Signal and membrane anchor functions overlap in the type II membrane protein I gamma CAT.
I gamma CAT is a hybrid protein that inserts into the membrane of the endoplasmic reticulum as a type II membrane protein. These proteins span the membrane once and expose the NH2-terminal end on the cytoplasmic side and the COOH terminus on the exoplasmic side. I gamma CAT has a single hydrophobic segment of 30 amino acid residues that functions as a signal for membrane insertion and anchoring. The signal-anchor region in I gamma CAT was analyzed by deletion mutagenesis from its COOH-terminal end (delta C mutants). The results show that the 13 amino acid residues on the amino-terminal side of the hydrophobic segment are not sufficient for membrane insertion and translocation. Mutant proteins with at least 16 of the hydrophobic residues are inserted into the membrane, glycosylated, and partially proteolytically processed by a microsomal protease (signal peptidase). The degree of processing varies between different delta C mutants. Mutant proteins retaining 20 or more of the hydrophobic amino acid residues can span the membrane like the parent I gamma CAT protein and are not proteolytically processed. Our data suggest that in the type II membrane protein I gamma CAT, the signals for membrane insertion and anchoring are overlapping and that hydrophilic amino acid residues at the COOH-terminal end of the hydrophobic segment can influence cleavage by signal peptidase. From this and previous work, we conclude that the function of the signal-anchor sequence in I gamma CAT is determined by three segments: a positively charged NH2 terminus, a hydrophobic core of at least 16 amino acid residues, and the COOH-terminal flanking hydrophilic segment.
Lipp
J
J
European Molecular Biology Laboratory, Heidelberg, Federal Republic of Germany.
Dobberstein
B
B
D000595:Amino Acid Sequence; D000818:Animals; D002474:Cell-Free System; D004252:DNA Mutational Analysis; D010450:Endopeptidases / Q000502:physiology*; D004721:Endoplasmic Reticulum / Q000502:physiology*; D006031:Glycosylation; D000949:Histocompatibility Antigens Class II / Q000502:physiology*; D066298:In Vitro Techniques; D008562:Membrane Glycoproteins / Q000145:classification / Q000502:physiology*; D008565:Membrane Proteins; D008969:Molecular Sequence Data; D011499:Protein Processing, Post-Translational; D011993:Recombinant Fusion Proteins / Q000502:physiology; D012697:Serine Endopeptidases; D012995:Solubility; D013329:Structure-Activity Relationship
D000949:Histocompatibility Antigens Class II; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D012697:Serine Endopeptidases; C026515:type I signal peptidase