Degradation of microinjected glycolytic enzymes in L6 myoblasts

Several glycolytic enzymes as well as horseradish peroxidase have been labelled by reductive methylation and microinjected into rat L6 myoblasts using the erythrocyte ghost fusion technique. Since endogenous protein in the recipient cells was labelled with [14C]leucine, the degradation of both injected and endogenous protein could be followed simultaneously. The experiments show that inhibitors of autophagy generally affect the catabolism of total endogenous protein to a greater extent than the injected proteins. Both lactate dehydrogenase and peroxidase are poorly susceptible to the inhibitors even though these two enzymes represent virtually the most and least stable, respectively, of the test proteins. Accordingly the results do not support the hypothesis that autophagy contributes more to the turnover of long half-life proteins. However, this interpretation should be treated cautiously because some of the injected proteins do not appear to be targeted to the cell cytosol by the fusion process, and the breakdown of such proteins is only poorly susceptible to inhibition by insulin, serum or cycloheximide.