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

effect was diminished by the deletion of RER1. In this context, their findings are consistent with our study, although the effect of the TMD was only partial in their work. In normal cells, the retention mechanism would mask the retrieval effect and vice versa. Successful dissection of two such signals has become possible by the systematic analysis of chimeric proteins and the use of the Arerl mutant in the present study. Further determination of the signals of Secl2p has been attempted at the level of amino acid residues. We have performed an extensive mutational analysis on the residues in the TMD and obtained a surprising result: Even the mutant TMD that has had all the polar and aromatic residues replaced by alanine (AIO of Fig. 12) can still localize the DSD molecule to the ER in an Rerlp-dependent manner. Since this TMD contains only alanine, valine, leucine, and isoleucine residues, one might argue that the recognition of the TMD signal by the retrieval system is not quite strict in terms of structure. However, we have also realized that the dependency on Rerlp is completely lost when most of these polar residues are replaced by leucine (L7 of Fig. 12) . Apparently, there is a difference between leucine and alanine residues in the Rerlp-dependent localization effect of the mutant TMD. One obvious difference between these two amino acids is the bulkiness of the side chain. In other words, they differ in the degree of hydrophobicity. By scrutinizing the Secl2p TMD and its mutant versions that retain the Rerlp dependency, we find that there is a gradient of hydrophobicity which peaks in the middle of the TMD. The hydrophobic core in the center that is flanked by less hydrophobic residues appears to be a feature of the Rerlp-dependent signals. From such a point of view, the Rerlp-dependent TMD of Sed4p has a similar profile, whereas the Rerlp-independent TMDs of Dap2p and Sec20p do not. In support of this idea, the artificial TMD consisting of 19 leucine residues does not show the Rerlp-dependent localization effect. Further experimental tests are possible and will be necessary to prove or disprove this:hypothesis.