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

ER localization information in the TMD completely~ although some mutations resulted in the decrease of efficiency. Interestingly, all these alanine mutants kept the Rerlp dependency. In the Arerl cells, the mutants produced halos as large as DSDm did. Upon this surprising result, we decided to construct another series of replacement using leucine instead of alanine. The result is also shown in Fig. 10 . N-L (N358L), Q-L (Q370L), and NQ-L (N-L plus Q-L) mutations formed larger halos than the corresponding alanine mutants, indicating that hydrophobicity of these residues is an important parameter. The Rerlp dependency was still seen with these mutants but became less apparent, especially with NQ-L. With the L7 mutant, in which serine, threonine, asparagine, and glutamine residues were all replaced by leucine, the Rerlp dependency finally disappeared. The difference between the alanine and leucine mutants suggests that the distribution of hydrophobicity in the TMD somehow affects its ability to localize the protein to the ER in the Rerlp-dependent manner. On the other hand, even with this L7 mutant, the secretion of s-factor was not as efficient as DDDm. We further proceeded to construct the LeuX19 mutant, which contains only 19 leucine residues in the TMD. This artificial TMD showed Rerlp-independent halo formation, but again the efficiency was not as good as DDDm. Since the expression levels of these mutant proteins were almost the same as DSDm and D D D m as determined by immunoblotting (data not shown), the decrease of the size of halos was not due to their reduced synthesis. In fact, immunofluorescence observation of the cells expressing L7 or LeuX19 showed staining of both the ER and the vacuoles (data not shown). The ER localization was not as strict as with the Secl2p TMD, but it was still obvious. The length of the stretch of leucine (LeuX13, LeuX15, LeuX17, LeuX21, LeuX23, and LeuX26) did not affect its localization property as long as we could test by the halo assay (M. Sato, unpublished data) . We also introduced some of these mutations in the TMD of SSSm and performed the halo assay. Similar tendencies were observed, though the halos were smaller because the defect was partly masked by the reten-