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

Mutations that changed residues 81-90 of DPAP A to other amino acids were also constructed by oligonucleotide mutagenesis of pCJRT1. The nomenclature used for the mutant proteins is Phes~ ~ Alags represented as F85A. EagI-BgllI fragments from derivatives of plasmid pCJRT1 containing the desired mutations were subcloned into the EagI-BgllI sites of pSN55. The following is a list of the resulting plasmids and the mutations contained within the A-ALP context: pSN196 (R81A), pSN197 (R82A), pSN198 (Eg3A), pSN199 ($84A), pSN99 (F85A), pSN105-S (F85S), pSN105-Y (F85Y), pSN105-D (F85D), pSNI05-G (F85G), pSN105-C (F85C), pSN105-R (F85R), pSN200 (Q86A), pSN106-R (Q86R), pSN106-K (Q86K), pSN137-W (Q86W), pSNI37-E (Q86E), pSN137-M (Q86M), pSN137-V (Q86V), pSN98 (F87A), pSN107-C (F87C), pSN107-V (F87V), pSNIO7-G (F87G), pSNI39 (F87Y), pSN174 (N88A), pSN175, (D89A), pSN176 (I90A), and pSN100 (F85A,F87A). The F85A and F87A mutations were incorporated into wild-type DPAP A by inserting SacI-MluI fragments (from pCJRT1 derivatives containing these mutations) into the SacI-MluI sites of pCJR106 resulting in the 2/~m, URA3 based plasmids pSN128 and pSNI27, respectively. All of the mutations described above were confirmed by restriction digest analysis (in cases where the point mutation or point of deletion fell within a restriction site) or by DNA sequence analysis.