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

The above conclusion has been obtained from the experiments using chimeric fusions between Sec12p and a reporter protein Dap2p. Since Dap2p, a vacuolar membrane protein, is believed to be innocent for its destination and is transported to the vacuole by default under normal conditions (Roberts et al., 1992) , the effects of the Sec12p domains on localization can be directly tested on chimeric proteins. The results of biochemical and morphological experiments demonstrate that either the TMD or the cytoplasmic domain of Sec12p causes ER localization when included in the chimera. The actions of these two domains are independent of each other. The TMD alone can localize the protein to the ER quite efficiently, while the cyto- Figure I1 . The effect of the TMDs of Sed4p and Sec20p on ER localization. (.4) The TMD of DDDm was replaced by that of Sed4p and Sec20p to construct D4Dm and D20Dm, respectively. Amino acid sequences of the junction regions are shown. (B) The secretion of mature a-factor was examined by the halo assay using wild-type (SNY9) and Arerl (SKY7) cells.