Recognition of a Transmembrane Domain: Another Role for the Ribosome?

Abstract

It has been known for some time that, in mammalian cells, most of the proteins that cross the membrane of the endoplasmic reticulum (ER) do so while they are being synthesized. For the past ten years or so, it has been clear that the “cotranslational” nature of the process reflected not so much a requirement for ongoing protein synthesis per se, but rather a requirement for the ribosome as a cofactor in the targeting and translocation reaction (see, for example,16Siegel V Walter P EMBO J. 1988; 7: 1769-1775Crossref PubMed Scopus (74) Google Scholar). Studies over the last several years have made it clear that the ribosome is crucially required for not just one but multiple steps in the translocation process. The ribosome was first shown to be a critical cofactor in the recognition of the signal sequences on ER-directed proteins by the signal recognition particle (SRP), a cytoplasmic ribonucleoprotein particle required for the targeting of most secretory and integral membrane proteins to the ER membrane (reviewed by18Walter P Johnson A.E Annu. Rev. Cell Biol. 1994; 10: 87-119Crossref PubMed Scopus (695) Google Scholar). SRP was found to bind to all ribosomes with moderate affinity and to ribosomes synthesizing secretory proteins with high affinity. The 54 kDa subunit of SRP can be cross-linked to the signal sequence of the secretory protein, but only when the signal sequence is exposed on the surface of the ribosome (as a ribosome–nascent chain complex). The involvement of the ribosome in the translocation of proteins across the ER membrane does not end with the recognition step. The ribosome also has binding sites at the ER membrane itself that are critical for translocation. The association of the ribosome with the ER membrane during translocation is very tight, as evidenced by the fact that nascent chains are rendered resistant to proteolysis even if detergents are added prior to protease treatment (1Connolly T Collins P Gilmore R J. Cell Biol. 1989; 108: 299-307Crossref PubMed Scopus (66) Google Scholar, 13Matlack K.E Walter P J. Biol. Chem. 1995; 270: 6170-6180Crossref PubMed Scopus (52) Google Scholar); indeed, the ribosome forms a tight seal with the ER membrane, as evidenced by the fact that small aqueous probes such as iodide ions cannot gain access to the nascent chain from the cytoplasmic side of the ER membrane (2Crowley K.S Liao S Worrell V.E Reinhardt G.D Johnson A.E Cell. 1994; 78: 461-471Abstract Full Text PDF PubMed Scopus (292) Google Scholar). The predominant ribosome binding protein at physiological salt concentrations is Sec61p (9Kalies K.-U Görlich D Rapoport T.A J. Cell Biol. 1994; 126: 925-934Crossref PubMed Scopus (144) Google Scholar), which is a major constituent of the translocon (4Görlich D Rapoport T.A Cell. 1993; 75: 615-630Abstract Full Text PDF PubMed Scopus (510) Google Scholar; 6Hanein D Matlack K.E.S Jungnickel B Plath K Kalies K.-U Miller K.R Rapoport T.A Akey C.W Cell. 1996; 87: 721-732Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar), the proteinaceous channel in the ER membrane through which secretory and integral membrane proteins move (17Simon S.M Blobel G Cell. 1991; 65: 371-380Abstract Full Text PDF PubMed Scopus (470) Google Scholar; reviewed by15Rapoport T.A Jungnickel B Kutay U Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (482) Google Scholar). Other ribosome binding components include p180 and p34, although it is controversial whether either of these proteins plays a role in translocation per se (for discussion, see15Rapoport T.A Jungnickel B Kutay U Annu. Rev. Biochem. 1996; 65: 271-303Crossref PubMed Scopus (482) Google Scholar). There also seems to be an additional ribosome binding activity associated with the ER membrane that can be detected when Sec61p is saturated (12Murphy III, E.C Zheng T Nicchitta C.V J. Cell Biol. 1997; 136: 1213-1226Crossref PubMed Scopus (22) Google Scholar). This site, which is also saturable, preferentially binds ribosome–nascent chain complexes (as opposed to inactive ribosomes) and may bind these complexes after targeting by SRP but before the association of the ribosome–nascent chain complex with Sec61p. Once the nascent chain reaches a certain length, the structure of the translocon changes, as evidenced by the fact that the nascent chain is now accessible to iodide ions on the lumenal side of the ER membrane. Because the lumenal side of the translocon is opened, the tight seal provided by the ribosome becomes critical, because it maintains the permeability barrier of the ER membrane. The pore itself is very large (EM structure of Sec61p in proteoliposomes gives an estimate of 20 Å6Hanein D Matlack K.E.S Jungnickel B Plath K Kalies K.-U Miller K.R Rapoport T.A Akey C.W Cell. 1996; 87: 721-732Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, while biophysical probes give an even larger estimate of 40–60 Å [Hamman et al., 1997]), and without the tight seal of the ribosome, not only ions but also quite large molecules would be expected to pass freely through it. It is thought that the ribosomal seal also provides a directionality to the translocation process, since nascent chains will only be able to exit the translocon on the lumenal side. Given that the ribosome binds tightly to the ER membrane, creating a sealed channel contiguous with the translocon through which the nascent secretory protein moves, the question arises as to whether the ribosome is simply an anchor helping to provide directionality to nascent chain movement, or whether the binding of the ribosome affects the translocon itself. Experiments using EDTA to release the ribosome have shown a reduction in the nascent chain cross-linking to Sec61, although the nascent chain is still able to translocate (14Nicchitta C.V Murphy III, E.C Haynes R Shelness G.S J. Cell Biol. 1995; 129: 957-970Crossref PubMed Scopus (34) Google Scholar). This suggests that, at least at later stages of translocation, release of the ribosome changes the translocon such that Sec61 no longer neighbors the nascent chain. Although it is clear that the ribosome stays associated with the membrane throughout translocation, there are conditions under which the ribosome no longer makes a tight seal with the membrane. For example, when the nascent chain contains a sequence that causes translocation to pause (known as a pause transfer sequence), the nascent chain transiently becomes accessible to proteases (7Hegde R.S Lingappa V.R Cell. 1996; 85: 217-228Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Thus, the association of the ribosome with the translocon seems to be regulated by the nascent chain. When secretory proteins are translocated across the ER membrane, the situation (mechanistically) is relatively simple. A tight seal has to be formed and then the nascent chain can be passed through the translocon to the lumenal side of the ER membrane. There are exceptions to the smooth transfer of the nascent chain, such as the case of pause transfer sequence briefly mentioned above, but in principle there needn't be any change to the translocation machinery or reorientation of the nascent chain with respect to the membrane. When integral membrane proteins are translocated across the ER membrane, the situation is much more complex. Cytoplasmic domains have to be left in the cytoplasm, lumenal domains have to be passed through the ER membrane, and transmembrane domains have to be properly oriented within the ER membrane. In order for these events to occur, it seems likely that the translocon changes as the nascent membrane protein is inserted. It would be extremely interesting to know whether and how the translocon changes to allow the insertion of a transmembrane domain and what components mediate these changes. In this issue of Cell, Art Johnson and colleagues (11Liao S Lin J Do H Johnson A.E Cell. 1997; 90 (this issue, 90,): 31-41Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar) have performed an elegant series of experiments aimed at addressing these questions. They synthesized a series of nascent chains of specific lengths that contained fluorescent probes at specific positions (in the example shown in Figure 1, they placed the fluorescent probe in the center of the transmembrane segment, as indicated by the diamond). They then asked whether the fluorescence from this probe could be quenched by the addition of iodide ions, which they added to the cytoplasmic side of the ER vesicles or to both sides (using a reagent that punched holes in the ER membrane). By quantitating the amount of quenching from one side or both sides, they could determine whether the nascent chain was exposed on the cytoplasmic or the lumenal side, or both. They monitored fluorescence quenching by iodide ions as a function of the length of the nascent integral membrane protein and of the position of the transmembrane segment relative to the translocon. In this minireview, we shall cover the case for only one of the integral membrane proteins they studied, but the results from other experiments are consistent with these results. First, consistent with previous work they had done with secretory proteins (2Crowley K.S Liao S Worrell V.E Reinhardt G.D Johnson A.E Cell. 1994; 78: 461-471Abstract Full Text PDF PubMed Scopus (292) Google Scholar), they found that once the nascent chain was long enough to be targeted to the ER membrane, the ribosome formed a tight seal with the translocon, sealing off the cytoplasmic side. When the chains were very short (or initially after targeting), the lumenal side of the translocon was also sealed. However, when the nascent chain reached a length of approximately 70 amino acids, the lumenal side of the translocon opened, presumably as a result of the signal sequence interacting with a component of the translocon. This state was maintained until shortly after the transmembrane domain was synthesized. Then an amazing thing happened (see Table 1 Figure 2).Table 1Exposure of the Nascent Chain to the Cytoplasm and ER Lumen Changes as the Transmembrane Segment Passes through the RibosomeDistance from Transmembrane Segment to tRNALumenal ExposureCytoplasmic Exposure2 amino acids+−4 amino acids−−7 amino acids−−9 amino acids−+ Open table in a new tab When the transmembrane segment was two amino acids from the tRNA, the situation was exactly as it was before (cytoplasmic side closed, lumenal side open). However, when the transmembrane segment was just four amino acids from the tRNA, the lumenal side of the translocon closed. Furthermore, when the transmembrane segment was nine amino acids from the tRNA, the cytoplasmic side of the translocon opened. The change in the state of the translocon was apparently due to the transmembrane segment itself because a half-transmembrane segment was not sufficient to induce any change in the accessibility of the nascent chain to iodide ions. These changes are remarkable for a number reasons. First, the regulation occurred with remarkable precision, and the translocon switched from being in a lumenal open–cytoplasm closed state to a lumen closed–cytoplasmic open state in fewer than ten amino acids. Furthermore, it did this in a defined order that maintained the permeability barrier of the ER membrane (note that if it had gone from open–closed to open–open instead of closed–closed, the permeability barrier would have been lost). Finally, and perhaps most remarkably, all of these changes in the translocon occurred less than ten amino acids after the transmembrane segment was synthesized. This is surprising because ∼35–40 amino acids are thought to be contained within the ribosome and therefore should not be accessible to components of the translocon. However19Wang S Sakai H Wiedmann M J. Cell Biol. 1995; 130: 519-528Crossref PubMed Scopus (100) Google Scholar found after high salt stripping of the ribosome and the removal of the nascent polypeptide-associated complex, sites as close as twelve amino acids from the peptidyl transferase center (which is one amino acid away from the amino acyl tRNA site) were accessible to protease. Although these experiments were done under conditions that were extremely nonphysiological, the results do leave open the possibility that components of the translocon might have access to the transmembrane segment even when it is within ten amino acids of the tRNA. One candidate for such a translocon component is Sec61β, which has been shown to cross-link to nascent bovine opsin prior to cross-linking of Sec61α (10Laird V High S J. Biol. Chem. 1997; 272: 1983-1989Crossref PubMed Scopus (53) Google Scholar). Alternatively, it is possible that some component of the ribosome recognizes the transmembrane segment and induces the changes in the translocon, perhaps by some conformational change. In order to address the question of how the transmembrane segment induces a change in the translocon, Johnson and colleagues asked what proteins associate with the transmembrane segment during the relevant stages. Instead of using a fluorescent probe, they placed a cross-linking agent into the nascent chain (the example I shall discuss contains a cross-linking agent in the middle of the transmembrane segment). Interestingly, they found a number of proteins that could be cross-linked to the transmembrane segment (and thus are in close proximity to it) and the cross-linking pattern changed during the crucial period when the cytoplasmic and lumenal sides of the translocon seemed to be opening and closing. For example, when the transmembrane segment was two amino acids away from the tRNA, a protein of ∼41 kDa was cross-linked to the transmembrane segment. When the chain moved five amino acids further along the nascent chain pathway (and the lumenal side of the translocon closed), a new protein of ∼20 kDa now was also cross-linked to the transmembrane segment. When the transmembrane segment moved to a position that elicited the opening of the cytoplasmic side, yet another target protein of ∼10 kDa was cross-linked in addition to p20 and p41. However, none of the cross-linked proteins seemed to be integral membrane proteins since they were extracted with carbonate; furthermore, they seemed to be components of the ribosome since the same proteins were cross-linked to the transmembrane segment when the experiments were performed in the absence of ER membranes. Because the pattern of ribosomal proteins in proximity to the transmembrane segment changed during this period, it is tempting to speculate that it is these same ribosomal proteins that mediate the changes in the translocon. In contrast, none of the known components of the translocon were cross-linked to the transmembrane segment at these stages. However, since not all proteins in proximity to the transmembrane segment will be cross-linked to it, it remains possible that some unknown component of the translocon reaches into the ribosome and interacts with the transmembrane segment and is thus directly involved in triggering the changes at the translocon. These results bring up two very interesting questions. First, why does transmembrane recognition occur so early? Second, why does the translocon change from a closed–open configuration to an open–closed configuration? Although there are no answers to these questions (and it still remains to be shown that these changes in the translocon are functionally linked to the proper insertion of transmembrane domains), one thing that might be worth keeping in mind is that the lumenal and cytoplasmic sides of the ER membrane are quite distinct, both in terms of soluble factors and in terms of the reducing environment (the lumen of the ER is an oxidizing environment, allowing the formation of disulfide bonds). It is possible that the configuration of the translocon switches simply so that transmembrane domains are inserted into the ER membrane in a reducing environment. It is also possible that cytoplasmic factors may play a role in orienting membrane proteins, as suggested by the authors (see11Liao S Lin J Do H Johnson A.E Cell. 1997; 90 (this issue, 90,): 31-41Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholarreferences therein). Although the experiments presented in the Liao et al. paper do not quite prove the point, they do suggest the possibility that the ribosome itself plays a role in recognizing the transmembrane segment. If this is true, it is possible that the ribosome also recognizes the pause transfer sequences mentioned earlier. Since these sequences cause the cytoplasmic side of the translocon to open, it seems likely that the lumenal side becomes closed so that the permeability barrier is maintained, in a gating mechanism similar to the one described by 11Liao S Lin J Do H Johnson A.E Cell. 1997; 90 (this issue, 90,): 31-41Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar. Just as we call the large and small ribosomal subunits part of the ribosome because they form an integral unit that functions as a translational machine, even though the two subunits are separate from each other in the absence of mRNA, should we also think of the ribosome as a of the translocon because it forms an integral part of the translocational machine, even though it is separate from the (other subunit of the) translocon in the absence of a translocating nascent chain? Viewed in this way, the ribosome would be the large subunit, since the mammalian ribosome is probably an order of magnitude larger than the (rest of the) translocon. Many experiments need to be done, of course, including identifying the specific factor or factors that mediate the changes in the translocon, and only time will tell whether the two subunits of the translocon communicate to mediate the response of the translocon to specific sequences within the nascent chain.