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 tried to determine which residues in the TMD of Secl2p are important for the ER localization. The TMD of Sec12p contains several polar and aromatic amino acid residues. These residues were first changed to alanine in a variety of combinations (Fig. 10) . The mutations were introduced into DSDm, whose ER localization was fulfilled only by virtue of the Sec12p TMD, and the effect was tested by the halo assay. F-A (F355A, F356A, and F359A) and SY-A ($366A, Y367A, and $372A) mutants did not produce any halo in the wild-type cells, indicating that they were completely retained in the ER/c/s-Golgi. FSY-A (F-A plus SY-A), N-A (N358A), and Q-A (Q370A) formed very small halos. The halo of NQ-A (N-A plus Q-A) was slightly larger than that of either N-A or Q-A. These mutants appear to be leaking out of the ER, but the majority is still localized to the ER. Even the A10 mutant, in which all of the polar and aromatic residues were replaced by alanine, is mostly located in the ER in the wild-type cells. It appears that none of these alanine mutants disrupts the The Journal of Cell Biology, Volume 134. 1996 ing Dap2p (B) were labeled with tran35S-label at 30°C for 10 min and chased for the indicated times. Secl2p and Dap2p were first immunoprecipitated with the respective antibodies. The immunoprecipitates were dissolved in 1% SDS, divided into three aliquots, and subjected to the second immunoprecipitation with the antibodies against Secl2p or Dap2p, etl--~6 mannosyl linkages (cd,6), and etl--~3 mannosyl linkages (cd,3). Each sample was further divided into two, treated with or without endo H, and analyzed by SDS-PAGE and radioimaging.