entirely into the ER lumen and is eventually secreted from the cell. These findings establish that the hydrophobic membrane-spanning a helix of the HGH receptor and of other type I proteins functions both as a stop-transfer sequence and 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 membrane-anchor sequence. Recall that type II and type III proteins have opposite orientations in the membrane (see Figure 16-10); 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 chain into the ER membrane so that the N-terminus of the chain faces the cytosol (Figure 16-12). The internal signal-anchor sequence is not cleaved and remains in the translocon while the C-terminal region of the growing chain is extruded into the ER lumen by co-translational translocation. During synthesis, the signalanchor sequence moves laterally between the protein sub-

units forming the translocon wall into the phospholipid bilayer, where it functions as a membrane anchor. Thus this function is similar to the anchoring function of the stop-transfer anchor sequence in type I proteins.

In the case of type III proteins, the signal-anchor sequence, which is located near the N-terminus, inserts the nascent chain into the ER membrane with its N-terminus facing the lumen, just the opposite of type II proteins. The signalanchor sequence of type III proteins also prevents further extrusion of the nascent chain into the ER lumen, functioning as a stop-transfer sequence. Continued elongation of the chain C-terminal to the signal-anchor/stop-transfer sequence proceeds as it does for type I proteins, with the hydropho-bic sequence moving laterally between the translocon sub-units to anchor the polypeptide in the ER membrane (see Figure 16-11).

One of the features of signal-anchor sequences that appears to determine their insertion orientation is a high density of positively charged amino acids adjacent to one end of the hydrophobic segment. For reasons that are not well understood these positively charged residues tend to remain on the cytosolic side of the membrane, thereby dictating the orientation of the signal-anchor sequence within the translo-con. Thus type II proteins tend to have positively charged

Nascent polypeptide chain

Nascent polypeptide chain

ER lumen


ER lumen


▲ FIGURE 16-12 Synthesis and insertion into the ER membrane of type II single-pass proteins. Step 1: After the internal signal-anchor sequence is synthesized on a cytosolic ribosome, it is bound by an SRP (not shown), which directs the ribosome/nascent chain complex to the ER membrane. This is similar to 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 chain becomes oriented in the translocon with its N-terminal portion toward the cytosol. This orientation is believed to be mediated 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 moves laterally out of the translocon and anchors the chain in the phospholipid bilayer. Step 3: Once protein synthesis is completed, the C-terminus of the polypeptide is released into the lumen, and the ribosomal subunits are released into the cytosol. [See M. Spiess and H. F Lodish, 1986, Cell 44:177, and H. Do et al., 1996, Cell 85:369.]

residues on the N-terminal side of their signal-anchor sequence, whereas type III proteins tend to have positively charged residues on the C-terminal side of their signalanchor sequence.

A striking experimental demonstration of the importance of the flanking charge in determining membrane orientation 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 neu-raminidase 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.

Multipass Proteins Have Multiple Internal Topogenic Sequences

Figure 16-13 summarizes the arrangements of topogenic sequences in single-pass and multipass transmembrane proteins. In multipass (type IV) proteins, each of the membrane-spanning a helices acts as a topogenic sequence in the ways that we have already discussed. Multipass proteins fall into one of two types depending on whether the N-terminus extends into the cytosol or the exoplasmic space (i.e., the ER lumen, cell exterior). This N-terminal topology usually is determined by the hydrophobic segment closest to the N-terminus and the charge of the sequences flanking it. If a type IV protein has an even number of transmembrane a helices, both its N-terminus and C-terminus will be oriented toward the same side of the membrane (see Figure 16-13d). Conversely, if a type IV protein has an odd number of a helices, its two ends will have opposite orientations (see Figure 16-13e).

Proteins with N-Terminus in Cytosol (Type IV-A) Among the multipass proteins whose N-terminus extends into the cy-tosol are the various glucose transporters (GLUTs) and most ion-channel proteins discussed in Chapter 7. In these proteins, the hydrophobic segment closest to the N-terminus initiates insertion of the nascent chain into the ER membrane with the N-terminus oriented toward the cytosol; thus this a-helical segment functions like the internal signal-anchor sequence of a type II protein (see Figure 16-12). As the nascent chain following the first a helix elongates, it moves through the translocon until the second hydrophobic a helix is formed. This helix prevents further extrusion of the nascent chain through the translocon; thus its function is similar to that of the stop-transfer anchor sequence in a type I protein (see Figure 16-11).

After synthesis of the first two transmembrane a helices, both ends of the nascent chain face the cytosol and the loop between them extends into the ER lumen. The C-terminus of the nascent chain then continues to grow into the cytosol, as it does in synthesis of type I and type III proteins. According to this mechanism, the third a helix acts as another type II signal-anchor sequence, and the fourth as another stop-transfer anchor sequence (see Figure 16-13d). Apparently, once the first topogenic sequence of a multipass polypeptide initiates association with the translocon, the ribosome remains attached to the translocon, and topogenic sequences that subsequently emerge from the ribosome are threaded into the translocon without the need for the SRP and the SRP receptor.

Experiments that use recombinant DNA techniques to exchange hydrophobic a helices have provided insight into the functioning of the topogenic sequences in type IV-A multipass proteins. These experiments indicate that the order of the hy-drophobic a helices relative to each other in the growing chain largely determines whether a given helix functions as a signal-anchor sequence or stop-transfer anchor sequence.

STA = Internal stop-transfer anchor sequence SA-II = Internal signal-anchor sequence SA-III = Internal signal-anchor sequence




Signal sequence

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