A search for novel genes that are up-regulated during development and differentiation of the epithelial cells of the intestinal mucosa led us to the isolation of the Dri 42 cDNA clone (Dri, differentially expressed in rat intestine). The nucleotide sequence of the full-length cDNA has shown that it encodes a 35.5-kDa protein with one consensus sequence for N-linked glycosylation and alternating hydrophilic and hydrophobic domains. To determine the intracellular localization of Dri 42 we have raised polyclonal antibodies in hens against a bacterially produced Dri 42-glutathione S- transferase fusion protein. Immunofluorescence detection with these antibodies has shown specific staining of the endoplasmic reticulum (ER) in the relatively undifferentiated fetal rat intestinal cell line FRIC B and in sections of rat small intestine. ER membrane localization of Dri 42 was confirmed by laser confocal microscopy of polarized Madin-Darby canine kidney cells overexpressing a Dri 42-chloramphenicol acetyltransferase (CAT) fusion protein by transfection. Pulse labeling experiments on transiently transfected cells demonstrated that the protein does not acquire Golgi modifications up to 4 h after synthesis, thus indicating that Dri 42 is an ER resident protein. The transmembrane disposition of Dri 42 was studied using in vitro insertion of Dri 42-CAT fusion proteins into microsomal membranes. The fusion proteins consisted of several different lengths of truncated Dri 42 and a reporter protein, CAT, that was linked in-frame after each hydrophobic segment. We found that hydrophobic segments H1, H3, and H5 had a signal/anchor function, and that membrane insertion of Dri 42 was achieved co-translationally by the action of a series of alternating insertion signals and halt transfer signals, resulting in the exposure of both termini of the protein to the cytosolic side. The functional implications of the structure and localization of Dri 42, whose primary sequence does not share significant homology to any previously described protein, are discussed.
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