Stimulation of the GTPase activity of the SRP–SR complex by RNC preparations. (A) GTPase assays were conducted in a physiological ionic strength buffer (buffer C, 150 mM K⁺) and contained pPL86 RNCs (all symbols) and were supplemented with 50 nM SR proteoliposomes (triangles and squares) and 50 nM SRP (squares). (B) RNCs and SRP–RNCs were assembled by translation of pPL86 mRNA in the absence or presence of SRP. The GTPase assays in buffer C (RNCs and SRP–RNCs) were supplemented with 25 nM SR proteoliposomes and 50 nM SRP as indicated. The assay designated LS lacked RNCs and was conducted in a low ionic strength buffer (buffer E, 50 mM K⁺). (C and D) GTPase assays in buffer C contained pG64 RNCs (C), ffluc77 RNCs (C), pPL86 RNCs (D), or mock-assembled RNCs (D) and were supplemented with 50 nM SR proteoliposomes and 50 nM SRP as indicated. (B–D) The GTPase activity was calculated using 0-, 3-, 6-, and 10-min time points.

Characterization of recombinant SR and SR subunit derivatives. (A) Diagrams of SRα, SRβ, and biotin and hexahistidine-tagged derivatives of SRα and SRβ. Segments of SRα are designated as follows: residues 1–151, red; residues 152–319, blue; N domain, yellow; G domain, magenta. Segments of SRβ are designated as follows: lumenal domain, white; TM span, black; G domain, magenta with diagonal hatching. The 13-kD biotinylation domain (bt) is designated by the green bar at the NH₂ terminus of the expression constructs. (B) Coomassie blue–stained gel lanes of (a) bt-SR (bt-SRβ + SRα), (b) bt-SRβ, (c) bt-SRα, (d) bt-SRαΔN, (e) His-SRβ, (f) His-SRβSRα₁₅₁, and (g) His-SRβSRα₃₁₉. Individual gel lanes were aligned relative to molecular mass markers. (C) Reconstitution of protein translocation activity of protease-digested microsomes (T₅K-RM) with bt-SR (d and e), bt-SRα (f and g), or bt-SRαΔN (h). The 12.5-μl reticulocyte lysate translation reactions were supplemented with either 0.5 eq of K-RM (lane b) or 0.5 eq of T₅K-RM (lanes c–h). The concentrations of the E. coli–expressed SR constructs was either 24 nM (lanes d, f, and h) or 72 nM (lanes e and g). pPL and prolactin (PL) were resolved by PAGE in SDS. (D) GTPase assays (1 μM GTP) of canine SR, bt-SR, bt-SRα, and bt-SRαΔN in buffer E (50 mM K⁺) were supplemented with 50 nM SRP as noted in the chart below the graphs. The concentration of the SR or SR subunits was 15 nM. (E and F) GTPase assays of canine SR, bt-SR, bt-SRα, bt-SRβ, and bt-SRαΔN in buffer C (150 mM K⁺) were supplemented with 50 nM SRP and/or 160 nM 80S ribosomes as noted in the chart below the graphs. The concentration of the SR or SR subunits was 40 nM.

Kinetics of ribosome binding to immobilized SR. (A–C) Binding of ribosomes or ribosomal subunits to the SR in buffer D (150 mM K⁺). (A) Association curves for binding of 1 nM 80S ribosomes to the following immobilized proteins: (a) bt-SR, (b) bt-SRα, (c) bt-SRβ, (d) bt-SRβ + 25 μM GTP, or (e) bt-SRαΔN. Binding of (f) 1 nM 80S ribosomes, (g) 1.7 nM 60S subunits, or (h) 2.4 nM 40S subunits to immobilized bt-SR. Ribosomes or ribosomal subunits were added at the 1-min time point. (B and C) Equilibrium binding of ribosomes (B) or 60S ribosomal subunits (C) to immobilized bt-SR. The insets show plots of k_on versus ligate concentration. (D) Cosedimentation of SR, SR subunits, and SRP with 80S ribosomes. Binding assays in buffer D were incubated for 5 min at 25°C and contained 50 nM SRP and/or 40 nM SR (or SR subunits) and 160 nM ribosomes as indicated. Individual assays contained the following proteins: (a) SRP plus bt-SR, (b) bt-SR, (c) SRP, (d) SRP plus bt-SR, (e and f) bt-SRαΔN, (g and h) bt-SRα, (i and j) bt-SRβ, (k and l) His-SRβ, (m and n) His-SRβSRα₃₁₉, or (o and p) His-SRβSRα₁₅₁. The assays were layered onto 50-μl cushions of 500 mM sucrose in buffer D and centrifuged for 10 min at 279,000 g_av using a TLA100 rotor. After centrifugation, the samples were separated into supernatant (S) and pellet (P) fractions and resolved by PAGE in SDS, and the SRP and SR were detected using antibodies to SRP54, SRα, or SRβ. Images from sequential immunoblots using polyclonal and monoclonal antibodies were combined in the bottom panel.

Inhibition of translocation activity by high ionic strength. (A) SRP–ribosome–op156 complexes were adjusted to final KOAc concentrations ranging between 50 and 500 mM and incubated for 40 min at 25°C with 1.2 eq (as defined in Walter et al., 1981) of PK-RM (top) or 1.2 eq of protease-digested PK-RM (C₁PK-RM) that lack intact SRα (bottom). After membrane-integrated forms of op156 were separated from nonintegrated op156 on alkaline sucrose gradients, the membrane pellets were analyzed by PAGE in SDS to resolve glycosylated (g-op156) and nonglycosylated (op156) polypeptides. (B) Membrane-integrated g-op156 was quantified with a phosphorimager and is expressed in units, where 100 U corresponds to the average of the 150 mM and 200 mM KOAc data points. The average and standard deviation were calculated using data from six experiments, one of which is shown in A.

80S ribosomes and 60S subunits stimulate the GTPase activity of the SRP–SR complex. (A–C) GTPase assays in buffer C (150 mM K⁺) were adjusted to 0.1% Nikkol. (A) GTPase assays either lacked (open squares) or contained pG64 RNCs (filled symbols) and were further supplemented with 50 nM SR (triangles and squares) and 50 nM SRP (squares). (B) GTPase assays contained 50 nM SR (circles) or 50 nM SR plus 50 nM SRP (squares), and the indicated concentration of 80S ribosomes. (C) GTPase assays contained 50 nM SR and were supplemented with 50 nM SRP, 160 nM 80S ribosomes, 120 nM 60S ribosomal subunits, or 220 nM 40S ribosomal subunits as indicated. (D) The KOAc concentration in buffer C (adjusted to 0.1% Nikkol) varied between 50 and 300 mM. The GTPase assays of 50 nM SRP and 50 nM SR were conducted in the presence (squares) or absence (triangles) of 160 nM 80S ribosomes. (B–D) The initial GTPase rates are based on 0-, 3-, 6-, and 10-min time points. GTPase rates for assays that contained an identical concentration of ribosomes or ribosomal subunits, but lacked both SRP and SR, were subtracted as background for each data point.

Kinetics of SRP binding to immobilized SR. (A) Association (a–e) and dissociation (a and b) curves for binding of SRP to the following proteins: (a) bt-SR, (b) bt-SRα, (c) bt-SRαΔN, (d) bt-SRβ, and (e) streptavidin. The association curves have been offset on the vertical axis for clarity. The biosensor cuvettes were equilibrated in buffer E (50 mM K⁺). Association curves were initiated at 0.5 min by the addition 22 nM SRP in the equilibration buffer, and data were collected for at least 6.5 min. Association curves for bt-SR and bt-SRα were truncated to show the dissociation curves. Dissociation curves for bt-SRα and bt-SR were initiated by replacing the SRP solution with equilibration buffer. (B) Equilibrium binding of SRP to immobilized bt-SR in buffer E containing 25 μM GTP. (C) Plots of k_on for SRP binding to bt-SR in the presence (solid symbols) or absence (open symbols) of 25 μM GTP. The binding experiments were conducted in buffer D (150 mM K⁺, triangles) or buffer E (squares). (D) Association and dissociation curves for binding of SRP to bt-SR in the presence of buffer E adjusted to (a) 25 μM GTP, (b) 25 μM GDP, or (c) 25 μM Gpp(NH)p. The curves have been offset on the vertical axis for clarity. Association curves were initiated at 1 min by the addition 60 nM SRP in the equilibration buffer, and data were collected for at least 6 min. Dissociation curves were initiated by replacing the SRP solution with equilibration buffer containing the appropriate nucleotide.

Binding of ribosomes to SR and Sec61 proteoliposomes. Increasing amounts of ¹²⁵I-labeled 80S ribosomes (0.03–0.65 pmol) were incubated with the SR proteoliposomes (A) or Sec61 proteoliposomes (B) in 50 mM TEA, 150 mM KOAc, 2.5 mM Mg(OAc)₂, 1 mM DTT. Proteoliposome-bound ribosomes were separated from free ribosomes by gel filtration chromatography as described in the Materials and methods.