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

We further examined whether any parts of other ER membrane proteins could act as a signal to localize Dap2p to the ER. Sed4p and Sec20p are also type II transmembrane proteins in the ER but have the H D E L sequence at their C O O H terminus, unlike Secl2p (Hardwick et al., 1992; Gimeno et al., 1995; Sweet and Pelham, 1992) . Sed4p shows a striking structural similarity to Secl2p in the NH2terminal cytoplasmic domain and the TMD (45% identical) but not in the COOH-terminal lumenal domain. Sec20p has no homology to Secl2p. It has been shown that the H D E L sequence of Sec20p is important for its ER localization (Sweet and Pelham, 1992) . We constructed D4Dm and D20Dm (Fig. 11 A) by replacing the TMD of Dap2p by that of Sed4p or Sec20p. The behavior of these chimeras in wild-type and Arerl cells was tested by the halo assay (Fig. 11 B) . D4Dm did not produce any halo in the wild-type cells, indicating that the TMD of Sed4p is sufficient for the ER localization. Subcellular localization of D4Dm was also examined by immunofluorescence, which showed strict ER staining similar to that of DSDm (Fig. 12 B) . This effect was dependent on Rerlp because D4Dm produced a halo in drerl (Fig. 11 B) . Thus, the Rerlp-dependent mechanism of ER localization commonly operates on the TMD of Secl2p and Sed4p. In contrast, D20Dm formed a large halo in either wild-type or Arerl cells (Fig. 11 B) . This clearly indicates that the Rerlp-dependent system is not effective to all transmembrane ER proteins. It apparently discriminates Sec20p from Secl2p and Sed4p. In the case of Sec20p, other motifs including the H D E L sequence might decide the localization.