Membrane protein retention in the yeast Golgi apparatus: dipeptidyl aminopeptidase A is retained by a cytoplasmic signal containing aromatic residues

Plasmids encoding the A-ALP fusion protein were constructed as follows: a 3.5-kbp KpnI-EcoRI fragment from pAL145, pBR322 carrying a 4-kbp BamHI fragment containing the entire PH08 gene (Kaneko et ai., 1987) , was inserted into the KpnI-EcoRI sites of pSK + resulting in the plasmid pSN8. Oligonucleotide mutngenesis was performed on pSN8 to introduce a silent mutation into the PH08 gene (A-G at position 570), which removes the BgllI site creating plasmid pSN9. PCR amplification from pSN9 resulted in a product consisting of nucleotides 97-1921 of the PH08 gene with additional nucleotides at the 5' and 3' ends encoding BglH and EcoRI sites, respectively. This PCR fragment was digested with BgllI and EcoRI, ligated to the l.l-kbp SalI-BgllI fragment from pCJRl6 (6-kpb XbaI-BamHI fragment of the ME/3 gene in pUC13; Roberts, C., and T. Stevens, unpublished observations) , and both fragments were inserted into the SalI-EcoRI sites of YCp50 via a three-way ligation creating pSN14. The protein sequence at the region of the fusion junction is (NH2...PEKRS-KIIV...) where the DPAP A sequence is underlined and the ALP sequence is not. Centromere-containing (CEN), HIS3 and URA3 based vectors carrying the STE13-PH08 gene fusion were constructed by inserting the EagI-EcoRI fragment from pSN14 into the EagI-EcoRI sites of plasmids pRS313 and pRS316 (Sikorski and Heiter, 1989) resulting in plasmids pSN54 and pSN55, respectively.