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

First, as a control, the cells harboring SEC12 or DAP2 on a multicopy plasmid were pulse-labeled for 10 min and chased for 0--60 min. Sec12p and Dap2p were immunoprecipitated with the anti-Secl2p or anti-Dap2p antibody and then subjected to the second immunoprecipitation with either the same antibody, anti-cd--~6 mannose antibody, or anti-ed---~3 mannose antibody (Fig. 8) . To avoid ambiguity due to heterogeneous glycosylation, a half of each sample was treated with endo H. After SDS-PAGE, the radioactivity of each band was quantified by radioimaging analysis. The relative amounts of al---~6 and etl---)3 mannosyl modifications are shown in Table III as percentage over the second Sec12p or Dap2p immunoprecipitate. As described previously (d 'Enfert et al., 1991; Nishikawa and Nakano, 1993) , Secl2p acquires a significant amount of ed---~6 mannose modification after synthesis, 18% at 60 min chase in this particular experiment (Fig. 8 A) . This modification is obvious, but its rate is much slower than those of typical secretory proteins. This indicates that the exit from the ER is rate limiting for Secl2p. Furthermore, modification by etl---~3 linkage was hardly detected even at 60 min chase, indicating that the retrieval process operates quite efficiently as well.