A temperature-sensitive Krp1 allows in vivo characterization of kexin activation

Members of the kexin family of processing enzymes are responsible for the c leavage of many proproteins during their transport through the secretory pa thway. The enzymes are themselves made as inactive precursors and we have i nvestigated the activation of Krp1, a kexin from the fission yeast Schizosa ccharomyces pombe. As Krp1 is essential for cell growth, we have used a krp 1(ts) strain to investigate the role of the prosequence in the activation p rocess. Mutations that reduce either the efficiency with which the proseque nce is released or the rate at which the released prosegment is subsequentl y cleaved at an internal site are less active when assayed in vivo. We also show that prosegments lacking an internal dibasic motif can act as autoinh ibitors and prevent activation of the catalytic fragment. Krp1 constructs c ontaining prosequences based on these inhibitors do not become active in vi tro. Surprisingly, the same constructs do become active in the intact cell and appear to suggest that alternative activation processes can be used by these enzymes.