Histidine-tagged ubiquitin substitutes for wild-type ubiquitin in Saccharomyces cerevisiae and facilitates isolation and identification of in vivo substrates of the ubiquitin pathway.
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.
Ling
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Section of Molecular and Cellular Biology, University of California, Davis, 1 Shields Avenue, Davis, California 95616, USA.
Colón
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Dahmus
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Callis
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D017360:Arabidopsis / Q000737:chemistry / Q000235:genetics; D002172:Canavanine / Q000494:pharmacology; D002852:Chromatography, Ion Exchange; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000378:metabolism*; D015151:Immunoblotting; D016297:Mutagenesis, Site-Directed; D009571:Nitrilotriacetic Acid / Q000031:analogs & derivatives / Q000737:chemistry; D009584:Nitrogen / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D012319:RNA Polymerase II / Q000737:chemistry / Q000302:isolation & purification; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics; D014162:Transfection; D014452:Ubiquitins / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology*; D015003:Yeasts / Q000737:chemistry / Q000235:genetics
D009942:Organometallic Compounds; D014452:Ubiquitins; C088321:nickel nitrilotriacetic acid; D002172:Canavanine; D006639:Histidine; D012319:RNA Polymerase II; D009571:Nitrilotriacetic Acid; D009584:Nitrogen