Control of translocation through the Sec61 translocon by nascent polypeptide structure within the ribosome - PubMed (original) (raw)

Control of translocation through the Sec61 translocon by nascent polypeptide structure within the ribosome

Colin J Daniel et al. J Biol Chem. 2008.

Abstract

During polytopic protein biogenesis, multiple transmembrane segments (TMs) must pass through the ribosome exit tunnel and into the Sec61 translocon prior to insertion into the endoplasmic reticulum membrane. To investigate how movement of a newly synthesized TM along this integration pathway might be influenced by synthesis of a second TM, we used photocross-linking probes to detect the proximity of ribosome-bound nascent polypeptides to Sec61alpha. Probes were inserted at sequential sites within TM2 of the aquaporin-1 water channel by in vitro translation of truncated mRNAs. TM2 first contacted Sec61alpha when the probe was positioned approximately 38 residues from the ribosome peptidyltransferase center, and TM2-Sec61alpha photoadducts decreased markedly when the probe was >80 residues from the peptidyltransferase center. Unexpectedly, as nascent chain length was gradually extended, photocross-linking at multiple sites within TM2 abruptly and transiently decreased, indicating that TM2 initially entered, withdrew, and then re-entered Sec61alpha. This brief reduction in TM2 photocross-linking coincided with TM3 synthesis. Replacement of TM3 with a secretory reporter domain or introduction of proline residues into TM3 changed the TM2 cross-linking profile and this biphasic behavior. These findings demonstrate that the primary and likely secondary structure of the nascent polypeptide within the ribosome exit tunnel can influence the timing with which topogenic determinants contact, enter, and pass through the translocon.

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Figures

FIGURE 1.

εANB-dependent cross-linking to Sec61α. A, AQP1 cDNA was truncated at codon 134, translated in reticulocyte lysate in the presence or absence of εANB-Lys-tRNA, and analyzed by SDS-PAGE and autoradiography. Samples were treated with puromycin (Puro) and UV irradiation as indicated. Major bands with apparent sizes of 14 and 17 kDa represent non-glycosylated and glycosylated polypeptides, respectively. The_downward arrowhead_ (lane 6) shows the ∼55-kDa photoadduct. The upward arrowheads (lane 1) indicate residual peptidyl-tRNA bands that survived SDS-PAGE. These bands were independent of UV light and eliminated by both puromycin (lanes 3 and_4_) and RNase digestion (data not shown). B, irradiated samples were denatured and immunoprecipitated with Sec61α antisera (lanes 1–4) or nonimmune sera (NIS; lanes 5–8). The faint 55-kDa band in lane 1 represents background cross-linking to Sec61 due to direct UV irradiation in the absence of the εANB probe. There was also a small but variable amount of nonspecific adsorption of translation products to protein A beads (17-kDa band in lanes 1–3). A diagram of the construct is indicated below the autoradiogram. The Site of _N_-linked glycosylation (Asn42 (N42)) is indicated.

FIGURE 2.

Site-specific TM2-Sec61α cross-linking. Photocross-linking was performed as described in the legend to Fig. 1 on truncated wild-type AQP1 (WT; lanes 1–4); a lysine-less AQP1 mutant (lanes 5–8); and constructs containing a single Lys residue within TM2 at positions 59–61 (lanes 9–20). All samples were translated in the presence of εANB-Lys-tRNA and treated with puromycin (Puro) and/or UV light as indicated. Photoadducts (downward arrowheads) were observed only in chains containing εANB-Lys prior to puromycin release (lanes 3, 10, 14, and_18_). The diagram shows the predicted architecture of the arrested translocation intermediate. The locations of probes (black circles) relative to the ribosome PTC and predicted locations of TM2 and TM3 within the ribosome-translocon complex are indicated.

FIGURE 3.

TM2 cross-linking to sequentially arrested integration intermediates. Truncated AQP1 cDNAs containing a Lys codon at position 59, 60, or 61 were translated in rabbit reticulocyte lysate to generate a series of arrested translocation intermediates. The truncation site for each construct is indicated above the autoradiograms. Arrowheads indicate UV light-dependent photoadducts. The diagrams below each autoradiogram represent a model for the predicted location of TM2 at each nascent chain length. The distance of TM2 from the ribosome PTC (in aa) is shown. Note that TM2 is shown exiting the translocon into the ER lumen based on previous studies demonstrating that only TM1, TM3, TM5, and TM6 co-translationally span the ER membrane (–33, 51).

FIGURE 4.

Specific phases of TM2-Sec61α cross-linking. A, AQP1-Sec61α photoadducts were immunoprecipitated and analyzed by SDS-PAGE and autoradiography as described under “Experimental Procedures.” The sites of εANB probe incorporation and the lengths of integration intermediates are shown above the autoradiograms. The doublet observed at truncation 118 is due to partial _N_-linked glycosylation at Asn42 (31). Glycosylated photoadducts for shorter truncations, particularly I60K, are visible only upon longer exposure because of the low glycosylation and cross-linking efficiencies. B, photoadducts from two independent experiments were quantified by phosphorimaging, and the relative signal intensity at each probe site was plotted as a function of nascent chain length. The data show distinct phases of TM2-translocon interactions and asymmetry of Sec61α cross-linking for nascent chain lengths of 118–143 residues.

FIGURE 5.

TM2 transient withdrawal from Sec61α during AQP1 synthesis. Photocross-linking, immunoprecipitation, and quantification were performed as described in the legend to Fig. 4. Probes were positioned in AQP1 at residues 56–58, and truncations were moved three residues toward the N terminus to maintain the number of aa between the probe and PTC as in Fig. 4. A, autoradiograms of immunoprecipitated Sec61α photoadducts. B, relative photocross-linking efficiencies for each probe site plotted against chain length. C, normalized Sec61α photocross-linking efficiencies at truncations 102, 107, 115, and 123 (n = 4 ± S.E.). *, significant difference from truncation 102 (p < 0.05; Student's t test).

FIGURE 6.

Nascent chain structure within the ribosome influences TM2 entry into Sec61α. A, AQP1 residues C-terminal to Pro77 were replaced with a secretory passenger domain derived from preprolactin (31), and Sec61α photoadducts to residues 56–58 were analyzed by SDS-PAGE. Truncation sites in the passenger domain (arrows) were chosen to maintain the same number of residues between the probes and the PTC as described in the legend to Fig. 5. B, photoadduct quantitation revealed that replacing TM2 C-terminal flanking residues within the ribosome exit tunnel markedly shortened the duration of TM2 cross-linking (truncations 102–124) and eliminated transient withdrawal of TM2 at truncation 107. C and D, experiments were performed as described for A and B except that truncated AQP1 constructs contained C102P and V103P mutations in TM3. Proline mutations eliminated TM2 withdrawal at truncation 107 but had little effect on the timing of TM2 movement away from Sec61α. E, shown is normalized Sec61α photocross-linking to F56K at closely spaced truncations 102, 105, 107, 110, and 114 or 115 in the AQP1-prolactin (Prl) fusion protein and proline mutant.

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Control of translocation through the Sec61 translocon by nascent polypeptide structure within the ribosome - PubMed (original) (raw)