H Heller

Occurrence of a polyubiquitin structure in ubiquitin-protein conjugates

In the ubiquitin-mediated pathway for the degradation of intracellular proteins, several molecules of ubiquitin are linked to the protein substrate by amide linkages. It was noted that the number of ubiquitin-protein conjugates and their apparent molecular size are higher than expected from the number of amino groups in the protein. When the amino groups of ubiquitin were blocked by reductive methylation, it was efficiently conjugated to lysozyme, but the higher-molecular-weight conjugates were not formed. This suggests that the higher-molecular-weight conjugates with native ubiquitin contain structures in which one molecule of ubiquitin is linked to an amino group of another molecule of ubiquitin. Methylated ubiquitin stimulated protein breakdown at about one half the rate obtained with native ubiquitin, and isolated conjugates of 125I-lysozyme with methylated ubiquitin were broken down by reticulocyte extracts. These findings indicate that the formation of polyubiquitin chains is not obligatory for protein breakdown, though it may accelerate the rate of this process.

MODE OF DEGRADATION OF ABNORMAL GLOBIN CHAINS IN RABBIT RETICULOCYTES

Publisher Summary This chapter discusses the mode of degradation of abnormal globin chains in rabbit reticulocytes. The reticulocyte contains a highly active and selective protein degrading system, but the biochemical characteristics of this system have not yet been defined. For SDS-polyacrylamide gel electrophoresis, samples of highly labeled reticulocytes were washed in ice-cold phosphate-buffered saline and boiled immediately for 5 min in 2% mercaptoethano1 and 2% SDS. The initial reaction(s) in the degradation of abnormal globin molecules is strongly rate-limiting and the subsequent proteolysis of intermediary cleavage fragments is much faster. One of the striking features of the degradation of analog-containing reticulocyte protein is its marked kinetic heterogeneity. A most important, and probably a most difficult question concerning the cell-free system is whether it is similar to that responsible for the degradation of abnormal globin in intact cells.

Alterations in components of the ubiquitin-protein ligase system following maturation of reticulocytes to erythrocytes

Previous studies have shown that the activity of the ubiquitin-mediated proteolytic system declines markedly following reticulocyte maturation, but the specific alterations responsible for this phenomenon have not been defined. We find that the rate of ATP-dependent degradation of 125I-albumin is reduced 20-fold in lysates of rabbit erythrocytes, as compared to reticulocyte lysates. The activity of the proteolytic system in erythrocyte extracts can be restored by supplementation with components of the ubiquitin-protein ligase system purified from reticulocytes by affinity chromatography. These components are the ubiquitin-carrier protein E2, the activity of which is nearly completely absent, and the ligase E3, the activity of which is partially reduced in erythrocytes. Erythrocyte extracts contain other ligases which attach a single, or a few ubiquitin molecules to proteins; these products are different from the multi-ubiquitin derivatives which are formed by the ligase system of protein breakdown. Mature red cells may thus serve to distinguish between different ubiquitin-protein ligase systems with presumably different functions.