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

Replacement of the transmembrane domain of DPAP A (residues 120-139) with the sequence L(LALV)5, was as follows: oligonucleotide mutagenesis of a plasmid pCJR71 (Roberts et al., 1992) consisting of the 0.65-kbp EagI-PstI STE/3 fragment (Flanagan, C. A., D. A. Barnes, M. C. Flessel, and J. Thorner, manuscript submitted for publication) in pKS + (Stratagene) was used to remove sequences encoding amino acids 120-139 while leaving an HpaI site at the in-frame fusion junction resulting in plasmid pSN113. Multiple tandem repeats of the linker (5"ACTAGCC,-CTAGT-31 were ligated into the HpaI site ofpSNll3. Sequence analysis showed that a resulting plasmid, pSNll8, contains five copies of this linker. The resulting DNA sequence encodes the following amino acids surrounding the transmembrane domain: (NH2...RSL(LALV)sTP...)__ where the wild-type residues are underlined. The SacI-MhiI fragment from pSNll8 was inserted into the SacI-MluI sites of pCJR106 (Roberts et al., 1992) creating pSN121 (A-X-A in a 2 #m plasmid). ~85-106-A-X-A was constructed by inserting an EagI-BsaI fragment from pSN60 (described below) into the EagI-BsaI sites of pSNll8 creating pSN119. The SacI-MluI fragment from pSNll9 was inserted into the SacI-MluI sites of pCJR106 resulting in pSN122 (A85-106-A-X-A in a 2 #m plasmid).