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 subcellular localization of these chimeras was examined in three different ways: halo assay, indirect immunofluorescence, and pulse-chase experiments. First, we un-dertook the halo assay method that was used to isolate rer mutants (Nishikawa and Nakano, 1993) . As illustrated in Fig. 2 , this method utilizes fusions containing Mfalp in the lumenal domain. If a particular fusion protein is transported to the late Golgi, Mfalp moiety would be processed by the Kex2 protease leading to the secretion of mature a-factor. Cells secreting a-factor form a halo when placed on an agar plate with a lawn of tester a strain as a result of the growth arrest of the a cells (Halo+). Accordingly, the Halo-phenotype should indicate that the fusion is retained in the ER or the early Golgi. Sec12-Mfalp (SSSm) behaves very much like the authentic Sec12p. It is mostly localized in the ER and does not yield a halo in the wild-type cells (Nishikawa and Nakano, 1993 We constructed a Dap2-Mfal fusion protein (DDDm) and tested whether the wild-type cells expressing this fusion (WT/DDDm) secrete a-factor by the halo assay. As shown in Fig. 4 , upper spot 2, WT/DDDm produced a large halo, indicating that this fusion protein was transported to the late Golgi quite efficiently. Furthermore, DDDm was found to be subject to degradation in the vacuole. Immunoblotting using the anti-Dap2p antibody showed that the amount of DDDm was higher than in the wild-type PEP4 cells when the major vacuolar proteolytic activities were disrupted (Apep4) (data not shown). Finally, the immunofluorescence observation of DDDm showed clear vacuolar staining in the Apep4 cells (Fig. 3 , C-E). These all indicate that DDDm is processed in the late Golgi, transported to the vacuole, and eventually degraded there (Fig. 2, gray arrows) . The presence or absence of the vacuolar proteolytic activities (PEP4 or Apep4) did not affect the size of halo produced from DDDm (data not shown). The radius of the halo is proportional to the logarithm of the amount of secreted a-factor and thus is a good measure of the passage of the fusion protein through the late Golgi compartment.