C-CYTOSOLIC AND TRANSMEMBRANE DOMAINS OF THE N-BENZOYL-L-TYROSYL-P-AMINOBENZOIC ACID HYDROLASE ALPHA-SUBUNIT (HUMAN MEPRIN ALPHA) ARE ESSENTIAL FOR ITS RETENTION IN THE ENDOPLASMIC-RETICULUM AND C-TERMINAL PROCESSING

N-benzoyl-L-tyrosyl-p-aminobenzoic acid hydrolase (PPH, human meprin) is a member of the astacin family of Zn-metalloendopeptidases and is h ighly expressed in the microvillus membrane of human small intestinal epithelial cells. It is a type I transmembrane protein consisting of d ifferentially processed glycosylated alpha and beta subunits. Biosynth esis experiments using transfected, metabolically labelled simian viru s 40 (SV40) transformed african green monkey kidney cells (COS-1) and Madin Darby canine kidney (MDCK) cells, have previously shown that PPH alpha was retained in the endoplasmic reticulum (ER) and that for sub sequent secretion removal of the alpha-tail was necessary [Grunberg, J ., Dumermuth, E., Eldering, J. A. & Sterchi, E. E. (1993) FEBS Lett. 3 35, 376-379]. We proposed an involvement of the alpha-tail in ER reten tion. To investigate the possible role of the transmembrane and/or the C-terminal domain of the alpha-subunit, tailswitch mutants were const ructed in which these domains were exchanged between the a and beta su bunits. Biosynthesis and post-translational processing of these mutant s were investigated in transiently transfected COS-1 cells. The beta/a lpha tailswitch mutant, in which the transmembrane and C-cytosolic par ts of PPH beta were substituted by the corresponding parts of the PPH alpha subunit, was transported much slower compared with the wild-type PPH beta subunit. In addition, fusion of the alpha-tail to a C-termin ally truncated secretory form of PPH alpha leads to its retention in t he ER. This mutant, but not the secretory form, coimmunoprecipitated w ith calnexin, indicating an involvement of this molecular chaperone in retaining PPH alpha in the ER. The alpha/beta tailswitch mutant, in w hich the transmembrane domain and the C-cytosolic part of PPH alpha we re substituted by the corresponding parts of PPH beta, was processed l ess efficiently in comparison with PPH alpha, resulting in a lower sec retion rate. Taken together these data suggest a role of the alpha-tai l in mediating association with ER-resident machinery, facilitating C- terminal processing.