Degradation of Ornithine Decarboxylase in Reticulocyte Lysate Is ATP-dependent but Ubiquitin-independent

Z Bercovich,Y Rosenberg-Hasson,A Ciechanover,C Kahana

doi:10.1016/s0021-9258(18)71571-8

Z Bercovich, Y Rosenberg-Hasson + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(18)71571-8

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Abstract

Reticulocyte lysate contains all the components of the ubiquitin-dependent proteolytic system. Several proteins are degraded in reticulocyte lysate in a ubiquitin-dependent manner. However, none of the proteins studied has a short intracellular half-life. We have investigated the degradation of ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells. ODC is efficiently degraded in reticulocyte lysate depleted of the ubiquitin activating enzyme, E1, in fraction II of reticulocyte lysate completely lacking ubiquitin, and in fraction II depleted of the entire complex of enzymes responsible for the ligation of ubiquitin to target proteins. The degradation of ODC is ATP dependent. Therefore, our results demonstrate that in addition to the ubiquitin-dependent proteolytic pathway, reticulocyte lysate contains at least one additional ATP-dependent proteolytic pathway. In vitro synthesized ODC served as a substrate in the present degradation study. Its successful utilization establishes a general strategy for investigating the degradation of short-lived proteins (for which a corresponding cDNA is available), that constitute a very small fraction of cellular proteins and for which purification is difficult or impossible. In contrast to ODC synthesized in vitro, that isolated from cells was not degraded by the reticulocyte lysate degradation system, suggesting that post-translational modifications may be involved in regulating ODC degradation.

Highlights

Cellular proteins are in a dynamic state of synthesis and degradation
9-l 40 a Degradation of ornithine decarboxylase (ODC) in Reticulocyte Lysate-ODC is present in low concentration in mammalian cells (16)
The resulting RNA was translated in reticulocyte lysate (Promega Biotec) in the presence of [35S]methionine.Three ODC polypeptides were synthesized, a predominant 53-kDa authentic ODC protein, and two minor polypeptides of 40 and 30 kDa, respectively, that presumably represent initiations at internal methionines

Summary

RESULTS

Degradation of ODC in Reticulocyte Lysate-ODC is present in low concentration in mammalian cells (16). Its quantitative purification presents a difficult task To overcome this difficulty and to establisha general method for investigating the degradation of short-lived proteins, we have utilized an in vitro translated product as substratefor degradation. In the absence of ATP and an ATP-regenerating system, ODC degradation was not observed (Fig. 1B). This result clearly demonstrates that in reticulocyte lysate, ODC degradation requires ATP. In contrast to theefficient degradation of ODC synthesized in vitro, the enzyme isolated from mammalian cells was completely stable during h of incubation in the reticulocyte lysate-based degradation mix (not shown).

ODC degradation was monitored in reticulocyte lysate degradation

DISCUSSION

In contrast tion vitro synthesized ODC that was efficiently