We have investigated the molecular details of the membrane insertion of the multiple-spanning membrane protein opsin. Using heterobifunctional crosslinking reagents the endoplasmic reticulum (ER) proteins adjacent to a series of defined translocation intermediates were determined. Once the nascent opsin chain reaches a critical minimum length Sec61␣ is the major ER component adjacent to the polypeptide. Using a homobifunctional reagent, the cross-linking partners from a single cysteine residue in the nascent chain were analyzed. This approach identified chain lengthdependent cross-linking products between nascent opsin and a 21-kDa ribosomal protein, followed by Sec61␤ and finally with Sec61␣. Our data support a model where the sequential transmembrane domains of a multiple-spanning membrane protein are integrated at an ER insertion site similar to that mediating the insertion of single-spanning membrane proteins.

Signal sequences for insertion of proteins into the endoplasmic reticulum induce translocation of either the C-or the N-terminal sequence across the membrane. The end that is translocated is primarily determined by the flanking charges and the hydrophobic domain of the signal. To characterize the hydrophobic contribution to topogenesis, we have challenged the translocation machinery in vivo in transfected COS cells with model proteins differing exclusively in the apolar segment of the signal. Homo-oligomers of hydrophobic amino acids as different in size and shape as Val 19 , Trp 19 , and Tyr 22 generated functional signal sequences with similar topologies in the membrane. The longer a homo-oligomeric sequence of a given residue, the more N-terminal translocation was obtained. To determine the topogenic contribution of all uncharged amino acids in the context of a hydrophobic signal sequence, two residues in a generic oligoleucine signal were exchanged for all uncharged amino acids. The resulting scale resembles a hydrophobicity scale with the more hydrophobic residues promoting N-terminal translocation. In addition, the helix breakers glycine and proline showed a positiondependent effect, which raises the possibility of a conformational contribution to topogenesis.Proteins destined for the endoplasmic reticulum (ER) 1 are synthesized with a hydrophobic signal sequence of typically 10 -20 uncharged, mainly apolar amino acids. This sequence is recognized by the signal recognition particle, which targets the nascent chain-ribosome complex via the signal recognition particle receptor to the ER membrane (1). The ribosome binds to the translocon, a gated pore made of several copies of the heterotrimeric Sec61 complex (2-4). The signal sequence inserts into the translocon, specifically contacting Sec61␣, in a manner that leads to translocation of either the C terminus or the N terminus across the membrane (5). Cleaved signals of secretory and type I membrane proteins (e.g. glycophorin) and signal anchor sequences of type II membrane proteins (e.g. transferrin receptor) translocate the C-terminal sequence, whereas the reverse signal anchors of cytochrome P-450, microsomal epoxide hydrolase, and opsin, for example, translocated the N-terminal sequence. The end of the signal that is translocated is determined by several factors. Charged residues flanking the apolar segment of the signal influence the insertion process in a manner that induces the more positive end to stay on the cytoplasmic side (6, 7). However, the charge distribution is not generally sufficient to determine the orientation and to generate a unique topology (8, 9). Hydrophilic sequences N-terminal of the signal may inhibit their translocation if they fold in the cytosol before targeting is completed (10). Similarly, we have recently observed that glycosylation at sites near the signal sequence can influence topogenesis by glycan attachment to polypeptide segments that are transiently exposed to the ER lumen (11).In addition, the apolar segment of the ...

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