The Yeast KEX-2-Processing Endoprotease Is Active in the Golgi Apparatus of Transfected NIH 3T3 Fibroblasts

Proteolytic processing of polyprotein precursors at pairs of basic amino acids is a prerequisite for the generation of bioactive peptide hormones. While the mammalian endoproteases responsible for these cleavages are yet to be identified, this function has been unequivocally asssigned in yeast to the product of the KEX-2 gene. To study the molecular mechanisms involved in polyprotein processing, we have transfected the yeast KEX-2 gene into mouse NIH 3T3 fibroblasts and established a new cell line (called 2N-DK) where the KEX-2 endoprotease is permanently expressed. Immunofluorescence studies show that the KEX-2 enzyme is retained within the Golgi of the 2N-DK cells. The evidence for this cellular location is supported by measurement of intracellular and extracellular KEX-2 enzyme activity. In this permanently transfected cell line, KEX-2 activity is exclusively intracellular, in contrast to the situation previously described in transiently infected cell lines, where extracellular KEX-2 activity was detec...