The Nterminal Variable Regions

In 1993 it was suggested that the variable N-terminal regions of S4-like ATPases are used to select substrates for degradation by the 26 S proteasome.39 The "helix-shuffle" hypothesis proposed that the N-terminal coiled-coils bound to unassembled substrates through their unpaired coiled-coil domains. For example, two known substrates of the 26 S proteasome, c-Fos and c-Jun, form heterodimers via leucine zippers. An S4-like ATPase could promote the degradation of c-Fos or c-Jun by binding unassembled monomers synthesized in excess. Recently, Wang et al demonstrated that c-Fos copurifies with 26 S proteasome particles and specifically binds the coiled-coil domain of

Fig. 7.2. Sequence alignment of regulatory complex S4-like ATPases. The amino acid sequences of the ATPase subunits of the RC were aligned using the program MegAlign (clustal method) of the DNASTAR™ package. Identical amino acids in at least four of the six sequences are shown white on black. Putative coiled-coil regions at the N-terminal portions of the proteins are shaded. The Walker-type consensi for nucleotide binding are labeled Box A and Box B (refer to text for details). Two regions of homology to DNA/RNA-dependent helicases are designated SAT and Helicase-like. A cysteine residue (CYS) near the C terminus is conserved in the sequences of S4-like ATPases of higher eukaryotes.

Fig. 7.2. Sequence alignment of regulatory complex S4-like ATPases. The amino acid sequences of the ATPase subunits of the RC were aligned using the program MegAlign (clustal method) of the DNASTAR™ package. Identical amino acids in at least four of the six sequences are shown white on black. Putative coiled-coil regions at the N-terminal portions of the proteins are shaded. The Walker-type consensi for nucleotide binding are labeled Box A and Box B (refer to text for details). Two regions of homology to DNA/RNA-dependent helicases are designated SAT and Helicase-like. A cysteine residue (CYS) near the C terminus is conserved in the sequences of S4-like ATPases of higher eukaryotes.

S8.53 This model, however, may oversimplify the mechanism of targeting and degradation of c-Fos by the 26 S proteasome. Tsurumi et al have reported that degradation of c-Fos is accelerated by c-Jun.54 This process requires both phosphorylation of c-Jun and an intact C-terminal PEST sequence in c-Fos.55,56 Once the signals for degradation have been added to the c-Fos/c-Jun dimer i.e., ubiquitin chains and phosphate groups, the dimer might be translocated to the 26 S proteasome.55,57 Subsequent interaction with the regulatory complex may involve a transient association between c-Fos and S8 and recognition of its PEST sequence. Thus, it is conceivable that the interaction of c-Fos and S8 represents an intermediate step in its degradation pathway or an alternate pathway restricted to unassembled c-Fos proteins.

The variable N-terminal regions in S4-like ATPases may serve another purpose. They could be involved in assembly of the RC by promoting the specific placement of the ATPase subunits in the complex. The six RC ATPases associate with one another in highly specific pairs. S4 binds S7, S6 binds S8, and S6' binds to S10b.33 We have also demonstrated that the N-terminal region of S4 is required for its specific binding to subunit 7.33 Progressive C-terminal deletions of S4 up to thr167 had no effect on its association with S7, but disruption of the putative coiled-coil region in S4 by removing 85 N-terminal amino acids abolished its binding to S7. Furthermore, we have constructed chimeric ATPases in which we have replaced the N-terminal region of S4 with the variable N-terminal regions of the remaining five ATPases in the complex (Fig. 7.3) (Gorbea, Taillandier, and Rechsteiner, in preparation). As predicted, the variable N-terminal region determined the binding specificity of the chimera towards a particular ATPase subunit. These findings coupled to the fact that specific interactions between ATPases persist after SDS-PAGE and transfer to nitrocellulose strongly suggest that coiled-coils are responsible for the specific association between ATPases. On the other hand, involvement of the N-terminal regions in assembly of the regulatory complex does not necessarily preclude their playing a role in substrate selection. Once they are incorporated into the RC, the N-terminal regions could become free to bind potential substrates, as postulated in the "helix-shuffle" hypothesis.

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