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

To demonstrate the secretion of the soluble forms of Kexlp, $86-16 (kex/-A2) was transformed with pKX1-8 (Kexlp) or pKXI-18AMS (Kexlp-AMS), and cells were then spheroplasted. The spheroplasts were radiolabeled (10 min), chased (30 min), and solubilized with SDS, and Kexlp was immunoprecipitated. Media, containing proteins exported beyond the plasma membrane, were concentrated and Kexlp was immunoprecipitated. Wild-type Kexlp remained associated with the spheroplasts, whereas Kexlp-AMS was shown to be secreted from the cell; the Kexlp-AMS signal associated with the cell fraction diminished with time while the signal from the medium showed the reverse trend (Fig. 5) . Thus, the results of the pulse-chase analysis correlated with the activity data and indicated that Kexlp-AMS, a soluble form of Kexlp, was secreted from the cell.