Linked Life Data

10860499

Source:http://linkedlifedata.com/resource/pubmed/id/10860499

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2000-10-10
pubmed:abstractText
A general method for purification of any substrate of the ubiquitin pathway, the major eukaryotic proteolytic pathway, should utilize the common characteristic of covalent linkage of ubiquitin to substrate lysyl residues. The utility of a N-terminal histidine-tagged ubiquitin (HisUb) for in vivo conjugation and isolation of ubiquitinated proteins by metal chelation chromatography is conditioned by the requirement that HisUb conjugate to the same set of proteins as wild-type ubiquitin. Stringent in vivo tests with Saccharomyces cerevisiae strains expressing ubiquitins only from plasmids were performed to show that HisUb could substitute for wild-type ubiquitin. The utility of HisUb as a method for purification of proteins ubiquitinated in vivo was demonstrated by metal chelation chromatography of yeast extracts expressing HisUb and immunoblotting for Rpb1, the largest subunit of RNA polymerase II. A fraction of Rpb1 was present in the ubiquitinated form in vivo. The ability to use HisUb expression in transgenic organisms that retain expression of their endogenous ubiquitin genes was demonstrated through transgenic Arabidopsis thaliana expressing HisUb or its variant HisUbK48R. UbK48R is a version of ubiquitin capable of conjugation to proteins, but cannot serve as an attachment site for ubiquitin via the major in vivo interubiquitin linkage. Whereas transgenic plants expressing HisUb showed insignificant enrichment of ubiquitinated proteins, transgenic Arabidopsis lines expressing HisUbK48R gave a much better yield.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Jun
pubmed:issn
0003-2697
pubmed:author
pubmed:copyrightInfo
Copyright 2000 Academic Press.
pubmed:issnType
Print
pubmed:day
15
pubmed:volume
282
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
54-64
pubmed:dateRevised
2006-11-15
pubmed:meshHeading
pubmed-meshheading:10860499-Arabidopsis, pubmed-meshheading:10860499-Canavanine, pubmed-meshheading:10860499-Chromatography, Ion Exchange, pubmed-meshheading:10860499-Dose-Response Relationship, Drug, pubmed-meshheading:10860499-Electrophoresis, Polyacrylamide Gel, pubmed-meshheading:10860499-Histidine, pubmed-meshheading:10860499-Immunoblotting, pubmed-meshheading:10860499-Mutagenesis, Site-Directed, pubmed-meshheading:10860499-Nitrilotriacetic Acid, pubmed-meshheading:10860499-Nitrogen, pubmed-meshheading:10860499-Organometallic Compounds, pubmed-meshheading:10860499-Plants, Genetically Modified, pubmed-meshheading:10860499-Plasmids, pubmed-meshheading:10860499-Protein Binding, pubmed-meshheading:10860499-RNA Polymerase II, pubmed-meshheading:10860499-Saccharomyces cerevisiae, pubmed-meshheading:10860499-Transfection, pubmed-meshheading:10860499-Ubiquitins, pubmed-meshheading:10860499-Yeasts
pubmed:year
2000
pubmed:articleTitle
Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway.
pubmed:affiliation
Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, California 95616, USA.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, Non-U.S. Gov't