Binding of signal recognition particle gives ribosome/nascent chain complexes a competitive advantage in endoplasmic reticulum membrane interaction

Abstract

Most secretory and membrane proteins are sorted by signal sequences to the endoplasmic reticulum (ER) membrane early during their synthesis. Targeting of the ribosome-nascent chain complex (RNC) involves the binding of the signal sequence to the signal recognition particle (SRP), followed by an interaction of ribosome-bound SRP with the SRP receptor. However, ribosomes can also independently bind to the ER translocation channel formed by the Sec61p complex. To explain the specificity of membrane targeting, it has therefore been proposed that nascent polypeptide-associated complex functions as a cytosolic inhibitor of signal sequence- and SRP-independent ribosome binding to the ER membrane. We report here that SRP-independent binding of RNCs to the ER membrane can occur in the presence of all cytosolic factors, including nascent polypeptide-associated complex. Nontranslating ribosomes competitively inhibit SRP-independent membrane binding of RNCs but have no effect when SRP is bound to the RNCs. The protective effect of SRP against ribosome competition depends on a functional signal sequence in the nascent chain and is also observed with reconstituted proteoliposomes containing only the Sec61p complex and the SRP receptor. We conclude that cytosolic factors do not prevent the membrane binding of ribosomes. Instead, specific ribosome targeting to the Sec61p complex is provided by the binding of SRP to RNCs, followed by an interaction with the SRP receptor, which gives RNC-SRP complexes a selective advantage in membrane targeting over nontranslating ribosomes.

Figure 1

SRP-independent membrane binding of ribosome/nascent chain complexes. (A) Photocrosslinking of pPL86 to SRP and membrane proteins. pPL86 was synthesized in the wheat germ system in the presence of modified lysyl-tRNA. Dog pancreatic SRP and PK-RM were added as indicated. The samples were UV-irradiated, separated by SDS-PAGE, and analyzed with a PhosphoImager. pPL86 × SRP54 stands for the cross-linked product containing SRP54. To identify the cross-links to membrane proteins (pPL86 × TRAM/Sec61α), immunoprecipitations with antibodies to Sec61α or TRAM, as well as control precipitations without antibodies were carried out (lanes 6–11). Arrowhead denotes a cross-linked product containing an unknown cytosolic protein. (B) Membrane targeting and translocation of pPL86. pPL86 synthesized in the wheat germ system was incubated with PK-RM and SRP as indicated. Some samples (lanes 2–4) were treated with proteinase K, the others (lanes 5–13) with puromycin. To test for translocation after puromycin treatment, the samples were incubated with proteinase K in the absence or presence of Triton X-100. pPL86-sp indicates the nascent chain fragment after signal peptide cleavage. Asterisk indicates the 30-amino acid fragment protected from proteolysis by the ribosome. (C) Determination of membrane targeting by flotation. pPL86 was incubated with different amounts of PK-RM in the absence of SRP. The samples were then layered under a sucrose gradient under low- (150 mM) or high- (500 mM) salt conditions and subjected to centrifugation. The floated and nonfloated fractions were analyzed by SDS-PAGE.

Figure 2

Inhibition of membrane binding of pPL86 by ribosomes. (A) Microsomal membranes were preincubated with increasing amounts of mock translation mixture lacking mRNA and amino acids. In a separate tube, pPL86 was synthesized in a wheat germ system and SRP was added where indicated. The two mixtures were then combined (the numbers indicate the fold excess of the mock translation mixture over that containing mRNA). After incubation, a protease protection assay was used to determine membrane targeting of pPL86. Lane 1 shows the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the position of the ribosome-protected fragment of about 30 residues. (B) A competition experiment similar to that in A was carried out. The mock translation mixture was separated into a ribosome pellet and a cytosolic supernatant. The original mixture and both subfractions were used in a sixfold excess over mRNA-containing translation mixture in the competition experiments.

Figure 3

Competition of RNCs with nontranslating ribosomes for membrane-binding sites. (A) Inhibition of SRP-independent targeting of isolated RNCs by ribosomes. pPL86 was synthesized in a wheat germ system and the RNCs were isolated under low- (150 mM) or high- (500 mM) salt conditions. As competitors, either a mock translation mixture was added or fractions containing the ribosome pellet or the cytosolic supernatant. In some experiments, ribosomes washed with high salt were used (high-salt ribosomes). Membrane binding to PK-RM was tested with a protease protection assay. Lanes 1 and 9 show the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the position of the ribosome-protected fragment of about 30 residues. (B) Dependence of targeting of RNCs on ribosomes and membrane-binding sites. pPL86 was synthesized in a wheat germ system and SRP was added where indicated. Some samples received a sixfold excess of a mock translation mixture. After addition of different amounts of PK-RM (given in Eq), membrane targeting was assayed by protease protection. Lane 1 shows the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the fragment of about 30 amino acids protected against the protease by the ribosome.

Figure 4

Ribosome inhibition of membrane targeting of RNCs with a mutated signal sequence in the absence and presence of SRP. A mutant form of pPL86 with a defective signal sequence (pPLΔ13–15) was synthesized in the wheat germ system. SRP and increasing amounts of a mock translation mixture (given as fold excess over the mRNA-containing mixture) were added as indicated. Membrane targeting was tested with a protease protection assay. Lane 1 shows the undigested pPL86 (total), all other samples were treated with proteinase K. The band designated with one asterisk is the ribosome-protected fragment of about 30 residues. The fragment indicated by two asterisks contains about 50 residues and is presumably an intermediate in the process of membrane insertion of pPL86.

Figure 5

SRP-independent targeting in the reticulocyte lysate system. PPL86 was synthesized in a reticulocyte lysate system and RNCs were isolated by sedimentation through a sucrose cushion containing a low-salt concentration. One-half of the sample was treated with NEM, the other remained untreated. Before membrane targeting, low-salt-washed reticulocyte ribosomes, which were also treated with NEM, were added in increasing amounts as indicated (given as fold excess over RNCs). Membrane targeting of the RNCs was tested with a protease protection assay. Lanes 1 and 7 show the undigested pPL86 (total), all other samples were treated with proteinase K. Asterisk indicates the ribosome-protected fragment of about 30 amino acids. Arrowheads denote a fragment slightly smaller than pPL86 that is presumably protected from proteolysis by a cytosolic protein.

Figure 6

Membrane targeting of RNCs with reconstituted proteoliposomes. (A) pPL86 was synthesized in the wheat germ system. Where indicated, SRP and increasing volumes of a mock translation mixture (given in fold excess over the mixture containing the RNCs) were added before addition of reconstituted proteoliposomes containing the Sec61p complex (Sec61p) and the SRP receptor (SR). Membrane targeting was tested with a protease protection assay. Lane 1 shows the undigested pPL86 (total), all other samples were treated with proteinase K. The band designated with an asterisk is the ribosome-protected fragment of about 30 residues. (B) An experiment similar to that in A was carried out with proteoliposomes containing only the Sec61p complex.