Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.
The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.
Spiess
M
M
Department of Biochemistry, University of Basel, Switzerland.
Handschin
C
C
D000595:Amino Acid Sequence; D037263:Asialoglycoprotein Receptor; D001483:Base Sequence; D002462:Cell Membrane / Q000276:immunology; D002872:Chromosome Deletion; D005796:Genes; D006801:Humans; D009154:Mutation; D010957:Plasmids; D014176:Protein Biosynthesis; D021382:Protein Sorting Signals / Q000378:metabolism; D011971:Receptors, Immunologic / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic
D037263:Asialoglycoprotein Receptor; D021382:Protein Sorting Signals; D011971:Receptors, Immunologic