REPLACEMENT OF ALL ALPHA-DOMAIN LYSINES WITH GLUTAMATES REDUCES METALLOTHIONEIN DETOXIFICATION FUNCTION

Mammalian metallothioneins (MTs) possess eight highly conserved lysine residues, including three in each of two metal binding domains. We us ed site-directed mutagenesis to replace these intradomain lysines in C hinese hamster ovary MT2 with glutamic acid and/or glutamine. These mu tant MTs were expressed in a metal sensitive yeast host. One mutant wh ich had all three lysines in the alpha-domain replaced by glutamates ( K43,51,56E) exhibited a reduced ability, relative to native MT, to pro tect yeast transformants against otherwise toxic levels of cadmium. Th is triply substituted mutant also exhibited anomalous migration on a n on-denaturing gel relative to wild type MT and other MT lysine mutants , suggesting that the intradomain lysines are important in maintaining the conformational integrity of MT. (C) 1994 Academic Press, Inc.