Jun 14, 1994 - Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse. 10, 13125 Berlin-Buch and lInstitute for Biochemistry, Humboldt-.
The EMBO Journal vol. 13 no. 17 pp.3973 - 3982, 1994
Systematic probing of the environment of a translocating secretory protein during translocation through the ER membrane Walther Mothes, Siegfried Prehn1 and Tom A.Rapoport2 Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Strasse 10, 13125 Berlin-Buch and lInstitute for Biochemistry, HumboldtUniversity Berlin, Hessische Strasse 3-4, 10115 Berlin, Germany 2Corresponding author Communicated by T.A.Rapoport
We have extended a previously developed photocrosslinking approach to systematically probe the protein environment of the secretory protein preprolactin, trapped during its transfer through the endoplasmic reticulum membrane. Single photoreactive groups were placed at various positions of nascent polypeptide chains of various length, corresponding to different stages of the transport process, and photocrosslinks to membrane proteins were analyzed. In all cases, the polypeptide segment extending from the ribosome was found to be located in a membrane environment that is formed almost exclusively from Sec6l1c, the multi-spanning subunit of the Sec6lp complex that is essential for translocation. At early stages of the translocation process, before cleavage of the signal sequence, almost the entire nascent chain emerged from the ribosome contacts Sec6la. The 'translocating chain-associating membrane' protein interacts mainly with the region of the signal sequence preceding its hydrophobic core. Our results suggest that the nascent chain is transferred directly from the ribosome into a protein-conducting channel, the major constituent of which is Sec6la. Key words: endoplasmic reticulum/photocrosslinking/ protein translocation/Sec6l/translocating chain-associating membrane protein
Introduction Protein transport across the endoplasmic reticulum (ER) membrane is initiated in general by an interaction of the signal sequence of a growing nascent polypeptide with the 54 kDa polypeptide component of the signal recognition particle (SRP54) [for a review see Rapoport (1992)]. Upon binding of the entire complex of ribosome, nascent chain and the SRP to the SRP receptor (docking protein) of the ER membrane, the ribosome becomes membranebound and the nascent polypeptide chain is transferred into the membrane. For secretory proteins and many membrane proteins it is assumed that the nascent chain is inserted in a loop structure into the membrane, with the N-terminus remaining in the cytoplasm and with one part of the hairpin being the signal sequence. The subsequent © Oxford University Press
movement of the C-terminal portion of the hairpin is postulated to occur through a hydrophilic protein-conducting channel that is formed at least in part from transmembrane proteins (Blobel and Dobberstein, 1975). The idea of a channel is supported by electrophysiological data that indicate the occurrence of large ion-conducting channels following the release of the nascent chains from membrane-bound ribosomes (Simon and Blobel, 1991). The inside of the channel may have a hydrophilic environment, as determined by measurement of the fluorescent life-time of probes incorporated into translocating polypeptide chains (Crowley et al., 1993). The translocation apparatus of the ER membrane seems to be surprisingly simple; in some cases only two protein components are required for the translocation of polypeptides into reconstituted proteoliposomes (Gorlich and Rapoport, 1993): (i) the SRP receptor (two subunits; Tajima et al., 1986), which is probably required only for the targeting of a nascent chain to the ER but not for its actual transfer through the membrane; and (ii) the Sec6lp complex (three subunits). The a-subunit of the Sec6lp complex is a good candidate to be a major component of the postulated protein-conducting channel. It probably spans the membrane 10 times and has in these putative membrane-spanning segments several hydrophilic and charged amino acids that could contribute to the formation of a hydrophilic channel (Gorlich et al., 1992b). Translocating polypeptides can be crosslinked to Sec6la both in yeast and mammalian microsomes at different stages of their membrane passage (Gorlich et al., 1992b; Musch et al., 1992; Sanders et al., 1992; High et al., 1993a). The translocation of most polypeptides also requires the presence of a third membrane component, the translocating chain associating membrane (TRAM) protein (Gorlich et al., 1992a; Gorlich and Rapoport, 1993). The TRAM protein may be in contact with nascent chains only at early phases of their transfer through the membrane before the signal sequence has been cleaved off (Gorlich et al., 1992a). Although the previous crosslinking experiments have shown that Sec6la and the TRAM protein are close to polypeptides which are in transit through the ER membrane, the exact molecular environment that polypeptides meet during their passage through the membrane is unknown. So far, in most experiments several sites of the polypeptide chain could be involved in crosslinking. Photocrosslinking experiments, with photoreactive lysine derivatives incorporated into nascent chains, generally employed translocation substrates which contained several lysine residues that could carry the probes. Crosslinking with bifunctional chemical reagents (Gorlich et al., 1990, 1992a; Kellaris et al., 1991) also lacked specificity with respect to the interacting site in the translocating polypeptide. It is therefore not even certain that it is the 3973
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membrane-inserted region of a translocating polypeptide that contacts Sec6la or the TRAM protein. To overcome these problems it would be desirable to position precisely photoreactive chemical groups in a translocating polypeptide chain. Two approaches may be considered. (i) One may use the 'classic' method, employing photoreactive lysine derivatives incorporated into a nascent polypeptide chain synthesized in vitro in the presence of modified lysyl-tRNA (Krieg et al., 1986; Kurzchalia et al., 1986) under conditions in which only one lysine codon is present in the mRNA. (ii) The probes may be incorporated at sites defined by stop codons in the mRNA, which are subsequently suppressed with suppressor-tRNA charged with a photoreactive amino acid derivative. The feasibility of the second approach has already been demonstrated by the incorporation of photoreactive phenylalanine derivatives at a few positions of short preprolactin chains of 86 amino acids (High et al., 1993b). The results indicated that the TRAM protein interacts with amino acid residues preceding the hydrophobic core of the signal sequence, whereas Sec6la interacts primarily with the core and with residues succeeding it. Further application of this method awaits the more general availability of the reagent. Also, there is some uncertainty as to whether stop codons can be suppressed with the same efficiency at every position of the polypeptide chain. Using the 'classic' photocrosslinking approach, we have now carried out a systematic analysis of the membrane protein environment of preprolactin chains which were trapped during their transfer through the ER membrane. Single lysine codons were placed in the mRNA at selected sites so that photocrosslinking could only occur from known positions of the polypeptide chain. Employing different translocation intermediates, it could be demonstrated that the region of the polypeptide chain preceding the ribosome-buried portion gives almost exclusively crosslinks to Sec6la, suggesting that the latter forms a protein-conducting channel. Our results also suggest that the nascent chain is inserted initially into the membrane in a loop structure in which the N-terminal part of the signal sequence contacts the TRAM protein, whereas the rest of the membrane-incorporated chain is in proximity to Sec6lac. Additional membrane proteins seem to contact the nascent polypeptide chain at the lumenal side of the membrane.
Results Experimental strategy To position precisely photoreactive lysine derivatives in preprolactin polypeptide chains trapped during translocation, we first removed all lysines of the wild type protein, up to position 154, yielding a 'lysine null' mutant (N) that is not expected to incorporate photoreactive groups (Figure lA). Subsequently, single lysine codons were introduced at various selected positions by site-directed mutagenesis (schemes in Figure lB and C). Thus, the photoreactive probes could be placed in a systematic manner into the nascent chain, starting with positions expected to be in the ribosome up to locations in the lumen of the ER membrane. In initial experiments, a late stage of translocation was
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