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First published online June 20, 2006
doi: 10.1242/10.1242/jcs.03018

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Active and passive displacement of transmembrane domains both occur during opsin biogenesis at the Sec61 translocon

Faculty of Life Sciences, University of Manchester, The Michael Smith Building, Oxford Road, Manchester, M13 9PT, UK

View larger version (63K): [in a new window]   Fig. 1. TM1 displays a biphasic association with the Sec61 complex. A schematic of ribosome-bound integration intermediates is presented above each set of products. The dashed line represents the transmembrane domain (TM), the solid line indicates the hydrophilic portion of the nascent chain and the star denotes the cysteine probe within TM1. Each integration intermediate was synthesised in a rabbit reticulocyte lysate translation system in the presence of [35S]Met/Cys and semi-permeabilised HT1080 cells. Membrane-associated integration intermediates were isolated by centrifugation and crosslinking performed by the addition of the homobifunctional reagent, BMH. Opsin-derived nascent chains were immunoprecipitated using either a monoclonal antibody specific for the N-terminus of the polypeptide (Op), or for the HA epitope on the C-terminus of the integration intermediates (HA). For each chain length, authentic doubly N-glycosylated opsin chains are indicated by an open arrowhead and their non-glycosylated counterparts by an open circle. Shorter opsin chains that lack the C-terminal HA epitope tag are indicated by asterisks. Adducts to Sec61 and Sec61ß were identified by immunoprecipitation with appropriate antisera (denoted and ß). P indicates adducts to an unidentified protein of 10 kDa, denoted PAT-10 (lanes 13, 18, 23 and 28). Vertical black lines indicate sets of samples resolved on different SDS-polyacrylamide gels. Positions of molecular size markers are indicated to the left.

View larger version (67K): [in a new window]   Fig. 2. The exit of TM1 from the ER translocon is facilitated by C-terminal TMs in the opsin nascent chain. A set of integration intermediates containing only opsin TM1 with a single cysteine probe [Cys56] were analysed by crosslinking with BMH as described for Fig. 1. The remainder of the opsin sequence including the C-terminal TMs was replaced with hydrophilic sequence derived from preprolactin. Nascent chain lengths identical to those studied with opsin were investigated, and all symbols are as defined in the legend to Fig. 1.

View larger version (103K): [in a new window]   Fig. 3. TM2 exit from the Sec61 complex. BMH-dependent crosslinking was performed using opsin integration intermediates of 140, 164 and 204 residues containing a single cysteine probe at position 89 within TM2 (Meacock et al., 2002). All symbols are as previously defined.

View larger version (93K): [in a new window]   Fig. 4. A scanning analysis reveals that TM3 is adjacent to ER translocon components in the OP164 intermediate. Membrane associated OP164 integration intermediates with a single cysteine probe at residues 115, 124 or 132 within TM3 were treated with BMH and the resulting adducts analysed following immunoprecipitation as previously described. Schematic representations of the integration intermediates with the relative location of the cysteine probe (indicated by an asterisk) are presented above the gels and all symbols are as previously stated.

View larger version (75K): [in a new window]   Fig. 5. TM3 moves out of the ER translocon upon chain extension. (A) Membrane-associated integration intermediates of OP150 to OP259 containing a single cysteine probe in TM3 (residue 115) were treated with BMH and the resulting adducts analysed following immunoprecipitation as previously described. An adduct with a putative ribosomal protein of 21 kDa was observed with OP150[Cys115] (panel A, lanes 2 and 3, labelled R) and in some cases adducts containing both Sec61 and Sec61ß were seen (labelled ß). All other symbols as previously defined. (B) Membrane-associated integration intermediates of OP204 containing a single cysteine probe in TM4 (residue 154) were analysed as for panel A. A novel adduct was observed with the OP204[Cys154] intermediate (panel B, lanes 2 and 3, filled circle), this remains to be fully characterised. Trace levels of radiolabelled Sec61 resulting from the translation of endogenous mRNA that sometimes remained after the nuclease treatment of the semi-intact cells (data not shown) can be seen in panel B, lane 5 (X).

View larger version (114K): [in a new window]   Fig. 6. Opsin TM3 exit from the ER translocon is independent of subsequent TM domains. OPTM1-3PPL was constructed by replacing the region of opsin located on the C-terminal side of TM3 with hydrophilic sequence derived from preprolactin. Membrane associated integration intermediates of 164 and 204 residues with a single cysteine probe located in TM3 (residue 115, cf. Fig. 5A) were then subjected to BMH-dependent crosslinking and the resulting adducts recovered by immunoprecipitation and analysed by SDS-PAGE. All symbols are as previously defined.

View larger version (105K): [in a new window]   Fig. 7. Double probe analysis of the OP164 and OP204 intermediates. Integration intermediates containing two cysteine probes, the first in TM1 (residue 56) and the second in TM3 (residue 115), were analysed by the BMH-dependent crosslinking of two previously characterised chain lengths (OP164 and OP204). Adducts were identified by immunoprecipitation, and for OP164 a novel crosslinking product corresponding to the opsin nascent chains simultaneously crosslinked to both Sec61ß and PAT-10 was observed (lane 5, ß+P). All other symbols are as previously defined.

View larger version (28K): [in a new window]   Fig. 8. Model for the integration of opsin TMs1-4. A schematic version of the view from the cytosolic face of the ER membrane showing the presumptive `open' conformation of the Sec61 translocon is presented, based on the structure of the archaeal complex (Van den Berg et al., 2004). Only the and ß subunits of the complex are represented because these are the components that can be crosslinked to various opsin integration intermediates, whereas the PAT-10 protein is as previously described (Meacock et al., 2002). Phase I crosslinking to Sec61 is equivalent to the point where adducts with Sec61 and Sec61ß are seen. Phase II crosslinking is displayed by TM1 and represents specific adducts with Sec61 that appear after initial exit from the Sec61 complex. The relative location of each TM (numbered circle) is indicated at different stages of the integration process as deduced from the crosslinking patterns of specific integration intermediates. The fates of TM1 and TM3 when synthesised in the absence of the respective C-terminal TMs of the wild-type opsin chain are also illustrated.

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