Ubiquitin-metallothionein fusion protein expression in yeast. A genetic approach for analysis of ubiquitin functions.
We have established a Saccharomyces cerevisiae genetic system that expresses the fusion protein ubiquitin-metallothionein. We have evaluated the effects of amino-terminal ubiquitination of metallothionein on the stability and function of metallothionein. The fusion protein of wild type ubiquitin and metallothionein was rapidly processed in vivo to release free ubiquitin and metallothionein. Site-directed mutants of ubiquitin-metallothionein expressed in yeast were used to study the specificity of the (alpha-NH2-ubiquitin) protein endopeptidases. The data suggest that amino-terminal ubiquitination is not a signal for the proteolysis of yeast metallothionein in yeast. We have also discovered that expression of selected ubiquitin mutants blocked the growth of yeast. The data suggest that in addition to its function as a proteolytic signal, ubiquitination of proteins plays multiple roles in the cell.
Butt
T R
TR
Department of Molecular Pharmacology, Smith Kline & French Laboratories, King of Prussia, Pennsylvania 19406-0939.
Khan
M I
MI
Marsh
J
J
Ecker
D J
DJ
Crooke
S T
ST
D003300:Copper / Q000494:pharmacology; D019327:Copper Sulfate; D004926:Escherichia coli / Q000235:genetics; D005822:Genetic Vectors; D007700:Kinetics; D008668:Metallothionein / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism; D014452:Ubiquitins / Q000235:genetics* / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014452:Ubiquitins; D003300:Copper; D008668:Metallothionein; D019327:Copper Sulfate