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

It is known that the ER retention of the proteins could also result from the quality control of the ER. The Dap2-Mfalp (DDDm) construct showed no enzymatic activity of dipeptidyl aminopeptidase. The Mfalp moeity might hinder correct folding of the lumenal domain of Dap2p and thus activate the potent quality control system in the ER for some of the chimeric proteins. Although the fact that DSDm forms a large halo in the Arerl ceils strongly argues against this possibility, we decided to test the ER localization effects of the TMD and the cytoplasmic domain in more native forms. We constructed DSD and SDD, which contain the complete COOH-terminal lumenal domain of Dap2p devoid of the Mfalp moiety. This version of the chimeric proteins expressed in the Adap2 cells showed full dipeptidyl aminopeptidase activities as the authentic Dap2p (data not shown). SDD is also completely functional as Secl2p since it could complement the Asec12 mutant on a single-copy plasmid. Immunofluorescence microscopy (Fig. 6, A and B) showed that, like DSDm, DSD was strictly localized to the ER. SDD was mainly localized to the ER, but weak vacuolar staining was also observed (Fig. 6, C-E) . This is consistent with the fact that SDDm had less ability of retention than DSDm as measured by the halo assay. Because DSD and SDD retain the full activity as Dap2p and SDD fucntions like the authentic Sec12p, it is unlikely that these proteins invoke the quality control of the ER as unfolded proteins.