Yeast Kex1p is a Golgi-associated membrane protein: deletions in a cytoplasmic targeting domain result in mislocalization to the vacuolar membrane

A number of carboxy-terminal truncations of Kexlp were produced to test the above possibilities. The construction of these mutations via site-specific mutagenesis or nonsense linker insertion is described in Materials and Methods and the predicted proteins are shown in Fig. 3 . Immunoprecipitations of the mutant Kexlp proteins were performed to confirm that they were correctly synthesized by the cell (Fig. 4) . All of the truncated proteins were smaller than Kexlp by the predicted amount and entered the secretory pathway as shown by the addition of Ash-linked glycosylation (data not shown). A concern was that the mutants designed to remain membrane associated may not attain a stable type I orientation and remain in the ER. The most severely truncated mutant in this class, Kexlp-Hpa, lacks the entire endogenous cytoplasmic domain; it was further analyzed and shown to receive Asn-linked glycosylation which was further modified within the Golgi apparatus (Fig. 4) . Previous sodium carbonate extractions of whole cell extracts demonstrated that Kexlp fractionated with the membrane containing pellet fraction whereas the soluble protein, Kexlp-AMS, fractionated with the supernatant (Cooper and Bussey, 1989) . Carbonate treatment of whole cell extracts showed that Kexlp-Hpa fractionated with the membrane pellet (Fig. 4 , lanes 5 and 6), consistent with it being an integral membrane protein. In addition, we have shown that CPY, a soluble Kexlplike protein, is fully soluble under these extraction conditions (data not shown).