Effects of denaturation and methylation on the degradation of proteins in cultured hepatoma cells and in reticulocyte cell‐free systems

Radioiodinated, native and denatured bovine serum albumin (albumin) β‐lactoglobulin and cytochrome c were introduced into hepatoma tissue culture cells by erythrocyte‐ghost‐mediated microinjection, and their rates of degradation were compared. Denatured albumin was degraded at 20% of the rate of undenatured albumin, denatured β‐lactoglobulin was degraded three times faster than undenatured β‐lactoglobulin, while denatured and undenatured cytochrome c were degraded at the same rate. Thus, denaturation does not affect the rates of intracellular breakdown of microinjected proteins in a simple predictable way. Exhaustive methylation did not inhibit the degradation of denatured β‐lactoglobulin or albumin, indicating that, like their undenatured counterparts, they are not degraded via the ubiquitin pathway. In reticulocyte lysates, in the presence of ATP, denatured albumin and β‐lactoglobulin were broken down at slightly slower rates than the parent proteins. Exhaustive methylation of both denatured and undenatured proteins completely abolished their ATP‐dependent breakdown. This inhibition is consistent with the hypothesis that free – NH₂ groups are required for the attachment of ubiquitin prior to degradation in this system. Removal of an ammonium sulfate fraction from reticulocyte lysates produces a proteolytic system markedly different from the whole lysate [Speiser, S. & Etlinger, J. D. (1983) Proc. Natl Acad. Sci. USA 80, 3577–3580]. In this system both denatured and undenatured albumin and β‐lactoglobulin were degraded essentially independently of ATP. Methylation only slightly decreased the breakdown of denatured proteins, suggesting that they are not degraded via the ubiquitin pathway. A possible explanation of these results is that removal of the ammonium sulfate fraction unmasks an ATP‐independent proteolytic system unrelated to the ubiquitin pathway.