Endoplasmic reticulum localization of Sec12p is achieved by two mechanisms: Rer1p-dependent retrieval that requires the transmembrane domain and Rer1p-independent retention that involves the cytoplasmic domain

The PstI to SalI fragment of MFal was subcloned into pBluescript II SK+ (pBal). The DAP2-MFal fusion was constructed by inserting the blunted BamHI to BbilI fragment of pGP3 (Roberts et al., 1989 ) (obtained from T. Stevens of the University of Oregon) into the Sinai site of pBcO (pMDI-1). An NcoI site was introduced at the start codon of DAP2-MFal by PCR-mediated, site-directed mutagenesis using the oligonucleotide, 5'-CCCCGGGCCATGGAAGGTGGCGAAGAAG-3' (D-l). The DAP2 promoter region (BamHI to NcoI) was replaced by the SEC12 promoter fragment (XbaI to BspHI) (Nishikawa et al., 1994) to give pDDD-1.