Cloning and Expression of a Yeast Ubiquitin-Protein Cleaving Activity in Escherichia Coli

Miller, Heather G.; Henzel, William J.; Ridgway, John Brady; Kuang, Wun-Jung; Chisholm, Vanessa; Liu, Chung-Cheng

doi:10.1038/nbt0789-698

Cited by 55 publications

(49 citation statements)

References 37 publications

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“…Recombinant bovine E2-25K harboring two catalytically silent mutations was expressed in Escherichia coli using the vector pET3d-C170S,F174L-25K and purified as described (30). Yeast ubiquitin hydrolase-1 was prepared by a slight modification of the method described previously (31). Ubal was prepared by either of two methods described previously (32,33).…”

Section: Methodsmentioning

confidence: 99%

Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

Piotrowski¹,

Beal²,

Hoffman³

et al. 1997

Journal of Biological Chemistry

213

211

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Ubiquitin is a covalent signal that targets cellular proteins to the 26 S proteasome. Multiple ubiquitins can be ligated together through the formation of isopeptide bonds between Lys 48 and Gly 76 of successive ubiquitins. Such a polyubiquitin chain constitutes a highly effective proteolytic targeting signal, but its mode of interaction with the proteasome is not well understood. Experiments to address this issue have been limited by difficulties in preparing useful quantities of polyubiquitin chains of uniform length. We report a simple method for large scale synthesis of Lys 48 -linked polyubiquitin chains of defined length. In the first round of synthesis, two ubiquitin derivatives (K48C-ubiquitin and Asp 77 -ubiquitin) were used as substrates for the well characterized ubiquitin-conjugating enzyme E2-25K. Diubiquitin blocked at the nascent proximal and distal chain termini was obtained in quantitative yield. Appropriately deblocked chains were then combined to synthesize higher order chains (tetramer and octamer in the present study). Deblocking was achieved either enzymatically (proximal terminus) or by chemical alkylation (distal terminus). Chains synthesized by this method were used to obtain the first quantitative information concerning the influence of polyubiquitin chain length on binding to the 26 S proteasome; this was done through comparison of different length (unanchored) polyubiquitin chains as inhibitors of ubiquitin-conjugate degradation. K 0.5 was found to decrease ϳ90-fold, from 430 to 4.8 M, as the chain was lengthened from two to eight ubiquitins. The implications of these results for the molecular basis of chain recognition are discussed.

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Section: Methodsmentioning

confidence: 99%

Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

Piotrowski¹,

Beal²,

Hoffman³

et al. 1997

Journal of Biological Chemistry

213

211

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“…To this aim, we therefore added an option relative to the overexpression of any favorite protein in the TermiNator2 prediction tool. If the protein has an N terminus associated with a risk of nonprocessing, various methods may be used to prevent Met retention: (i) adjusting expression conditions to optimize MAP activity according to the rate of protein synthesis in vivo (58), (ii) in vitro processing with purified wild-type or engineered MAP (59 -62), (iii) dual co-overproduction of MAP and the protein of interest in vivo (25,63,64), (iv) processing in vitro or in vivo of the N terminus with another amino-or endoprotease (65-67), or (v) fusion of the ORF to a propeptide or ubiquitin sequence and use of endogenous signal peptidase specificity (68,69). This last strategy has also proved successful for residues that cannot be unmasked by NME (this study, Fig.…”

Section: Nme Prediction Of Natural Bacterial Substrates and Recombinamentioning

confidence: 99%

The Proteomics of N-terminal Methionine Cleavage

Frottin

Martinez

Peynot

et al. 2006

Molecular & Cellular Proteomics

344

350

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“…The nascent polyubiquitin proteins are proteolyticalty processed to active monomers. Activities capable of cleaving m-linked ubiquitin in polyubiquitin or ubiquitin extension protein fusions have been described (Baker et al, 1992;Jonnalagadda et al, 1989;Miller et aL, 1989;Sullivan et aL, 1990).…”

Section: Introductionmentioning

confidence: 99%

Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes

Sun

Callis²

1997

The Plant Journal

122

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SummaryThe highly conserved protein ubiquitin is encoded by five polyubiquitin genes in Arabidopsis thaliana ecotype Columbia that have been divided into two subtypes, the UBQ31UBQ4 subtype and the UBQ101UBQ111UBQ14 subtype. Northern analysis using gene-specific oligonucleotides as hybridization probes and enzyme activity measurements from transgenic plants expressing J~-glucuronidase (GUS) under the control of individual polyubiquitin 5' flanking regions were used to determine the developmental and environmental regulation of polyubiquitin transcription and mRNA accumulation. Polyubiquitin mRNA levels within and between subtypes were independently modulated. UBQ3 mRNA levels were threefold higher than UBQ4 mRNA levels in vegetative organs, but only two-thirds of the UBQ4 mRNA levels in flowers. UBQ3 mRNA was modulated by dark/light treatments, while mRNAs from UBQ4 and all members of the other subtype were unaffected. Similarly, within the UBQIO/ UBQ11/UBQ14 subtype, UBQ11/UBQ14 mRNAs were modulated differently in seedlings after a two-hour heatshock treatment. Among all the polyubiquitin genes, UBQIO mRNA level was the most constant in all organs and environmental conditions examined. Transgenic plants transformed with a UBQIO 5' flanking region::GUS gene contained higher levels of GUS activity than transgenic plants expressing GUS under the control of UBQ3 5' flanking regions. In conclusion, the relative abundance of different Arabidopsis polyubiquitin mRNAs, even those produced from highly similar genes within a subtype, appears to be modulated independently in response to developmental and environmental cues.

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Cloning and Expression of a Yeast Ubiquitin-Protein Cleaving Activity in Escherichia Coli

Cited by 55 publications

References 37 publications

Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

Inhibition of the 26 S Proteasome by Polyubiquitin Chains Synthesized to Have Defined Lengths

The Proteomics of N-terminal Methionine Cleavage

Independent modulation of Arabidopsis thaliana polyubiquitin mRNAs in different organs and in response to environmental changes

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