario for SNARE complexes that do not contain a SNAP-25 family member is slightly different. Although they are also likely to comprise four parallel -helical bundles, recent evidence suggests that such complexes are formed by four different proteins rather than three (at least one of which is a syntaxin family member). These four protein complexes comprise three Q-SNAREs and one R-SNARE where a single SNARE (either a Q- or R-SNARE) is contributed by one membrane and the remaining three proteins are contributed by the other membrane. It is not yet clear whether there is a strict three Q- and one R-SNARE requirement for quaternary complexes in cells.
Is there sufficient complexity for vesicle targeting specificity?

Although mammalian SNARE-complex assembly is nonselective (at least for the soluble forms of these proteins), when yeast

N
SNARESNARE binding domain or 'core domain' Variable N-domain Regulatory domain

Cytosol Membrane C Syntaxin SNAREs (Q-SNAREs) Lumen

C
Non-syntaxin SNAREs (Q- or R-SNAREs)
Ti BS

SNARE categorization (as well as the accompanying nomenclature) can often be the source of confusion even for SNARE enthusiasts! Some SNAREs are more

Fig. 1. A schematic diagram of the structural organization of SNAREs.

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TRENDS in Biochemical Sciences Vol.26 No.1 January 2001

Q- or R-SNAREs (light chains) vSNARE (Q- or R-SNARE)

Syntaxin (Q-SNARE)

SNARESNARE interactions, coupled with a specific asymmetric requirement for particular SNAREs on vesicles and target organelle membranes, could potentially generate a sufficient number of complexes to allow one SNARE complex to act in only one transport step (see Fig. 2). A strategy organisms such as yeast, which have a limited repertoire of SNARE proteins, might very probably exploit. These new findings reconcile conceptual difficulties arising from the observation that a single SNARE can participate in more than one trafficking step, as a given SNARE might function differently in different complexes.
Is there anything else that should be considered?

engagement. Once these combinations are known, the somewhat more difficult task of sorting out the physiological roles of these complexes can begin. In addition, several important questions remain to be addressed. Among them is the question of how cells localize and sort SNAREs, thus ensuring that the necessary intracellular asymmetric distributions of these proteins are established and maintained.
Further reading 1 McNew, J.A. et al. (2000) Compartmental specificity of cellular membrane fusion encoded in SNARE proteins. Nature 407, 153159 2 Parlati, F. et al. (2000) Topological restriction of SNARE-dependent membrane fusion. Nature 407, 194198 3 Fukuda, R. et al. (2000) Functional architecture of an intracellular membrane t-SNARE. Nature 407, 198202 4 Fasshauer, D. et al. (1999) Mixed and non-cognate SNARE complexes. Characterization of assembly and biophysical properties. J. Biol. Chem. 274, 1544015446 5 Yang, B. et al. (1999) SNARE interactions are not selective. Implications for membrane fusion specificity. J. Biol. Chem. 274, 56495653 6 Pelham, H.R.B. (1999) SNAREs and the secretory pathway-lessons from yeast. Exp. Cell Res. 247, 18 7 Waters, G.M. and Hughson, F.M. (2000) Membrane tethering and fusion in the secretory and endocytic pathways. Traffic 1, 588597

Target organelle Sed5p; Q-SNARE Bos1p; Q-SNARE Sec22p; R-SNARE Vam3p; Q-SNARE Vam7p; Q-SNARE Vti1p; Q-SNARE

Transport vesicle Bet1p; Q-SNARE

Nyv1p; R-SNARE
Ti BS

Fig. 2.The formation of fusogenic yeast SNARE complexes displays a requirement for asymmetrically distributed SNAREs.

Additional factors probably contribute to the specificity of SNARE complex formation in vivo by regulating both the spatial as well as the temporal formation of SNARE complexes. These components include so-called tethering or vesicle docking factors, as well as GTPases of the Rab family (Ypt proteins, in yeast).
Whats next?

SNAREs are reconstituted into two different liposome populations only certain combinations appear to be able to mediate membrane fusion between them, suggesting that SNAREs can indeed pair specifically. Thus, a significant degree of complexity could be achieved by using different combinations of SNAREs. Combinatorial

It now appears that several of the significant criticisms regarding the role of SNAREs in transport vesicle targeting specificity can be reconciled. What remains is the identification of the precise combinations of SNAREs that can mediate membrane fusion reactions as well as to determine the rules of

David K. Banfield Dept of Biology,The Hong Kong University of Science and Technology, Clearwater Bay, Kowloon, Hong Kong, China. e-mail: bodkb@ust.hk

Historical Perspective

Aging and the biochemistry of life

Robin Holliday
Living organisms extract energy and chemicals from the environment, create ordered structures and reproduce themselves. In an environment where resources are abundant, reproduction can lead to an exponential increase in numbers, but it is obvious that no environment can sustain such an increase indefinitely. The limit to resources means that organisms compete with each other to maintain themselves and produce offspring. Most complex animals, having developed to adulthood and reproduced, subsequently survive for only a finite length of time, as active adult life is replaced by senescence, aging and death. The complex biochemical systems that underlie the normal features of the animal therefore eventually return to disorder. It is not complexity per se which gives rise to aging and death, because many organisms, particularly plants, can survive indefinitely by vegetative propagation.
Theories of aging

It is only in fairly recent years that the reasons for the evolution of aging in complex animals have become apparent. For most of the 20th century, aging was regarded as an unsolved problem. During this period many theories of aging were proposed, and it was commonly asserted that there was one major reason for the finite survival time of animals. Thus, it was proposed that somatic mutations or

chromosome damage lead to senescence; or that protein became progressively crosslinked or altered in other ways; or that the integrity of mitochondria could not be maintained; or that aging was simply the running down of the genetic program for development; or that the generation of oxygen free radicals produced irreversible damage in DNA, proteins and membranes. My own interest stemmed from another theory of aging, proposed in 1963 by Leslie Orgel1. He suggested that the machinery for protein synthesis might be unstable. The specificity of biochemical reactions is never perfect, and in assembling polypeptide chains, the organism must

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