The signal recognition particle (SRP) is a ribonucleoprotein required for targeting a subset of nascent pre-secretory proteins to the endoplasmic reticulum membrane. Of the six SRP polypeptides, the most highly conserved is Srp54p, a modular protein consisting of an amino-terminal (N) domain of unknown function, a central GTPase (G) domain, and a carboxyl-terminal (M) domain implicated in the recognition of both signal sequences and SRP RNA. To identify regions of Srp54p that interact with other SRP subunits or regulatory proteins, we carried out systematic mutagenesis of the fission yeast homolog, principally using a "clustered charged-to-alanine" strategy. Of the 35 alleles examined, 13 are unable to support growth, two confer cold-sensitivity, five confer heat-sensitivity, and 15 produce no discernible phenotype. The lethal and conditional mutations map throughout the protein to several conserved regions, confirming that these motifs play critical roles in Srp54p function. The effects of the amino-acid substitutions are analyzed with reference to the recently determined tertiary structures of the N/G domain and the intact protein from a thermophilic bacterium.