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

Abstract

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 approximately 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.

Figure 1.

SDS-PAGE of purified human SRP68 separated on a 10% Tricine gel (left) and of polypeptides 68a, 68a′, 68b, 68c, 68c′, 68d′, 68e, and 68e′ separated on a 12.5% Tricine gel (right). Molecular mass markers (kDa) are indicated.

Figure 2.

Analysis of human SRP68 on 10%–40% sucrose gradients. (A) SRP68 prepared in urea (black bars) or SLS (gray bars). (B) Comigration of SRP68 (prepared in SLS; gray bars), recombinant SRP72 (white bars), and human SRP RNA (solid line). Fractions are numbered from 2 to 16. Proteins and RNA in the various fractions were analyzed by gel electrophoresis followed by measuring the intensities of the stained bands. The positions of the molecular weight marker proteins Fibrinogen (FBG, 340 kDa), Bovine Serum Albumin (BSA, 78 kDa), and Lysozyme (LYZ, 16 kDa) are indicated. Other arrowheads show the mobilities of the free SRP68/72 heterodimer (68/72) and of purified SRP72 (72) that were analyzed in parallel. (C) Secondary structures diagrams of human SRP RNA (left) and the Δ35nwt mutant RNA (right) (Larsen and Zwieb 1991; Iakhiaeva et al. 2005). Helices are numbered from 2 to 8; letters denote helical sections. Indicated are the positions of the two hinges (Halic et al. 2004; Zwieb et al. 2005) as well as the regions corresponding to the small (Alu) and the large (S) domain. Residues are numbered in increments of 10.

Figure 3.

RNA binding activities of the various SRP68 fragments as measured by sucrose gradient centrifugation. The gray bars indicate the distribution of polypeptides 68a′, 68c, 68c′, 68d′, 68e, and 68e′ in the presence of the Δ35nwt RNA (solid lines). The migration of free 68a′ is shown in the top left panel by the black bars.

Figure 4.

The RNA binding region of SRP68. (Top panel) Mild digestion of fragments 68a (left) and 68a′ (right) with V8 protease followed by the separation of the fragments on 15% polyacrylamide Tricine gels; the migration distances of 68a and fragments 68-1 to 68-7 are indicated. The arrowhead marks the 68-7 fragment of 6.5 kDa generated by the primary V8 protease cut. (Lower left panel) Comigration of fragments 68a (black bars) and 68a-1 (gray bars) with human SRP RNA (black line) during sucrose gradient centrifugation. (Lower right panel) Collective distribution of the proteolytic fragments 68-2 to 68-7 (gray bars) generated by V8 protease. As in the lower left panel, the black line indicates the mobility of human SRP RNA.

Figure 5.

Interactions of the protein binding site of SRP68 with fragments of SRP72. (A) The 68c polypeptide was mixed with SRP72 fragments 72a′, 72b′, 72c, or 72d and analyzed by sucrose gradient centrifugation. Fraction aliquots were analyzed by SDS-PAGE. (B) Testing the interaction between 68c′ and 72b′. (C) Testing for binding between the RNA binding domain of SRP68 (fragment 68a bound to human SRP RNA, indicated as 68a-R) and 72b′. Gradient fractions 3–15 are indicated in top of each panel.

Figure 6.

Binding of the N-terminal region of SRP72 to fragments of SRP68. (A) Fragments 68d, 68e, 68e′, 68f, fused to thioredoxin, and 72b′, separated on a 15% polyacrylamide Tris-glycine gel. Lanes labeled with plus signs indicate TEV protease digested samples. The black arrowhead marks the liberated 72b′ fragment; white arrowhead indicates the various SRP68 fragments fused to thioredoxin. Weak bands in the 28-kDa range correspond to the TEV protease. The right panel outlines the four fusion constructs indicating the start and stop codons of the plasmid as well as the position of the TEV cleavage site. Analysis of the TEV-cleaved fusion proteins by sucrose gradient centrifugation followed by the SDS-PAGE of the fractions showed degrees of binding as expressed in percent bound/free 72b′. (B) Pull-down assays with GST–72b′ and Thx–68e′. E. coli lysates analyzed on 10% Tris-Tricine SDS gels containing expressed GST–72b′ (lane 1) and Thx–68e′ (lane 2). (Lanes 3–5) Proteins in the wash of the GST-Sepharose column; (lane 6) coelution of GST–72b′ and Thx–68e′ using 20 mM glutathione; (lane 7) control reaction lacking GST–72b′ proving that Thx–68e′ has no intrinsic affinity for GST-Sepharose.

Figure 7.

The protein binding region of SRP72. Separation of a mixture of polypeptide 68c with fragments generated by mild digestion of 72b′ with chymotrypsin followed by analysis of the sucrose gradient fractions by SDS-PAGE. Fractions 4–15 are indicated on the top. Molecular weight markers in kDa are indicated on the left.

Figure 8.

Features of SRP68/72. For both proteins, amino acid positions are given in increments of ∼50 residues. Amino acid residues that are invariant or highly conserved in the multiple sequence alignments (provided as Supplemental Material Sup1-SRP68.pdf and Sup2-SRP72.pdf) are shown above the lines. Helices (red) and β-structures (green) as predicted by jpred (Cuff and Barton 2000) are shown below the lines. Annotated in blue are the glycine cluster at the N terminus of SRP68 and the four predicted TPR-like motifs (Blatch and Lassle 1999) with their divisions into helices a and b. The wavy brackets designate the regions responsible for binding to SRP RNA and for the binding of the two proteins to each other.