Hydrophobic start-transfer and stop-transfer signal sequences both act to fix the topology of the protein in the membrane by locking themselves into the membrane as membrane-spanning α helices; and they can do this in either orientation. Whether a given hydrophobic signal sequence functions as a start-transfer or stop-transfer sequence must depend on its location in a polypeptide chain, since its function can be switched by changing its location in the protein by using recombinant DNA techniques. Thus, the distinction between start-transfer and stop-transfer sequences results mostly from their relative order in the growing polypeptide chain. It seems that the SRP begins scanning an unfolded polypeptide chain for hydrophobic segments at its N-terminus and proceeds toward the C-terminus, in the direction that the protein is synthesized. By recognizing the first appropriate hydrophobic segment to emerge from the ribosome, the SRP sets the “reading frame” for membrane integration: after the SRP initiates trans-location, the translocator recognizes the next appropriate hydrophobic segment in the direction of transfer as a stop-transfer sequence, causing the region of the polypeptide chain in between to be threaded across the membrane. A similar
Figure 12–43 Integration of a single-pass transmembrane protein with an internal signal sequence into the ER membrane. an internal er signal sequence that functions as a start-transfer signal can bind to the translocator in one of two ways, leading to a membrane protein that has either its C-terminus (pathway a) or its n-terminus (pathway
B) in the er lumen. proteins are directed into either pathway by features in the polypeptide chain flanking the internal start-transfer sequence: if there are more positively charged amino acids immediately preceding the hydrophobic core of the start-transfer sequence than there are following it, the membrane protein is inserted into the translocator in the orientation shown in pathway a, whereas if there are more positively charged amino acids immediately following the hydrophobic core of the start-transfer sequence than there are preceding it, the membrane protein is inserted into the translocator in the orientation shown in pathway B. Because translocation cannot start before a start-transfer sequence appears outside the ribosome, translocation of the n-terminal portion of the protein shown in (B) can occur only after this portion has been fully synthesized.
note that there are two ways to insert a single-pass membrane-spanning protein whose n-terminus is located in the er lumen: that shown in figure 12–42 and that shown here in (B).
scanning process continues until all of the hydrophobic regions in the protein have been inserted into the membrane as transmembrane α helices.
Because membrane proteins are always inserted from the cytosolic side of the ER in this programmed manner, all copies of the same polypeptide chain will have the same orientation in the lipid bilayer. This generates an asymmetrical ER membrane in which the protein domains exposed on one side are different from those exposed on the other side. This asymmetry is maintained during the many membrane budding and fusion events that transport the proteins made in the ER to other cell membranes (discussed in Chapter 13). Thus, the way in which a newly synthesized protein is inserted into the ER membrane determines the orientation of the protein in all of the other membranes as well.
When proteins are extracted with detergent from a membrane and then reconstituted into artificial lipid vesicles, a random mixture of right-side-out and inside-out protein orientations usually results. Thus, the protein asymmetry observed in cell membranes seems not to be an inherent property of the proteins, but instead results solely from the process by which proteins are inserted into the ER membrane from the cytosol.
Figure 12–44 Integration of a double-pass transmembrane protein with an internal signal sequence into the ER membrane. in this protein, an internal er signal sequence acts as a start-transfer signal (as in figure 12–43) and initiates the transfer of the C-terminal part of the protein. at some point after a stop-transfer sequence has entered the translocator, the translocator discharges the sequence laterally into the membrane.