Internal Stop-Transfer Anchor and Signal-Anchor Sequences Determine Topology of Single-Pass Proteins

We begin our discussion of how membrane protein topology is determined with the insertion of integral membrane proteins that contain a single hydrophobic membrane-spanning segment. As we will see, three main types of topogenic sequences are used to direct proteins to the ER membrane and to orient them within it. We have already introduced one, the N-terminal signal sequence. The other two, introduced here, are internal sequences known as stop-transfer anchor sequences and signal-anchor sequences. Unlike signal sequences, these two types of internal topogenic sequences end up in the mature protein as membrane-spanning segments. However, the two types differ in their final orientation in the membrane.

Type I Proteins In addition to an N-terminal signal sequence that targets them to the ER, all type I transmembrane proteins possess an internal hydrophobic sequence of approximately 22 amino acids that becomes the membrane-spanning α helix. The N-terminal signal sequence of a nascent type I protein, like that of a soluble secretory protein, initiates cotranslational translocation of the protein through the combined action of the SRP and SRP receptor. Once the N-terminus of the growing polypeptide enters the lumen of the ER, the signal sequence is cleaved, and the growing polypeptide chain continues to be extruded across the ER membrane. However, when the sequence that will become a transmembrane domain enters the translocon, it stops transfer of the protein through the channel by allowing the transmembrane segment to move laterally from the channel into the membrane (Figure 13-11). The gating mechanism that allows lateral movement is the same as that for the opening of the translocon to accept a signal sequence: two five-helix bundles of Sec61α hinge open to allow the hydrophobic transmembrane segment to move laterally past the hydrophobic signal-sequence binding site through the opened edge of the translocon (see Figure 13-8). When the peptide exits the translocon in this manner, the hydrophobicity of the transmembrane segment anchors it in the hydrophobic interior of the membrane. Because such a sequence functions both to stop passage of the polypeptide chain through the translocon and to become a hydrophobic transmembrane segment in the membrane bilayer, it is called a stop-transfer anchor sequence.

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FIGURE 13-11 Membrane insertion and orientation of type I single-pass transmembrane proteins. Step 1: After the nascent polypeptide chain–ribosome complex becomes associated with a translocon in the ER membrane, the N-terminal signal sequence is cleaved. This process occurs by the same mechanism as the one for soluble secretory proteins (see Figure 13-6). Steps 23: The chain is elongated until the hydrophobic stop-transfer anchor sequence is synthesized and enters the translocon, where it prevents the nascent chain from extruding farther into the ER lumen. Step 4: The stop-transfer anchor sequence moves laterally through a hydrophobic cleft between translocon subunits and ultimately becomes anchored in the phospholipid bilayer. At this time, the translocon probably closes. Step 5: As synthesis continues, the elongating chain may loop out into the cytosol through the small space between the ribosome and translocon. Step 6: When synthesis is complete, the ribosomal subunits are released into the cytosol, leaving the protein free to diffuse laterally in the membrane. See H. Do et al., 1996, Cell 85:369, and W. Mothes et al., 1997, Cell 89:523.

Once translocation is interrupted, translation continues at the ribosome, which is still anchored to the now unoccupied and closed translocon. As the C-terminus of the protein chain is synthesized, it loops out on the cytosolic side of the membrane. When translation is complete, the ribosome is released from the translocon, and the C-terminus of the newly synthesized type I protein remains in the cytosol.

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Support for this mechanism has come from studies in which cDNAs encoding various mutant receptors for human growth hormone (HGH) were expressed in cultured mammalian cells. The wild-type HGH receptor, a typical type I protein, is transported normally to the plasma membrane. However, a mutant receptor that has charged residues inserted into the single membrane-spanning segment, or that is missing most of this segment altogether, is translocated entirely into the ER lumen and is eventually secreted from the cell as a soluble protein. These kinds of experiments have established that the hydrophobic membrane-spanning segment of the HGH receptor, and of other type I proteins, functions both as a stop-transfer sequence and as a membrane anchor that prevents the C-terminus of the protein from crossing the ER membrane.

Type II and Type III Proteins Unlike type I proteins, type II and type III proteins lack a cleavable N-terminal ER signal sequence. Instead, both possess a single internal hydrophobic signal-anchor sequence that functions as both an ER signal sequence and a membrane anchor. Recall that type II and type III proteins have opposite orientations in the membrane (see Figure 13-10); this difference depends on the orientation that their respective signal-anchor sequences assume within the translocon. The internal signal-anchor sequence in type II proteins directs insertion of the nascent polypeptide chain into the ER membrane so that the N-terminus of the chain faces the cytosol, using the same SRP-dependent mechanism described for signal sequences (Figure 13-12a). However, the internal signal-anchor sequence is not cleaved and eventually moves laterally from the signal-sequence binding site at the edge of the translocon directly into the phospholipid bilayer, where it functions as a membrane anchor. As elongation continues, the C-terminal region of the growing chain is extruded through the translocon into the ER lumen by cotranslational translocation.

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FIGURE 13-12 Membrane insertion and orientation of type II and type III single-pass transmembrane proteins. (a) Type II proteins. Step 1: After the internal signal-anchor sequence is synthesized on a cytosolic ribosome, it is bound by an SRP (not shown), which binds the SRP receptor on the ER membrane. This process is similar to the targeting of soluble secretory proteins except that the hydrophobic signal sequence is not located at the N-terminus and is not subsequently cleaved. The nascent polypeptide chain becomes oriented in the translocon with its N-terminal portion toward the cytosol. This orientation is dictated by the positively charged residues shown N-terminal to the signal-anchor sequence. Step 2: As the chain is elongated and extruded into the lumen, the internal signal-anchor sequence moves laterally through a hydrophobic cleft between translocon subunits and anchors the chain in the phospholipid bilayer. Step 3: Once protein synthesis is complete, the C-terminus of the polypeptide is released into the lumen, and the ribosomal subunits are released into the cytosol. (b) Type III proteins. Step 1: Insertion is by a process similar to that of type II proteins, except that positively charged residues on the C-terminal side of the signal-anchor sequence cause the transmembrane segment to be oriented within the translocon with its C-terminal portion toward the cytosol and the N-terminal end in the ER lumen. Steps 23: Elongation of the C-terminal portion of the polypeptide chain is completed in the cytosol, and the ribosomal subunits are released. See M. Spiess and H. F. Lodish, 1986, Cell 44:177, and H. Do et al., 1996, Cell 85:369.

In the case of type III proteins, the signal-anchor sequence, which is located near the N-terminus, directs insertion of the nascent chain into the ER membrane with its N-terminus facing the lumen, in an orientation opposite to that of the signal-anchor in type II proteins. The signal-anchor sequence of type III proteins also functions like a stop-transfer sequence and prevents further extrusion of the elongating chain into the ER lumen (Figure 13-12b). Continued elongation of the chain C-terminal to the signal-anchor sequence proceeds as it does for type I proteins, with the hydrophobic sequence eventually moving laterally out of the translocon to anchor the polypeptide in the ER membrane (see Figure 13-11).

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The key difference between type II and type III proteins is the orientation of the hydrophobic transmembrane segment as it binds to the hydrophobic signal-sequence binding site at the edge of Sec61α. The most important feature of signal-anchor sequences that determines their orientation is a high density of positively charged amino acids adjacent to one end of the hydrophobic segment. These positively charged residues tend to remain on the cytosolic side of the membrane, rather than traversing the membrane into the ER lumen. Thus the position of the charged residues dictates the orientation of the signal-anchor sequence within the translocon as well as whether the rest of the polypeptide chain continues to pass into the ER lumen: type II proteins tend to have positively charged residues on the N-terminal side of their signal-anchor sequence, orienting the N-terminus in the cytosol and allowing passage of the C-terminal side into the ER (see Figure 13-12a), whereas type III proteins tend to have positively charged residues on the C-terminal side of their signal-anchor sequence, which restrict the C-terminus to the cytosol (see Figure 13-12b). Note that the hydrophobic segment of a type II signal-anchor sequence assumes the same orientation in Sec61α as the signal sequence of a secreted protein, and that in most respects these signal-anchor sequences behave exactly like signal sequences, although they are not cleaved.

A striking experimental demonstration of the importance of the flanking charge in determining orientation in the membrane is provided by neuraminidase, a type II protein in the surface coat of influenza virus. Three arginine residues are located just N-terminal to the internal signal-anchor sequence in neuraminidase. Mutation of these three positively charged residues to negatively charged glutamate residues causes neuraminidase to acquire the reverse orientation. Similar experiments have shown that other proteins, with either type II or type III orientation, can be made to “flip” their orientation in the ER membrane by mutating charged residues that flank the internal signal-anchor segment.

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FIGURE 13-13 Insertion of tail-anchored proteins. For C-terminal tail-anchored proteins, the hydrophobic C-terminus is not available for membrane insertion until protein synthesis is complete and the protein has been released from the ribosome. Step 1: Get3 in an ATP-bound state binds to the hydrophobic C-terminal tail of the protein. This binding reaction is facilitated by a complex of three other proteins, Sgt2, Get4, and Get5, which sequester the hydrophobic C-terminal tail before transferring it to Get3·ATP (not shown). Step 2: The ternary complex Get3·ATP bound to the protein docks onto the dimeric Get1/Get2 receptor, which is embedded in the ER membrane. Step 3: In succession, ATP is hydrolyzed and ADP is released from Get3. At the same time, the hydrophobic C-terminal tail is released from Get3 and ultimately becomes embedded in the ER membrane in a process that is facilitated by Get1/Get2. Step 4: Get3 binds to ATP and Get3·ATP is released from Get1/Get2 in a soluble form, ready for another round of binding to a hydrophobic C-terminal tail.

Tail-Anchored Proteins For all the topological classes of proteins we have considered so far, membrane insertion begins when the SRP recognizes a hydrophobic topogenic sequence as it emerges from the ribosome. Recognition of tail-anchored proteins, which have a single hydrophobic topogenic sequence at the C-terminus, presents a unique challenge because the hydrophobic C-terminus becomes available for recognition only after translation has been completed and the protein has been released from the ribosome. Insertion of tail-anchored proteins into the ER membrane does not employ an SRP, SRP receptor, or translocon, but instead depends on a pathway dedicated to this purpose, as depicted in Figure 13-13. This pathway involves an ATPase known as Get3, which binds to the C-terminal hydrophobic segment of a tail-anchored protein. The complex of Get3 bound to a tail-anchored protein is recruited to the ER by a dimeric integral membrane receptor known as Get1/Get2. The tail-anchored protein is released from Get3, and the transmembrane portion of Get1/Get2 participates in the insertion of the tail-anchor into the ER membrane. This process is mechanistically similar to the targeting of type II and type III signal-anchor sequences to the ER by the SRP and SRP receptor. A major difference between the two targeting processes is that Get3 couples targeting and transfer of tail-anchored proteins to ATP hydrolysis, whereas SRP couples protein targeting to GTP hydrolysis. Moreover, the SRP receptor recruits the SRP-ribosome complex to the ER, and in a separate step, the translocon inserts the signal-anchor sequence into the membrane, whereas Get1/Get2 evidently performs both functions, recruiting Get3 to the ER membrane and catalyzing insertion of the tail-anchor into the membrane bilayer.