Demonstration of Specific Receptors of the Rough Endoplasmic Membrane for the Signal Sequence of Carp Preproinsulin

Abstract

1 Evidence is given for the cotranslational processing in a wheat germ cell-free system of carp preproinsulin to proinsulin by dog pancreatic rough endoplasmic membranes stripped of ribosomes by EDTA. Concomitantly with processing there is a translocation of the protein across the membrane as shown by the accessibility to proteases. Conversion of preproinsulin to proinsulin occurs post-translationally only in the presence of detergent. 2 Stripped rough endoplasmic membranes bind a significant proportion of the cell-free translation products synthesized in a wheat germ system in response to poly(A)-rich RNA from carp islets. Among the bound material there is completed preproinsulin. The bound material is accessible to proteases. It is located on the cytoplasmic side of the membranes. Peptides lacking a signal sequence, such as globin or proinsulin, are not bound. The binding can be abolished by pretreatment of the rough membranes with proteases. Neither smooth endoplasmic membranes from dog pancreas nor erythrocyte plasma membranes from rabbits are able to bind signal peptides. The binding sites on the rough membranes can be saturated with cell-free products coded by carp islet RNA and are then no longer able to process preproinsulin to proinsulin in a cotranslational assay. The amount of membranes necessary to bind a certain amount of cell-free products post-translationally corresponds roughly to the amount required for complete conversion of preproinsulin to proinsulin cotranslationally. The binding phenomenon occurs independently of the presence of ribosomes and is not sensitive to high ionic strength. A hydrophobic peptide (Ac-Lys-Phe-Phe-Gly-Leu-Nle-NH₂) did not compete with signal peptides. 3 The results show that stripped rough endoplasmic membranes possess specific proteincontaining receptors for signal sequences on their cytoplasmic side. The recognition of the signal by the endoplasmic membranes occurs without participation of the ribosomes. The latter are only required for the translocation of the nascent peptide into the lumen of the membrane.