Insertion of a multispanning membrane protein occurs sequentially and requires only one signal sequence.
To study the insertion of multispanning membrane proteins into the endoplasmic reticulum, we constructed novel proteins on the cDNA level by repeating, up to four times, the internal signal-anchor domain of the asialoglycoprotein receptor H1. Upon in vitro translation in the presence of microsomes, these polypeptides are indeed inserted as polytopic membrane proteins. The first hydrophobic domain functions as a signal and the second as a stop-transfer sequence, while the third initiates a second translocation process, halted again by the fourth. We were able to demonstrate that insertion occurs sequentially, starting with the first apolar segment from the amino terminus. By replacing the original signal-anchor domains by a mutant sequence not recognized by signal recognition particle (SRP), it was shown that only the first hydrophobic domain needs to be a signal sequence and that the second translocation event does not require SRP.
Wessels
H P
HP
Department of Biochemistry, University of Basel, Switzerland.
Spiess
M
M
D000818:Animals; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D006031:Glycosylation; D008565:Membrane Proteins / Q000032:analysis*; D010957:Plasmids; D014176:Protein Biosynthesis; D011971:Receptors, Immunologic / Q000032:analysis*; D012261:Ribonucleoproteins / Q000032:analysis / Q000235:genetics; D018271:Signal Recognition Particle
D037263:Asialoglycoprotein Receptor; D008565:Membrane Proteins; D011971:Receptors, Immunologic; D012261:Ribonucleoproteins; D018271:Signal Recognition Particle