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

Site-direct mutagenesis was performed to introduce XbaI and AfllI sites at the 354th and the 373rd codons of SEC12. A BspHI site was created at the start codon of SEC12 (Nishikawa et al., 1994) . SEC12-MFal bearing these three restriction sites was subcloned into pBluescript II SK+ whose BspHI site was destroyed by blunting and religation. The resulting plasmid was named pMS2-3 (also called pSSS-1). The start to the 29th codon fragment of DAP2 was made by PCR using primers D-1 and 5'-CCTCTAGACCITATGAGCITATCTAACAAATG-3' (D-5), digested by NcoI and XbaI and used to replace the BspHl to XbaI region of pSSS-1, and thus produced pDSS-1. For pDDS-1, PCR was performed using primers D-1 and 5'-GCCTTAAGTATACTTITFAGCAACAAAAC-3' (D-3). The resulting fragment (start to the 48th codon of DAP2) was digested by NcoI and AfllI and inserted into the BspHI and AfllI sites of pSSS-I. For pSDS-1, a fragment encoding the transmembrane domain (TMD) 1 of Dap2p was synthesized by PCR using primers D-3 and 5'-GCT-CTAGAGTCGGAATAATCCTTGTAC-3' (D-4). This fragment was digested by XbaI and AfllI and introduced into the XbaI and Aflll sites of pSSS-1. An AfllI site was created just before the 48th codon of DAP2-MFcU by PCR-mediated mutagenesis and subcloned into pBluescript II SK+ (named pMD3-3). The AfllI to SacI (in vector) fragment of pMD3-3 was used to replace the corresponding fragments of pSSS-1, pDSS-1, and pSDS-1 to produce pSSD-1, pDSD-1, and pSDD-1, respectively. These chimeric genes were also subcloned into pSQ326 to yield pXXX-2, where XXX stands for SSS, DDD, SDS, DDS, DSS, DSD, SDD, or SSD. The plasmids for the mutational analysis of the Secl2p TMD were constructed by PCR-mediated mutagenesis using oligonucleotides containing mutations corresponding to each mutant. The Xbal to Aflll region of pDSD-1 was replaced by the XbaI/AfllI mutated fragments made by PCR. All mutant genes were subcloned into pSQ326.