Publication Information
Saracci et al., 2009
No external accession available
Abstract
Plant J. 2009 Jul;59(2):344-58. doi: 10.1111/j.1365-313X.2009.03862.x. Epub 2009
Mar 9.
Tandem affinity purification and mass spectrometric analysis of ubiquitylated
proteins in Arabidopsis.
Saracco SA(1), Hansson M, Scalf M, Walker JM, Smith LM, Vierstra RD.
Author information:
(1)Department of Genetics, University of Wisconsin-Madison, Madison, WI
53706-1574, USA.
Protein ubiquitylation is a central regulatory mechanism that controls numerous
processes in plants, including hormone signaling, developmental progression,
responses to biotic and abiotic challenges, protein trafficking and chromatin
structure. Despite data implicating thousands of plant proteins as targets, so
far only a few have been conclusively shown to be ubiquitylated in planta. Here
we describe a method to isolate ubiquitin-protein conjugates from Arabidopsis
that exploits a stable transgenic line expressing a synthetic poly-UBQ gene
encoding ubiquitin (Ub) monomers N-terminally tagged with hexahistidine.
Following sequential enrichment by Ub-affinity and nickel chelate-affinity
chromatography, the ubiquitylated proteins were trypsinized, separated by
two-dimensional liquid chromatography, and analyzed by mass spectrometry. Our
list of 54 non-redundant targets, expressed by as many as 90 possible isoforms,
included those predicted by genetic studies to be ubiquitylated in plants (EIN3
and JAZ6) or shown to be ubiquitylated in other eukaryotes (ribosomal subunits,
elongation factor 1alpha, histone H1, HSP70 and CDC48), as well as candidates
whose control by the Ub/26S proteasome system is not yet appreciated. Ub
attachment site(s) were resolved for a subset of these proteins, but
surprisingly little sequence consensus was detected, implying that specific
residues surrounding the modified lysine are not important determinants for
ubiquitylation. We also identified six of the seven available lysine residues on
Ub itself as Ub attachment sites, together with evidence for a branched
mixed-linkage chain, suggesting that the topologies of Ub chains can be highly
complex in plants. Taken together, our method provides a widely applicable
strategy to define ubiquitylation in any tissue of intact plants exposed to a
wide range of conditions.
DOI: 10.1111/j.1365-313X.2009.03862.x
PMCID: PMC3639010
PMID: 19292762 [Indexed for MEDLINE]
