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.