ENDOPROTEOLYTIC PROCESSING OF RECOMBINANT PROALBUMIN VARIANTS BY THE YEAST KEX2 PROTEASE

The yeast Kex2 protease is regarded as the prototype of the eukaryotic family of subtilisin-like serine proteases involved in processing aft er dibasic amino acid sequences. Here we investigate the specificity o f Kex2 using recombinant human proalbumin variants. Proalbumins with t he processing site sequences Arg-Arg and Lys-Arg were cleaved after th e dibasic sequence at approximately the same rate by Kex2 in vitro, an d yeast expressing either of these sequences secreted mature albumin i nto the culture medium. As expected, the Arg-Gly-Val-Phe-His-Arg-album in (proalbumin Lille) was not a substrate for Kex2 and neither was the Arg-Gly-Arg-Phe-His-Arg-albumin. In contrast to the mammalian endopro teases furin and the hepatic proalbumin convertase, the Kex2 protease was adversely affected by a P4 arginine. There was an 85% decrease in the cleavage of Arg-Gly-Arg-Phe-Arg-Arg-albumin compared with normal; also chicken proalbumin with an Arg-Phe-Ala-Arg processing site sequen ce was not a substrate for Kex2. A P1' arginine had a marked negative effect on processing and N-terminal sequence analysis confirmed that c leavage was occurring at the P1-P1' bond. The sequence context surroun ding the classical dibasic site is critical in determining susceptibil ity to cleavage by the Kex2 protease.