Specific disulfide cleavage is required for ubiquitin conjugation and degradation of lysozyme.
Both ubiquitin conjugation and ubiquitin-dependent degradation of chicken egg white lysozyme in a reticulocyte lysate depend on the presence of a reducing agent. We present evidence that the reduction of a specific disulfide bond, namely that at Cys6-Cys127, facilitates ubiquitination and is a prerequisite to the formation of a multiubiquitin chain on one of at least four chain initiation sites on lysozyme. The Cys6-Cys127 disulfide bond in lysozyme can be specifically reduced, and the modified protein can be isolated after carboxymethylation of the 2 resulting cysteines. This modified lysozyme no longer requires the presence of a reducing agent for ubiquitin conjugation and degradation. Inhibition of ubiquitination by the dipeptide Lys-Ala revealed that this modified lysozyme, like the unmodified protein, is recognized via the binding of the ubiquitin protein ligase, E3, to the substrate's N-terminal lysyl residue. Both the rate and the extent of ubiquitin-lysozyme conjugation, however, are significantly higher with this modified substrate. Likewise, ubiquitin-dependent degradation of 6,127-reduced/carboxymethylated lysozyme was 2-4-fold faster than degradation of the unmodified counterpart. These results are consistent with an interpretation that the modified lysozyme mimics an intermediate formed at the rate-limiting step of the degradation of lysozyme in the reticulocyte lysate. Reduction of the Cys6-Cys127 disulfide bond is expected to unhinge the N-terminal region of lysozyme, and we propose that the recognition of this otherwise stable protein by the ubiquitin pathway is due to facilitated binding of E3 that results from such a conformational transition.
Dunten
R L
RL
Department of Chemistry and Biochemistry, University of California, Los Angeles 90024.
Cohen
R E
RE
Gregori
L
L
Chau
V
V
D002852:Chromatography, Ion Exchange; D002942:Circular Dichroism; D004531:Eggs; D004591:Electrophoresis, Polyacrylamide Gel; D009113:Muramidase / Q000737:chemistry*; D010084:Oxidation-Reduction; D013056:Spectrophotometry, Ultraviolet; D013438:Sulfhydryl Compounds / Q000737:chemistry*; D014452:Ubiquitins / Q000737:chemistry*
D013438:Sulfhydryl Compounds; D014452:Ubiquitins; D009113:Muramidase