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Protein SRP68 of human signal recognition particle: Identification of the RNA and SRP72 binding domains

Department of Molecular Biology, University of Texas Health Science Center at Tyler, Tyler, Texas 75708, USA

(RECEIVED September 23, 2005; FINAL REVISION February 8, 2006; ACCEPTED February 27, 2006)

The signal recognition particle (SRP) plays an important role in the delivery of secretory proteins to cellular membranes. Mammalian SRP is composed of six polypeptides among which SRP68 and SRP72 form a heterodimer that has been notoriously difficult to investigate. Human SRP68 was purified from overexpressing Escherichia coli cells and was found to bind to recombinant SRP72 as well as in vitro-transcribed human SRP RNA. Polypeptide fragments covering essentially the entire SRP68 molecule were generated recombinantly or by proteolytic digestion. The RNA binding domain of SRP68 included residues from positions 52 to 252. Ninety-four amino acids near the C terminus of SRP68 mediated the binding to SRP72. The SRP68–SRP72 interaction remained stable at elevated salt concentrations and engaged 150 amino acids from the N-terminal region of SRP72. This portion of SRP72 was located within a predicted tandem array of four tetratricopeptide (TPR)-like motifs suggested to form a superhelical structure with a groove to accommodate the C-terminal region of SRP68.

Keywords: signal recognition particle; SRP; protein secretion; RNA binding domain; protein–protein interaction

Reprint requests to: Christian Zwieb, Department of Molecular Biology, The University of Texas Health Science Center at Tyler, 11937 US Highway 271, Tyler, TX 75708-3154, USA; e-mail: zwieb{at}uthct.edu; fax: (903) 877-5731.

Article published online ahead of print. Article and publication date are are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051861406.

Abbreviations: DTT, dithiothreitol; EDTA, ethylenediaminetetraacetic acid; EST, expressed sequence tag; ER, endoplasmic reticulum; IPTG, isopropyl--D-thiogalactopyranoside; LB, Luria-Bertani medium; MALDI-TOF, matrix assisted laser desorption/ionization time of flight; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; SDS, sodium dodecyl sulfate; SLS, sodium lauryl sarcosinate; SRP, signal recognition particle; TCA, trichloroacetic acid; TEV, tobacco etch virus; TFA, trifluoroacetic acid; TPR, tetratricopeptide repeat; Tricine, N-tris(hydroxymethyl)methylglycine.

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