Transmembrane segments of nascent polytopic membrane proteins control cytosol/ER targeting during membrane integration

Abstract

During cotranslational integration of a eukaryotic multispanning polytopic membrane protein (PMP), its hydrophilic loops are alternately directed to opposite sides of the ER membrane. Exposure of fluorescently labeled nascent PMP to the cytosol or ER lumen was detected by collisional quenching of its fluorescence by iodide ions localized in the cytosol or lumen. PMP loop exposure to the cytosol or lumen was controlled by structural rearrangements in the ribosome, translocon, and associated proteins that occurred soon after a nascent chain transmembrane segment (TMS) entered the ribosomal tunnel. Each successive TMS, although varying in length, sequence, hydrophobicity, and orientation, reversed the structural changes elicited by its predecessor, irrespective of loop size. Fluorescence lifetime data revealed that TMSs occupied a more nonpolar environment than secretory proteins inside the aqueous ribosome tunnel, which suggests that TMS recognition by the ribosome involves hydrophobic interactions. Importantly, the TMS-triggered structural rearrangements that cycle nascent chain exposure between cytosolic and lumenal occur without compromising the permeability barrier of the ER membrane.

Figure 1.

Nascent chain exposure to cytosol and lumen. In a quenching experiment, the initial net emission intensity (F₀) of a sample of ER microsome-bound RNCs with an NBD dye (red) located inside the ribosomal tunnel is measured (A) after purification by gel filtration. After addition of KI/KCl (B), the net intensity (F) is measured again to quantify the extent of collisional quenching by cytosolic I⁻. MLT is then added to create pores in the ER membrane (C), and the net intensity is remeasured to quantify the extent of quenching by I⁻ in both cytosol and lumen. (D) Ion flow through the aqueous translocon pore is prevented on the cytosolic side of the membrane by an ion-tight ribosome–translocon junction, and on the lumenal side of the membrane by BiP and a J-domain protein (Alder et al., 2005), acting directly (i) and/or indirectly (ii). (E) Nascent chain exposure to cytosolic I⁻ may result from conformational changes in the RTC (i) and/or by the dissociation of an RTC-associated protein(s) (ii). Depicted species are not drawn to scale.

Figure 2.

TMS2 control of RTC structure. (A) Protein primary structures are depicted to show the locations of topogenic sequences and the single lysine codon (red) in each. TMS1 = VSVG (green); TMS2 = opsin 2 (yellow); SS = pPL signal sequence (orange). (B) The entry of TMS1 into the ribosome tunnel opens the RTC junction and closes the lumenal end of the pore (i). When TMS2 moves into the tunnel (ii), does the RTC junction close and the lumenal end of the pore open? The cytosolic loop sequence after TMS1 is shown in red for the full-length PMP (iii). (C–F) Collisional quenching data for RTCs with the indicated nascent chains obtained before (red ▴) or after (black •) MLT addition. (G and H) Bar graphs showing the ΔK_sv values for the indicated nascent chain lengths of the indicated proteins. Standard deviations and n values are shown in Table I. Error bars indicate SD.

Figure 3.

TMS3 control of RTC structure. (A) The entry of TMS2 into the ribosome tunnel closes the RTC junction and opens the lumenal end of the pore (i). When TMS3 moves into the tunnel, does the RTC junction open and the lumenal end of the pore close (ii)? The lumenal loop sequence after TMS2 is shown in black, whereas the cytosolic loop sequences after TMS1 and TMS3 are shown in red for the full-length PMP (iii). (B and G) Protein primary structures are depicted to show the locations of topogenic sequences and the single lysine codon (red) in each. TMS3 = opsin 3 (magenta); others are as in Fig. 2 A. (C and D) Collisional quenching data for RTCs with the indicated nascent chains obtained before (red ▴) or after (black •) MLT addition. The straight lines coincide in D. (E, F, H, I, and J) Bar graphs show the ΔK_sv values for the indicated nascent chain lengths of the indicated proteins. Standard deviations and n values are shown in Table II. Error bars indicate SD.

Figure 4.

One-half of TMS2 did not integrate. Full-length 2INV_L53K1.5 was translated in the presence of ER microsomes, SRP, and [³⁵S]Met, then analyzed by SDS-PAGE. The insolubility in pH 11.5 carbonate shows that the proteins are integrated. Their orientation is N_lum-C_cyt because the signal sequence is cleaved, but the absence of higher-mass EndoH-sensitive bands shows that the three glycosylation sites in the C-terminal domain are not glycosylated and hence are in the cytosol.

Figure 5.

Integration of 2TM variants. Full-length 2INV_L64K2 (A) and 2DUP_L64K2 (B) were translated in the presence of ER microsomes, SRP, and [³⁵S]Met, then analyzed by SDS-PAGE. The insolubility in pH 11.5 carbonate shows that the proteins are integrated. The sensitivity of higher-mass bands to EndoH shows that the C-terminal domain was glycosylated and hence in the lumen.

Figure 6.

Cotranslational PMP integration. After RNC targeting to a translocon, a nascent signal-cleaved PMP in the aqueous ribosomal tunnel and translocon pore is sealed off from the cytosol by an ion-tight junction between the ribosome and translocon/membrane (i). The synthesis and entry of TMS1 into the tunnel triggers BiP-mediated closure of the lumenal end of the pore and the opening of the RTC junction (ii), thereby making the nascent chain inside the tunnel accessible to cytosolic but not lumenal I⁻. The entry of TMS2 into the ribosome tunnel reverses the RTC conformational and/or compositional changes elicited by TMS1. Hence, the tight RTC junction is reestablished and the lumenal end of the pore is opened (iii), thereby exposing the nascent chain inside the tunnel to lumenal but not cytosolic I⁻. The appearance of TMS3 in the tunnel reverses the changes elicited by TMS2, and the nascent chain in the tunnel becomes accessible to cytosolic but not lumenal I⁻ (iv). The lumenal loop sequence following TMS2 is shown in black, whereas the cytosolic loop sequences following TMS1 and TMS3 are shown in red for the full-length PMP (v).