Subcellular localization of ubiquitin and ubiquitinated proteins in Arabidopsis thaliana.
Ubiquitin is a highly conserved, 76-amino acid, eukaryotic protein. Its widely accepted role as a proteolytic cofactor depends on its unique ability to covalently ligate to other cellular proteins. While there is good evidence for the existence of such ubiquitinated proteins in the cytosolic and nuclear compartments, relatively little is known about the presence of free ubiquitin and ubiquitinated proteins in other subcellular compartments. This is especially true of higher plants, which have not previously been the subject of extensive biochemical subcellular localizations of ubiquitinated proteins. We extracted cell wall proteins and purified nuclei, vacuoles, chloroplasts, and microsomes from chlorophyllous tissues of Arabidopsis. Immunoblot analyses were used to compare the profiles of ubiquitinated proteins from purified subcellular fractions to those from unfractionated extracts. Purified nuclei contained, in addition to a complex mixture of high molecular mass ubiquitinated proteins, a strongly immunoreactive 28-kDa protein. In the apoplastic extract, we did not detect any ubiquitinated proteins enriched above the background level of those due to cytosolic contamination. Vacuoles appeared to contribute significantly to the ubiquitinated proteins present in the whole protoplast extract. At least three high molecular mass ubiquitinated proteins were unique to the vacuolar extract. Chloroplast stromal proteins did not react specifically with anti-ubiquitin antibodies. When microsomal ubiquitinated proteins were compared to those found in a whole protoplast extract, a distinct pattern was evident. Microsomal ubiquitinated proteins were not visible in the 10,000 x g supernatant used to prepare the 100,000 x g pellet, indicating that they were probably low abundance proteins in the protoplast extract.
Beers
E P
EP
Department of Biochemistry and Biophysics, University of California, Davis 95616.
Moreno
T N
TN
Callis
J
J
D002458:Cell Fractionation; D002467:Cell Nucleus / Q000737:chemistry; D002736:Chloroplasts / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D008861:Microsomes / Q000648:ultrastructure; D008970:Molecular Weight; D010940:Plant Proteins / Q000032:analysis* / Q000302:isolation & purification; D010944:Plants / Q000737:chemistry*; D013347:Subcellular Fractions / Q000737:chemistry; D014452:Ubiquitins / Q000032:analysis* / Q000302:isolation & purification; D014617:Vacuoles / Q000737:chemistry
D010940:Plant Proteins; D014452:Ubiquitins