Control of ubiquitination of proteins in rat tissues by ubiquitin conjugating enzymes and isopeptidases.
The activity of the ubiquitin-dependent proteolytic system in differentiated tissues under basal conditions remains poorly explored. We measured rates of ubiquitination in rat tissue extracts. Accumulation of ubiquitinated proteins increased in the presence of ubiquitin aldehyde, indicating that deubiquitinating enzymes can regulate ubiquitination. Rates of ubiquitination varied fourfold, with the highest rate in the testis. We tested whether ubiquitin-activating enzyme (E1) or ubiquitin-conjugating enzymes (E2s) could be limiting for conjugation. Immunodepletion of the E2s UBC2 or UBC4 lowered rates of conjugation similarly. Supplementation of extracts with excess UBC2 or UBC4, but not E1, stimulated conjugation. However, UBC2-stimulated rates of ubiquitination still differed among tissues, indicating that tissue differences in E3s or substrate availability may also be rate controlling. UBC2 and UBC4 stimulated conjugation half-maximally at concentrations of 10-50 and 28-44 nM, respectively. Endogenous tissue levels of UBC2, but not UBC4, appeared saturating for conjugation, suggesting that in vivo modulation of UBC4 levels can likely control ubiquitin conjugation. Thus the pool of ubiquitin conjugates and therefore the rate of degradation of proteins by this system may be controlled by E2s, E3s, and isopeptidases. The regulation of the ubiquitin pathway appears complex, but precise.
Rajapurohitam
Venkatesh
V
Department of Medicine, McGill University, Montreal, Canada H3A 2B2.
Bedard
Nathalie
N
Wing
Simon S
SS
D000818:Animals; D019759:Carbon-Nitrogen Lyases / Q000378:metabolism*; D007700:Kinetics; D008025:Ligases / Q000032:analysis / Q000378:metabolism; D008297:Male; D009928:Organ Specificity; D011506:Proteins / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D025801:Ubiquitin / Q000378:metabolism*; D044763:Ubiquitin-Conjugating Enzymes
D011506:Proteins; D025801:Ubiquitin; D044763:Ubiquitin-Conjugating Enzymes; C063109:ubiquitin-conjugating enzyme UBC4; D019759:Carbon-Nitrogen Lyases; C034300:isopeptidase; D008025:Ligases