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

Abstract

Radioiodinated, native and denatured bovine serum albumin (albumin) P-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 8-lactoglobulin was degraded three times faster than undenatured p-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 P-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 P-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, 3577-35801. In this system both denatured and undenatured albumin and P-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.

Effect of heat shock on protein degradation in mammalian cells: involvement of the ubiquitin system.

“…Criteria for selective proteolysis by the ubiquitin system appear to include free N-terminal amino groups (Hershko et al, 1984), the identity of the N-terminal amino acid residue (Bachmair et al, 1986;Ferber and Ciechanover, 1986), oxidation of methionine residues (Hersko et al, 1986), amino acid analogue substitution and heatdenaturation (this paper). Earlier work in our laboratory showed that the degradation of some proteins micro-injected into HTC cells was accelerated if they were first denatured (Katznelson and Kulka, 1985). The denaturation of other proteins inhibited their degradation (Katznelson and Kulka, 1985).…”

Skeletal muscle proteasome can degrade proteins in an ATP-dependent process that does not require ubiquitin.

“…However, in reticulocyte extracts (8,20) and intact hepatocytes (21) proteins modified so that they cannot be conjugated to ubiquitin are still degraded by an ATP-activated process; probably the proteasome catalyzes this process. In MEL cells Waxman et al (22) failed to demonstrate ubiquitin-dependent proteolysis but did find a high-molecular weight ATP-dependent protease that functioned independently of ubiquitin; its properties resemble those of the proteasome purified here from muscle.…”

“…Several observations have suggested that a ubiquitinindependent system may also exist in the mammalian cytosol (8,20,21). For example, certain proteins modified so that they cannot be ligated to ubiquitin are still degraded in an ATP-stimulated fashion (8,20,21).…”

“…Several observations have suggested that a ubiquitinindependent system may also exist in the mammalian cytosol (8,20,21). For example, certain proteins modified so that they cannot be ligated to ubiquitin are still degraded in an ATP-stimulated fashion (8,20,21). In extracts of murine erythroleukemia cells, Waxman et al (22) failed to find a ubiquitin-dependent proteolytic process but demonstrated an ATP-requiring proteolytic activity of high molecular mass (>600 kDa) that functioned independently of ubiquitin.…”

The Regulation of Human Cyclin B Protein Levels by the Ubiquitin Proteolytic Pathway

“…Perhaps the simplest explanation is that nonubiquitinated p21 is directly recognized by the proteasome. Unstructured proteins can be directly recognized and degraded by proteasomes in vitro and in vivo without ubiquitination (Katznelson and Kulka, 1985;Wenzel and Baumeister, 1993;Michalek et al, 1996). Moreover, free p21 does not have a well-defined tertiary structure (Kriwacki et al, 1996(Kriwacki et al, , 1997.…”