Two translocating hydrophilic segments of a nascent chain span the ER membrane during multispanning protein topogenesis

Abstract

During protein integration into the endoplasmic reticulum, the N-terminal domain preceding the type I signal-anchor sequence is translocated through a translocon. By fusing a streptavidin-binding peptide tag to the N terminus, we created integration intermediates of multispanning membrane proteins. In a cell-free system, N-terminal domain (N-domain) translocation was arrested by streptavidin and resumed by biotin. Even when N-domain translocation was arrested, the second hydrophobic segment mediated translocation of the downstream hydrophilic segment. In one of the defined intermediates, two hydrophilic segments and two hydrophobic segments formed a transmembrane disposition in a productive state. Both of the translocating hydrophilic segments were crosslinked with a translocon subunit, Sec61alpha. We conclude that two translocating hydrophilic segment in a single membrane protein can span the membrane during multispanning topogenesis flanking the translocon. Furthermore, even after six successive hydrophobic segments entered the translocon, N-domain translocation could be induced to restart from an arrested state. These observations indicate the remarkably flexible nature of the translocon.

Figure 1.

Two intermediates of N-domain translocation generated by SBP-tag trapping. (A) The SBP-tag (SBP) and the glycosylation sequence were fused to the N terminus of SytII (S-I). A 38-residue spacer sequence was inserted between the glycosylation probe sequence and SytII (S-38-I). Glycosylation sites are indicated by open circles. Truncated mRNAs encoding to Arg²⁰⁰ of SytII in the fusion proteins were used for in vitro translation. Numbers indicate the amino acid residues within the indicated regions. The amino acid sequence of the SBP-tag is indicated. (B) The truncated mRNA for the S-I fusion protein was translated in the cell-free system in the absence (−) or presence (+) of RM, SAv, and biotin for 60 min, and then further incubated for 15 min in the presence of CHX. Some aliquots translated in the presence of SAv were further incubated in the presence (+) or absence (−) of biotin for 60 min. Lane 4 shows the translation products in the presence of SAv and biotin (SAv +/B). An aliquot was treated with EndoH (EndoH + lane). Filled circles indicate the nonglycosylated form. Single and double open circles indicate monoglycosylated and diglycosylated forms, respectively. Diglycosylation efficiencies (%) are indicated. (C) The fusion proteins were synthesized in the presence of RM and SAv. After CHX treatment, N-domain translocation was chased for the indicated time in the presence of biotin. Diglycosylation efficiencies (%) are indicated in the panel. (D) N-domain translocations of S-I and S-38-I proteins were arrested at different stages by SAv. Neither glycosylation site of S-I was glycosylated, whereas the second site of S-38-I was glycosylated. In both cases, N-domain translocation resumed during the biotin chase. In the S-38-I intermediate, the SA-I sequence and the preceding hydrophilic segment spanned the membrane (advanced stage). Insertion of the S-I protein was arrested at an earlier stage than S-38-I protein (earlier stage). Open and filled circles indicate the glycosylated and nonglycosylated potential sites, respectively.

Figure 2.

Earlier intermediate does not influence the next insertion. (A) The SA-I sequence in the S-I protein was followed by the second H-segment (II) of the TM3 in human Na⁺/H⁺ exchanger isoform 6, the glycosylation probe sequence (third open circle), and a hydrophilic sequence from bovine prolactin (PL) (S-I-II). The numbers of residues within the indicated regions are shown. In the S-I-II(2G) protein, the second glycosylation site was silenced. (B) The truncated RNA was translated and N-domain translocation was chased as described in Fig. 1. Filled circles indicate nonglycosylated forms. Single, double, and triple open circles indicate mono-, di-, and triglycosylated forms, respectively. The diglycosylation efficiencies of S-I-II(2G) are indicated in the panel. (C) While the N-domain translocation was in the earlier stage, the second H-segment (II) and the following hydrophilic sequence spanned the membrane. The N-domain translocation was chased despite the presence of the downstream translocating polypeptide chain.

Figure 3.

Two translocating hydrophilic segments span the membrane. (A) The SBP-tag of S-I-II was separated from SA-I by a 38-residue spacer (S-38-I-II). As a third H-segment, 15 residues in the indicated region were replaced with 15 leucine residues (S-I-II-15L). Because the 15-leucine segment is 50 residues away from the truncation site, it should be in the translocon. (B) Translation and the translocation chase were performed as described in Figs. 1 and 2. (C) In the presence of SAv, two hydrophilic segments of S-38-I-II protein spanned the membrane and the two glycosylation sites, other than the N-terminal site, were glycosylated (open circles). Insertion of the hydrophobic 15-leucine segment did not interfere with the resumption of N-domain translocation.

Figure 4.

Productivity of the translocation intermediates. (A) The fourth glycosylation site was generated 40 residues away from the C-terminal truncation site of (4G) constructs. (B) After translation was performed for 60 min, the reaction was terminated by CHX or Puro and further incubated for 10 min. A biotin chase was then performed in the presence of CHX or Puro for 60 min. Translocations of both the N-domain and C-terminal segments resumed after release from the ribosome. The tetraglycosylated form was observed (four open circles) after Puro treatment. The third H-segment (15L) interrupts the translocation of the C-terminal segment, so that the fourth site is not accessible to the glycosylation enzyme. (C) S-I-II and S-38-I-II proteins were synthesized in the presence of SAv, and the N-domain translocation was chased in the presence of CHX or Puro. Triglycosylation efficiency (%) after the chase reaction is indicated. The translocation chase of the N-domain of the S-I-II protein was influenced by Puro treatment, whereas that of the S-38-I-II protein was not.

Figure 5.

Two translocating polypeptides flank Sec61α. (A and B) For site-specific chemical cross-linking, two Cys residues were created by point mutations at the indicated positions (numbered) using a Cys-less mutant of S-38-I-II. (C) The Cys mutants were synthesized in the presence of RM and SAv, and then subjected to chemical cross-linking using homobifunctional cross-linkers, BMH (+ lanes), whose cross-linking distances are 16.1 Å. Proteins cross-linked with the Sec61α were immunoprecipitated (IP Sec61α + lanes). Downward arrowheads indicate the products cross-linked with Sec61α. Asterisk indicates free probe products. Relative cross-linking efficiency was calculated by the formula: (immunoprecipitated band) × 100/(translation product). The efficiencies were normalized with that of position 3 and indicated in the panel. (D) Effects of Puro treatment and biotin chase on the cross-linking reactions were examined with positions-3 and -7 Cys mutants. After CHX, Puro, and/or biotin treatment, a cross-linking reaction was performed with BMOE. Immunoprecipitated bands were quantitated and normalized values were indicated in the panel.

Figure 6.

Insertion of six TM segments did not compete with N-domain translocation. (A) Six TM segments of rhodopsin were fused after the SA-I (S-I-Rhod). The endogenous glycosylation site in SytII was silenced. In the G-loop constructions, the glycosylation sequence (G-loop) was inserted either between TM2 and TM3 or TM4 and TM5 of rhodopsin. (B) The mRNAs truncated at the C-terminal residue (Ala³⁴⁸) of rhodopsin were translated in the presence of RM and SAv. The biotin chase was then performed. (C) Effect of Puro on the N-domain translocation resumption. Puro was used to terminate translation instead of CHX and then the biotin chase was performed. The glycosylated and nonglycosylated forms were quantitated and the glycosylation efficiencies were calculated. The experiments were performed more than three times, and the average and standard deviations are indicated in the figure. (D) Schematic of translocation intermediates. The arrest of N-domain translocation did not affect insertion of the following TM segments and insertion of six TM segments did not influence the resumption of N-domain translocation.

Figure 7.

Working models of possible arrangements of two translocating hydrophilic segments in the translocon. Vertical views of the translocon pore from the cytoplasmic side are shown. (A) Two Sec61 pores cooperate to accommodate polypeptide chains. Translocating polypeptide chains (a and b) are in different Sec61 pores. The two insertion signals (I and II) are recognized by different sites and the translocating chains do not compete with each other. Although the front-to-front dimer model is represented in the figure, the back-to-back configuration is equally possible. (B) A single Sec61 pore accommodates two polypeptide chains (a and b). The hydrophilic environment is enlarged.