Characterization of the SRP68/72 interface of human signal recognition particle by systematic site‐directed mutagenesis

Abstract

The signal recognition particle (SRP) is a ribonucleoprotein complex which is crucial for the delivery of proteins to cellular membranes. Among the six proteins of the eukaryotic SRP, the two largest, SRP68 and SRP72, form a stable SRP68/72 heterodimer of unknown structure which is required for SRP function. Fragments 68e 0 (residues 530 to 620) and 72b 0 (residues 1 to 166) participate in the SRP68/72 interface. Both polypeptides were expressed in Escherichia coli and assembled into a complex which was stable at high ionic strength. Disruption of 68e 0 /72b 0 and SRP68/72 was achieved by denaturation using moderate concentrations of urea. The four predicted tetratricopeptide repeats (TPR1 to TPR4) of 72b 0 were required for stable binding of 68e 0 . Site-directed mutagenesis suggested that they provide the structural framework for the binding of SRP68. Deleting the region between TPR3 and TPR4 (h120) also prevented the formation of a heterodimer, but this predicted alpha-helical region appeared to engage several of its amino acid residues directly at the interface with 68e 0 . A 39-residue polypeptide (68h, residues 570-605), rich in prolines and containing an invariant aspartic residue at position 585, was found to be active. Mutagenesis scanning of the central region of 68h demonstrated that D585 was solely responsible for the formation of the heterodimer. Coexpression experiments suggested that 72b 0 protects 68h from proteolytic digestion consistent with the assertion that 68h is accommodated inside a groove formed by the superhelically arranged four TPRs of the N-terminal region of SRP72.

A Novel Histone H4 Arginine 3 Methylation-sensitive Histone H4 Binding Activity and Transcriptional Regulatory Function for Signal Recognition Particle Subunits SRP68 and SRP72

“…SRP68 and SRP72 are part of the SRP, a particle critically important for targeting secretory and membrane proteins to ER. SRP68 and SRP72 were previously shown to form heterodimers independent of the SRP complex and are released from the SRP as a stable SRP68/72 that is essential for SRP-mediated protein targeting (27,29). Our pulldown and gel filtration assays provide compelling evidence that the SRP68/72 heterodimers, but not the SRP complex, binds the H4 tail.…”

“…The CFP-Lac-H4t plasmid was generated by cloning 2 tandem copies of oligonucleotides encoding the first 20 amino acids of human H4 N-terminal tail. The plasmids for in vitro synthesis of [ 35 S]Met-labeled SRP54, SRP68, and SRP68 and their respective deletion mutants have been described previously (27)(28)(29). To express SRP68 or SRP72 and their deletion mutants as Gal4(DBD) fusion proteins, the corresponding cDNAs were cloned into pCMV-Gal4(DBD) vector.…”

“…The SRP68/72 Heterodimer but Not the SRP Complex Binds to the H4 Tail-The observed binding of SRP68 and SRP72 to the H4 N-terminal tail peptide raised the question if these proteins bind to the H4 tail in the form of heterodimers or associated with the SRP complex (25,27). Western blot analysis demonstrated that, unlike SRP68 and SRP72, SRP9, SRP14, SRP19, and SRP54 did not bind to the H4 peptide (Fig.…”

“…The resulting targeting complex then docks to ER via interaction with the SRP receptor in a GTP-dependent manner (26). Previous studies have shown that SRP68 and SRP72 exist predominantly as a stable SRP68/72 heterodimer that is essential for SRP-mediated ER-targeting of proteins (27).…”

A Novel Terminator Primer and Enhancer Reagents for Direct Expression of PCR-Amplified Genes in Mammalian Cells

“…Despite many alternative procedures and improvements for E. coli cloning techniques, DNA sequencing and functional confirmation of the clones still requires laborious work and cost. Currently, studies require DNA manipulations of numerous genes in a genome, or numerous systematic mutations in a gene in molecular genetic studies, called systematic or genomewide approaches [34][35][36][37]. To meet these demands of systematic DNA manipulations, E. coli cloning techniques are certainly unsatisfactory.…”