SECRETION OF A TYPE-II INTEGRAL MEMBRANE-PROTEIN INDUCED BY MUTATION OF THE TRANSMEMBRANE SEGMENT

Signal peptide/membrane anchor (SA) domains of type II membrane protei ns initiate the translocation of downstream polypeptides across the en doplasmic reticulum (ER) membrane. In contrast with signal peptides, h owever, SA domains are not cleaved by signal peptidase and thus anchor the protein in the membrane. In the present study we have introduced mutations in the SA domain of neprilysin (neutral endopeptidase-24.11; NEP) to identify structural elements that would favour the processing of SA domains by signal peptidase. Mutants of full-length and truncat ed (without cytoplasmic domain) protein were constructed by substituti on of the sequences SQNS, QQTT or YPGY for VTMI starting at position 1 5 of the NEP SA domain. In addition, a Pro residue was substituted for Thr at position 16 of the SA domain. The rationale for the use of the se sequences was decided from our previous observation that substituti on in the NEP SA domain of the sequence SQNS, which is polar and has a -helix-breaking potential, could promote SA domain processing under ce rtain conditions (Roy, Chatellard, Lemay, Crine and Boileau (1993) J. Biol. Chem. 268, 2699-2704; Yang, Chatellard, Lazure, Crine and Boilea u (1994) Arch. Biochem. Biophys. 315, 382-386). The QQTT sequence is p olar but, according to secondary structure predictions, is compatible with the alpha-helix structure of the NEP SA domain, The YPGY sequence and single Pro residue are less polar and have alpha-helix-breaking p otential. The predicted effects of these mutations on the structure of the NEP SA domain were confirmed by CD analysis of 42-residue peptide s encompassing the hydrophobic segment and flanking regions. Wild-type and mutated proteins were expressed in COS-1 cells and their fate (me mbrane-bound or secreted) was determined by immunoblotting and by endo glycosidase digestions. Our biochemical and structural data indicate t hat: (1) the cytosolic domain of NEP restricts the conformation of the SA domain because mutants not secreted in their full-length form are secreted in their truncated form; (2) alpha-helix-breaking residues ar e not a prerequisite for cleavage; (3) the presence, in close proximit y to a putative signal peptidase cleavage site, of a polar sequence th at maintains the alpha-helical structure of the SA domain is sufficien t to promote cleavage. Furthermore pulse-chase studies suggest that cl eavage is performed in the ER by signal peptidase and indicate that cl eavage is not a limiting step in the biosynthesis of the soluble form of the protein.