THE N-DOMAIN OF THE SIGNAL RECOGNITION PARTICLE 54-KDA SUBUNIT PROMOTES EFFICIENT SIGNAL SEQUENCE BINDING

The signal recognition particle 54-kDa subunit (SRP54) binds to the si gnal sequences of nascent presecretory and transmembrane proteins. Pre vious studies have shown that signal sequences bind to the C-terminal methionine-rich domain of the protein (M-domain), but have raised the possibility that either the N-terminal domain (N-domain) or the centra l guanosine triphosphatase module (GTPase-domain) also contribute to s ignal-sequence-binding activity. We have generated a series of N-domai n and GTPase-domain mutants to investigate this issue further. Mutatio ns in a conserved N-domain motif (ALLEADV) produced significant defect s in signal sequence binding that correlate with the severity of the m utation. The magnitude of the defect was independent of the preprotein substrate, which suggested that the mutations do not alter the specif icity of signal sequence recognition. The N-domain mutants also showed defects in promoting the translocation of presecretory proteins acros s the membrane of microsomal vesicles, but these defects appeared to b e a direct consequence of the reduction in signal-sequence-binding act ivity and not separate effects of the mutations. By contrast, mutation s in the guanosine triphosphatase consensus sequence had no effect on signal sequence binding, but instead severely impaired protein translo cation activity. These results indicate that a principal function of t he SRP54 N-domain is to promote efficient signal sequence binding. The se data also suggest that the SRP54 GTPase regulates the cycle of sign al sequence binding and release, perhaps by modulating the relative or ientation of the N- and M-domains.