Rescue of the Complex Temperature-sensitive Phenotype of Chinese Hamster Ovary E36ts20 Cells by Expression of the Human Ubiquitin-activating Enzyme CDNA

Overview

Journal Biochem J

Specialty Biochemistry

Date 1994 Dec 15

PMID 7818464

Citations 12

Authors

J S Trausch-Azar

A Ciechanover

A L Schwartz

Affiliations

Soon will be listed here.

Abstract

The ubiquitin conjugation system is a multi-step pathway in which ubiquitin is activated and conjugated to acceptor proteins, one function of which is to target acceptor proteins for rapid degradation within the cell. The conjugation system is involved in many aspects of cellular functions, including the cell cycle. Several cell-cycle arrest mutant cell lines have been characterized and appear to harbour a mutant ubiquitin-activating enzyme, E1, as their primary defect. One such cell line is ts20, which is derived from Chinese hamster ovary E36 cells. This cell line has been used to characterize some of the potential functions of the ubiquitin conjugation system in vivo, such as its involvement in the maturation of autophagic vacuoles. The present study describes the complete rescue of the complex ts20 phenotype following the expression of the cDNA for human E1. Stable transfectants expressing the human E1 cDNA in the CMVneo expression vector were measured for ubiquitin-conjugation activity, protein degradation and growth in culture at the nonpermissive temperature. This rescue confirms that the phenotype observed in the ts20 cells is due to a defect in the E1 enzyme. Thus, the ts20 cell line will serve as a useful tool to delineate the functions of the ubiquitin system in vivo.

Citing Articles

Shared and distinct mechanisms of UBA1 inactivation across different diseases.

Collins J, Magaziner S, English M, Hassan B, Chen X, Balanda N EMBO J. 2024; 43(10):1919-1946.

PMID: 38360993 PMC: 11099125. DOI: 10.1038/s44318-024-00046-z.

Novel Somatic UBA1 Variant in a Patient With VEXAS Syndrome.

Stiburkova B, Pavelcova K, Belickova M, Magaziner S, Collins J, Werner A Arthritis Rheumatol. 2023; 75(7):1285-1290.

PMID: 36762418 PMC: 10821773. DOI: 10.1002/art.42471.

Estimated Prevalence and Clinical Manifestations of UBA1 Variants Associated With VEXAS Syndrome in a Clinical Population.

Beck D, Bodian D, Shah V, Mirshahi U, Kim J, Ding Y JAMA. 2023; 329(4):318-324.

PMID: 36692560 PMC: 10408261. DOI: 10.1001/jama.2022.24836.

The evolving role of ubiquitin modification in endoplasmic reticulum-associated degradation.

Preston G, Brodsky J Biochem J. 2017; 474(4):445-469.

PMID: 28159894 PMC: 5425155. DOI: 10.1042/BCJ20160582.

Roles for the ubiquitin-proteasome pathway in protein quality control and signaling in the retina: implications in the pathogenesis of age-related macular degeneration.

Shang F, Taylor A Mol Aspects Med. 2012; 33(4):446-66.

PMID: 22521794 PMC: 3417153. DOI: 10.1016/j.mam.2012.04.001.

References

Laemmli U . Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970; 227(5259):680-5. DOI: 10.1038/227680a0. View

Michalek M, Grant E, Gramm C, GOLDBERG A, Rock K . A role for the ubiquitin-dependent proteolytic pathway in MHC class I-restricted antigen presentation. Nature. 1993; 363(6429):552-4. DOI: 10.1038/363552a0. View

Ciechanover A, Elias S, Heller H, Hershko A . "Covalent affinity" purification of ubiquitin-activating enzyme. J Biol Chem. 1982; 257(5):2537-42. View

Haas A, WARMS J, Hershko A, Rose I . Ubiquitin-activating enzyme. Mechanism and role in protein-ubiquitin conjugation. J Biol Chem. 1982; 257(5):2543-8. View

Hershko A, Heller H, Elias S, Ciechanover A . Components of ubiquitin-protein ligase system. Resolution, affinity purification, and role in protein breakdown. J Biol Chem. 1983; 258(13):8206-14. View

Parker C, Topol J . A Drosophila RNA polymerase II transcription factor contains a promoter-region-specific DNA-binding activity. Cell. 1984; 36(2):357-69. DOI: 10.1016/0092-8674(84)90229-0. View

Finley D, Ciechanover A, Varshavsky A . Thermolability of ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. Cell. 1984; 37(1):43-55. DOI: 10.1016/0092-8674(84)90299-x. View

Ciechanover A, Finley D, Varshavsky A . The ubiquitin-mediated proteolytic pathway and mechanisms of energy-dependent intracellular protein degradation. J Cell Biochem. 1984; 24(1):27-53. DOI: 10.1002/jcb.240240104. View

Pickart C, Rose I . Functional heterogeneity of ubiquitin carrier proteins. J Biol Chem. 1985; 260(3):1573-81. View

10.

Yarden Y, Escobedo J, Kuang W, Yang-Feng T, Daniel T, Tremble P . Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986; 323(6085):226-32. DOI: 10.1038/323226a0. View

11.

Chomczynski P, Sacchi N . Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987; 162(1):156-9. DOI: 10.1006/abio.1987.9999. View

12.

Chen C, Okayama H . High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987; 7(8):2745-52. PMC: 367891. DOI: 10.1128/mcb.7.8.2745-2752.1987. View

13.

KULKA R, Raboy B, Schuster R, Parag H, Diamond G, Ciechanover A . A Chinese hamster cell cycle mutant arrested at G2 phase has a temperature-sensitive ubiquitin-activating enzyme, E1. J Biol Chem. 1988; 263(30):15726-31. View

14.

Mayer A, Gropper R, Schwartz A, Ciechanover A . Purification, characterization, and rapid inactivation of thermolabile ubiquitin-activating enzyme from the mammalian cell cycle mutant ts85. J Biol Chem. 1989; 264(4):2060-8. View

15.

Zacksenhaus E, Sheinin R . Molecular cloning, primary structure and expression of the human X linked A1S9 gene cDNA which complements the ts A1S9 mouse L cell defect in DNA replication. EMBO J. 1990; 9(9):2923-9. PMC: 552008. DOI: 10.1002/j.1460-2075.1990.tb07483.x. View

16.

Hatfield P, Callis J, Vierstra R . Cloning of ubiquitin activating enzyme from wheat and expression of a functional protein in Escherichia coli. J Biol Chem. 1990; 265(26):15813-7. View

17.

Kudo M, Sugasawa K, Hori T, Enomoto T, Hanaoka F, Ui M . Human ubiquitin-activating enzyme (E1): compensation for heat-labile mouse E1 and its gene localization on the X chromosome. Exp Cell Res. 1991; 192(1):110-7. DOI: 10.1016/0014-4827(91)90164-p. View

18.

Scheffner M, Werness B, Huibregtse J, Levine A, Howley P . The E6 oncoprotein encoded by human papillomavirus types 16 and 18 promotes the degradation of p53. Cell. 1990; 63(6):1129-36. DOI: 10.1016/0092-8674(90)90409-8. View

19.

Glotzer M, Murray A, Kirschner M . Cyclin is degraded by the ubiquitin pathway. Nature. 1991; 349(6305):132-8. DOI: 10.1038/349132a0. View

20.

Ciechanover A, DiGiuseppe J, Bercovich B, Orian A, Richter J, Schwartz A . Degradation of nuclear oncoproteins by the ubiquitin system in vitro. Proc Natl Acad Sci U S A. 1991; 88(1):139-43. PMC: 50765. DOI: 10.1073/pnas.88.1.139. View