Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

Pickart, Cecile M.; Graziani, Lynne A.; Dosch, Susan F.

doi:10.1016/0003-9861(89)90201-4

Cited by 13 publications

(6 citation statements)

References 29 publications

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“…This observation is consistent with an expanded dependence on alternate targeting pathways in erythroid cells which is inferred from the greater variety and content of other E2 family isozymes in reticulocytes (29,34). The Ubc2 activity reported here for rabbit reticulocytes agrees with earlier estimates by immunological methods (53,54), confirming the validity of Ubc2 quantitation by direct thiolester formation.…”

Section: Hsubc2bc88a and Hsubc2bc88s-ubiquitin Oxyester Are Competitisupporting

confidence: 80%

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Siepmann

Bohnsack

Tokgöz

et al. 2003

Journal of Biological Chemistry

190

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Rate studies have been employed as a reporter function to probe protein-protein interactions within a biochemically defined reconstituted N-end rule ubiquitin ligation pathway. The concentration dependence for E1-catalyzed HsUbc2b/E2 14kb transthiolation is hyperbolic and yields K m values of 102 ؎ 13 nM and 123 ؎ 19 nM for high affinity binding to rabbit and human E1/Uba1 orthologs. Competitive inhibition by the inactive substrate and product analogs HsUbc2bC88A (K i ‫؍‬ 104 ؎ 15 nM) and HsUbc2bC88S-ubiquitin oxyester (K i ‫؍‬ 169 ؎ 17 nM), respectively, indicates that the ubiquitin moiety contributes little to E1 binding. Under conditions of rate-limiting E3␣-catalyzed conjugation to human ␣-lactalbumin, HsUbc2b-ubiquitin thiolester exhibits a K i of 54 ؎ 18 nM and is competitively inhibited by the substrate analog HsUbc2bC88S-ubiquitin oxyester (K i ‫؍‬ 66 ؎ 29 nM). In contrast, the ligase product analog HsUbc2bC88A exhibits a K i of 440 ؎ 55 nM with respect to the wild type HsUbc2b-ubiquitin thiolester, demonstrating that ubiquitin binding contributes to the ability of E3␣ to discriminate between substrate and product E2. A survey of E1 and E2 isoform distribution in selected cell lines demonstrates that Ubc2 isoforms are the predominant intracellular ubiquitin carrier protein. Intracellular levels of E1 and Ubc2 are micromolar and approximately equal based on in vitro quantitation by stoichiometric 125 I-ubiquitin thiolester formation. Comparison of intracellular E1 and Ubc2 pools with the corresponding ubiquitin pools reveals that most of the free ubiquitin in cells is present as thiolesters to the components of the conjugation pathways. The present data represent the first comprehensive analysis of protein interactions within a ubiquitin ligation pathway.The majority of short-lived cellular proteins are targeted for degradation by the 26 S proteasome in response to assembly of degradation signals on their surface comprising chains of ubiquitin moieties covalently linked through specific lysine residues (1). Target protein specificity for this process is determined in part by a large family of diverse ubiquitin-protein isopeptide ligases (E3) 1 that recognize specific features of the native structure (2-4), transposable trans-acting amino acid sequences (5-7), or exposed regions of non-native conformation (8). The activated ubiquitin required to drive isopeptide bond formation is donated by specific ubiquitin carrier proteins (E2/ Ubc), also termed ubiquitin-conjugating enzymes, in which the ubiquitin carboxyl terminus is bound as a thiolester to a conserved cysteine (9, 10). The apparent specificity of different isopeptide ligases for recognizing a single or limited number of E2 isozymes accounts for the large cohort of related carrier protein isoforms (9 -11). Some E2 moieties may contribute to the substrate specificity of their cognate E3 isozyme because they are able to associate with targets in the absence of ligase (12)(13)(14). In addition, a subset of E2 isozymes catalyzes formation of polyubiquiti...

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Section: Hsubc2bc88a and Hsubc2bc88s-ubiquitin Oxyester Are Competitisupporting

confidence: 80%

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Siepmann

Bohnsack

Tokgöz

et al. 2003

Journal of Biological Chemistry

190

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“…Others have reported tissue-specific expression of a component or several components of the ubiquitin conjugation/deconjugation pathway: for example, the neuronal-specific expression of a ubiquitin-dependent deconjugating enzyme, PGP 9.5 (30), the human spermspecific expression of an isozyme of ubiquitin activating enzyme (El) (31,32), and changes in conjugating enzyme (E2) composition during erythrocyte maturation (33)(34)(35). Developmentally regulated increases in ubiquitin gene expression have also been documented.…”

Section: Discussionmentioning

confidence: 99%

Developmentally regulated loss of ubiquitin and ubiquitinated proteins during pollen maturation in maize.

Callis¹,

Bedinger²

1994

Proc. Natl. Acad. Sci. U.S.A.

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Eukaryotic cells typically contain 0.2-1.0% of their total protein as t highly conserved protein ubiqui, which exists both free and covalently attached to chlr proteins. (7). Thus, ubiquitin conjugation plays multiple fundamental roles in cellular growth and physiology in diverse eukaryotes. In higher plants, production of gametophytes (the iN generation) is highly regulated and requires precise control of gene expression (8-10). The identification of numerous pollen-specific genes as well as effects of mitochondrial genome variation specifically on pollen development indicate that unique and specialized physiological processes occur in gametogenesis in higher plants (11-13

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“…Extracts were analyzed directly for E2-25K (see below). Stepwise anion-exchange fractionation was carried out to enrich for E2-20K and E2-230K before analysis, by a modification of a published procedure (24). Soluble extracts were applied to Q-Sepharose, and the loaded columns were washed with 4 vol of buffer containing 20 mM Tris-HCl (pH 7.5), 0.2mM EDTA, and protease inhibitors as above, followed by 3 vol of the same buffer containing 0.2 M NaCl.…”

Section: Methodsmentioning

confidence: 99%

Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation.

Wefes

Mastrandrea

Haldeman

et al. 1995

Proc. Natl. Acad. Sci. U.S.A.

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A global cellular reorganization occurs during the reticulocyte stage of erythroid differentiation. This reorganization is accomplished partly through programmed protein degradation. The selection of proteins for degradation can be mediated by covalent attachment of ubiquitin. We have cloned cDNAs encoding two ubiquitin-conjugating (E2) enzymes, E2-20K and E2-230K, and found their genes to be strongly induced during the differentiation of erythroblasts into reticulocytes. Induction ofthe E2-20K and E2-230K genes is specific, as transcript levels for at least two other ubiquitinating enzymes fall during erythroblast differentiation. In contrast to most proteins induced in reticulocytes, E2-20K and E2-230K enzymes are present at strongly reduced levels in erythrocytes and thus decline in abundance as reticulocyte maturation is completed. This result suggests that both enzymes function during the reticulocyte stage, when enhanced protein degradation has been observed. These data implicate regulated components of the ubiquitin conjugation machinery in erythroid differentiation.

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Murine erythroleukemia cells possess an active ubiquitin- and ATP-dependent proteolytic pathway

Cited by 13 publications

References 29 publications

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Protein Interactions within the N-end Rule Ubiquitin Ligation Pathway

Developmentally regulated loss of ubiquitin and ubiquitinated proteins during pollen maturation in maize.

Induction of ubiquitin-conjugating enzymes during terminal erythroid differentiation.

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