Protein translocation across the ER requires a functional GTP binding site in the alpha subunit of the signal recognition particle receptor.
The signal recognition particle (SRP)-mediated translocation of proteins across the RER is a GTP dependent process. Analysis of the primary amino acid sequence of one protein subunit of SRP (SRP54), as well as the alpha subunit of the SRP receptor (SR alpha), has indicated that these proteins contain predicted GTP binding sites. Several point mutations confined to the GTP binding consensus elements of SR alpha were constructed by site specific mutagenesis to define a role for the GTP binding site in SR alpha during protein translocation. The SR alpha mutants were analyzed using an in vitro system wherein SR alpha-deficient microsomal membranes were repopulated with SR alpha by in vitro translation of wild-type or mutant mRNA transcripts. SRP receptors containing SR alpha point mutants were analyzed for their ability to function in protein translocation and to form guanylyl-5'-imidodiphosphate (Gpp[NH]p) stabilized complexes with the SRP. Mutations in SR alpha produced SRP receptors that were either impaired or inactive in protein translocation. These SRP receptors were likewise unable to form Gpp(NH)p stabilized complexes with the SRP. One SR alpha point mutant, Thr 588 to Asn 588, required 50- to 100-fold higher concentrations of GTP relative to the wild-type SR alpha to function in protein translocation. This mutant has provided information on the reaction step in protein translocation that involves the GTP binding site in the alpha subunit of the SRP receptor.
Rapiejko
P J
PJ
Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester 01655.
Gilmore
R
R
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001693:Biological Transport, Active / Q000235:genetics; D016384:Consensus Sequence / Q000235:genetics; D004285:Dogs; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005136:Eye Proteins / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism*; D006165:Guanylyl Imidodiphosphate / Q000378:metabolism; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010179:Pancreas / Q000378:metabolism; D014176:Protein Biosynthesis; D011956:Receptors, Cell Surface / Q000235:genetics*; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D017299:Rod Opsins; D014158:Transcription, Genetic
D005136:Eye Proteins; D046911:Macromolecular Substances; D011956:Receptors, Cell Surface; D018160:Receptors, Cytoplasmic and Nuclear; D018000:Receptors, Peptide; D017299:Rod Opsins; C036318:signal peptide receptor; D006165:Guanylyl Imidodiphosphate; D006160:Guanosine Triphosphate