AAA proteins

Members o f the AAA family o f ATPases (ATPases Associated with diverse cellular Activities) include metalloproteases, proteins involved in vesicle and organelle biogenesis, cell-cycle regulators, transcription factors and components o f the 26S proteasome (Confalonieri and Duguet, 1995; Patel and Latterich, 1998). They are found in eukaryotes, prokaryotes and archeabacteria, revealing their ancient origin and central role in all life forms.

AAA proteins are characterised by a consensus sequence, the AAA cassette consisting o f -2 3 0 amino acids, which contains three highly conserved boxes: the Walker motifs A (ATP-binding) and B (ATP-hydrolysis) (Walker et al., 1982) which are part o f the ATP binding site and the AA A -m otif (Patel and Latterich, 1998). AAA proteins contain either one (type I) or two (type II) AAA cassettes. The AAA cassettes are highly conserved among the family members. The AAA cassette contains strongly conserved regions, which discriminates the AAA family members into multiple subfamilies (Beyer, 1997). The N-terminal domains are highly variable and they act as adaptors for diverse arrays o f binding partners.

The AAA proteins are believed to function in a manner analogous to that o f the cellular chaperones (i.e., the GroEL-GroES complex), that have been implicated in folding polypeptide chains into their correct three-dimensional structure. In many cases, such as for NSF and p97, the AAA domains assemble into oligomeric rings, which are likely to change their structure during the ATPase cycle. This nucleotide-dependent conformational switch may generate a force that drives the remodelling, or alters the oligomerisation state o f effector molecules by using the free energy generated by ATP- hydrolysis (Patel and Latterich, 1998). This idea was supported by the finding that the trimeric AAA domain o f Ymel (residues 250-525), a mitochondrial protease, binds unfolded polypeptides and suppresses their aggregation, indicating that this AAA domain indeed has a chaperone-like activity (Leonhard et al., 1999).

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1.5.1 N S F (N-ethylmaleimide-sensitive factor)

NSF (SeclSp in yeast) was the first member o f the AAA family to be implicated in the process o f membrane fusion (Block et a l, 1988). It was purified on the basis o f its ability to support transport between Golgi cistemae (M alhotra et al., 1988; Wilson et al., 1989) and then shown to be involved in many other vesicle-mediated transport steps on the exocytic and endocytic pathways in organisms from yeast to man (Rothman, 1994).

1.5.1.1 Structure/function relationship of NSF

Limited proteolysis o f NSF demonstrated that each subunit is divided into three distinct domains: the N-terminal (N) domain (residues 1-205) and two homologous ATP binding domains, the D1 domain in the middle o f the protein (206-477) and the D2 domain at the C-terminus (478-744) (Tagaya et al., 1993). The N domain is required for interactions with SNAP-SNARE complexes but must be contigous with the D1 or D2 domain (Nagiec et al., 1995). The D1 domain is involved in remodelling these complexes, whereas the D2 domain determines the hexameric state o f the protein (Nagiec et al., 1995; Whiteheart et al., 1994).

The structure/function relationship o f the conserved boxes within the AAA cassette o f the protein, namely the Walker motifs A and B (Walker et al., 1982) and the AAA- m otif (Beyer, 1997), were further illuminated using NSF mutants with targeted residues in these motifs. Analysis o f NSF mutants with mutations in the Walker A m otif (K266A) and Walker B m otif (E329Q) o f the D1 domain demonstrated that A TP binding by this domain is necessary for interaction with SNAP-SNARE complexes, whereas ATP hydrolysis is essential for the dissociation o f SNAP-SNARE complexes (M atveevaet al., 1997; Nagiec et al., 1995). D l-N SF mutants were unable to promote intra-Golgi transport (Whiteheart et al., 1994) and inhibited endosomal fusion (Colombo et al., 1996) and intra-Golgi transport (Nagiec et al., 1995; Sumida et al.,

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1994) almost completely. Interestingly, membrane fusion can be rescued by addition o f w tN SF, however, only when added at early times during the fusion reaction (Colombo et al., 1996; Sumida et al., 1994). Together, this demonstrates that NSF function, e.g. remodelling SNAP-SNARE complexes, is controlled by the dynamics o f its D1 domain.

Analysis o f NSF mutants with mutations in the D2 domain revealed that ATP binding, but not hydrolysis is necessary for the hexamerisation o f NSF (Whiteheart et al.,

1994). A mutation in the Walker A m otif (K549A, K557M/Q) or the Walker B m otif (D604Q) o f this ATP binding site inhibited membrane fusion slightly (Colombo et al.,

1996; Sumida et al., 1994).

A mutation in the A A A -m otif o f NSF {secl8-109; T394P) causes abnormal membrane trafficking in yeast and revealed that the AA A -m otif may be involved in regulating ATP hydrolysis o f NSF as mutants were defective in hydrolysing ATP (Steel et al.,

2000).

N SF’s two homologous ATP-binding domains, the D1 and D2 domains, differ in their nucleotide-binding properties: the D2 domain has a high affinity for ATP whereas the ATP-affmity o f the D1 domain is 1000-fold lower (Matveeva et al., 1997). NSF is a slow, intrinsic, NEM -sensitive ATPase with a specific activity o f ~4pmol/hr/mg at p H 9.0 (Tagaya et al., 1993). A mutation in the Walker B box m otif o f the D1 (E329Q) or D2 domain (D604Q) o f the protein causes a decreased ATPase activity o f 69% and 28% respectively, suggesting that the D1 domain accounts for the major portion o f N SF’s ATPase activity (Whiteheart et al., 1994). SNAP proteins have been shown to stimulate the ATPase activity o f NSF suggesting that they serve as N SF activators. For instance, a/y-SNAPs that are pre-bound to plastic stimulate the ATPase activity o f NSF by increasing the affinity for ATP (decreasing the Km) o f the D1 domain -100- fold to a value well below the cellular ATP level (Morgan et al., 1994; Painter et al., 1989; Steel and Morgan, 1998), which would sensitise the D1 domain o f NSF to physiological ATP concentrations. A complex consisting o f a-SN A P and the synaptic t-SNARE syntaxin-1 further stimulates the ATP-hydro lysis rate o f NSF (Haynes et

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al., 1998). Intriguingly however, a SNAP-SNARE complex containing v- and t- SNAREs was unable to enhance N SF’s activity (Matveeva and Whiteheart, 1998). Interestingly, it has been shown that the stimulation o f N SF’s ATPase activity, e.g. ATP-binding by the D1 domain o f NSF, correlates with the dynamics o f SNAP- SNARE complexes and membrane fusion. This suggests that, transmitted via SNAP proteins, the D1 domain o f NSF is responsible for regulating N SF’s activity (Barnard et al., 1997; Matveeva et al., 1997; Nagiec et al., 1995).