Characterization of a temperature-sensitive mutant of a ubiquitin-conjugating enzyme and its use as a heat-inducible degradation signal.
The ubiquitin/proteasome pathway is a highly conserved mechanism of proteolysis in all eukaryotes. Ubiquitin (Ub) is conjugated to proteolytic substrates through the sequential action of ubiquitin-activating (E1/Uba) and ubiquitin-conjugating (E2/Ubc) enzymes. The mechanism of substrate recognition and ubiquitination is an area of active investigation, and we have begun a site-directed mutagenesis approach to define the biochemical and biophysical properties of ubiquitin-conjugating enzymes. We have characterized a specific mutation in Ubc4 (Ubc4(P62S)) which was previously shown to cause a temperature-sensitive growth defect in several other Ubc's. Ubc4(P62S) was rapidly degraded in vivo, contributing to the loss of function. However, reconstitution experiments revealed that the catalytic activity of Ubc4(P62S) was reversibly inactivated at 37 degrees C, demonstrating that the primary defect of Ubc4(P62S) is its inability to form a ubiquitin thioester bond at high temperature. The in vivo defect is compounded by increased susceptibility of Ubc4(P62S) to degradation by the ubiquitin/proteasome pathway. We have exploited the temperature-dependent degradation of the P62S mutant to destabilize an otherwise stable test protein (glutathione S-transferase). The use of this mutant may provide a useful cis-acting temperature-inducible degradation signal.
Department of Biochemistry, Robert Wood Johnson Medical School-UMDNJ, 675 Hoes Lane, Piscataway, New Jersey 08854, USA.
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Tongaonkar
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Beck
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Shinde
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Madura
D019943:Amino Acid Substitution; D002942:Circular Dichroism; D003001:Cloning, Molecular / Q000379:methods; D003545:Cysteine; D004795:Enzyme Stability; D006358:Hot Temperature; D007700:Kinetics; D008025:Ligases / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D016133:Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D013816:Thermodynamics; D044763:Ubiquitin-Conjugating Enzymes
D011993:Recombinant Fusion Proteins; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D008025:Ligases; D003545:Cysteine