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Bioinformatics for protein sequence, structure and function

Membrane proteins in the ER: escaping from the protein-conducting channel

In eukaryotic cells, most integral membrane proteins destined for one or other of the membranes along the secretory pathway are initially inserted into the ER membrane, from which they are further routed by vesicular transport (Rothman, 1996). A machinery related to the bacterial Sec complex is responsible for translocating secretory proteins into the lumen of the ER (Jungnickel et al., 1994; Schatz and Dobberstein, 1996), and is also utilized by the integral membrane proteins (High, 1995; Oliver et al., 1995). The only clear example of "Sec-independent" insertion into the ER is for the tail-anchored protein synaptobrevin, in which a short C-terminal tail at the end of its only transmembrane segment is translocated independently of the normal targeting and translocation machineries (Kutay et al., 1995; Whitley et al., 1996).

Both secretory and membrane proteins are targeted to the ER through an initial interaction with the cytoplasmic signal recognition particle (SRP) that primes the ribosome-nascent chain complex for further interactions with the SRP receptor on the cytoplasmic face of the ER followed by transfer to the Sec61p translocation complex (Rapoport, 1992). The ordered binding and recognition steps along this pathway are controlled by GTP binding, hydrolysis, and GDP-GTP exchange (Connolly and Gilmore, 1993; Miller et al., 1993; Rapiejko and Gilmore, 1994; Bacher et al., 1996).

Once the ribosome-nascent chain complex is bound to the Sec61p translocase, the chain is translocated co-translationally to the lumen where it is received by chaperones such as the hsp70 protein BiP that may help to pull the chain across the membrane (Schatz and Dobberstein, 1996). In addition to BiP, folding and oligomerization in the ER often depend on other chaperones such as the calnexin/calreticulin system (Helenius, 1994; Pind et al., 1994; Gelman et al., 1995; Peterson et al., 1995).

As for Sec-dependent membrane protein assembly in E. coli, the insertion of integral membrane proteins into the ER is controlled by four kinds of topogenic signals: signal peptides, signal-anchor sequences, reverse signal-anchor sequences, and stop-transfer sequences, Fig. 11. Signal peptides target proteins to the ER and initiate translocation of the following portion of the nascent chain, signal-anchor sequences have the same function but are not cleaved and end up anchoring the protein to the bilayer, reverse signal-anchor sequences also target to the ER but become oriented with their N-terminus in the lumen, and stop-transfer sequences abort ongoing translocation and then become embedded in the bilayer. Proteins with more than one transmembrane segment are thought to be weaved into the membrane starting from the most N-terminal topogenic signal in a co-translational, sequential process (Wessels and Spiess, 1998; Lipp et al., 2001), at least when the loops connecting the transmembrane segments are long (Gafvelin et al., 1996). Hydrophobic segments and positively charged flanking residues are the major features also of the eukaryotic topogenic signals, and the topological effects of charged residues are similar though not identical in the ER and in E. coli (von Heijne and Manoil, 1990; Gafvelin et al., 1996).

Recently, the steps through which a stop-transfer sequence is released from the translocase into the surrounding lipid have been studied in some detail using site-directed photochemical crosslinking (Do et al., 1996). An ordered sequence of steps has been postulated, starting with the hydrophobic stretch entering the putative Sec61a-channel, then being progressively moved out of the channel into a location where it contacts the TRAM protein (and possibly also phospholipids), and finally being released into the bilayer upon termination of translation. The possibility to extract ribosome-bound nascent chains but not fully synthesized proteins containing multiple transmembrane segments from the ER with urea has also been taken as an indication that integral membrane proteins are not fully released into the lipid bilayer until the ribosome disassembles from the translocase (Borel and Simon, 1996).

Generally speaking, insertion of membrane proteins into the ER is thus in many ways analogous to Sec-dependent insertion into the inner membrane of E. coli. In fact, sequences that as act as stop-transfer signals in one system often do also in the other (SŠŠf et al., 1996), and heterologously expressed membrane proteins often adopt the same topology in E. coli and in the ER (Hennessey et al., 1993). Whether there is a distinction in eukaryotic cells similar to the one between Sec-dependent and Sec-independent insertion in E. coli is not clear, except for the tail-anchored proteins mentioned above. One difference between prokaryotic and eukaryotic membrane proteins that may be relevant to this question is the observation that positively charged residues in lumenal loops in eukaryotic proteins are not as strongly selected against as in bacterial proteins (Wallin and von Heijne, 1995), suggesting that lumenal loops may depend on the Sec61p machinery for translocation.