Conjugation of [125I]ubiquitin to cellular proteins in permeabilized mammalian cells: comparison of mitotic and interphase cells.

Abstract

[125I]Ubiquitin introduced into permeabilized hepatoma tissue culture (HTC) cells rapidly forms conjugates with endogenous proteins. A characteristic pattern of low mol. wt conjugates is obtained which includes the ubiquitinated histone, uH2A, and unknown molecular species with MrS of 14, 23, 26 (two bands) and 29 kd. A broad spectrum of higher mol. wt conjugates is also produced. The formation of all conjugates is absolutely dependent on ATP, and upon depletion of ATP they are rapidly broken down. The 14, 23 and 29 kd species are found in all subcellular fractions examined. uH2A is located exclusively in the nuclear fraction. The pair of 26 kd bands is specifically associated with the ribosome fraction. A considerable percentage of the higher mol. wt conjugates sediments with the small particle (100,000 g) fraction in the ultracentrifuge but is solubilized with deoxycholate, indicating that there are many membrane‐associated conjugates. The pattern of ubiquitin conjugation in interphase and metaphase cells was compared. The incorporation of ubiquitin into uH2A was markedly reduced in metaphase cells whereas its incorporation into other low mol. wt conjugates and into high mol. wt conjugates was affected slightly, if at all. This shows that the known decrease of uH2A levels in metaphase is due to a specific effect on histone ubiquitination and not to a general decrease in ubiquitination activity or increase of isopeptidase activity. Changes in the levels of uH2A during mitosis measured by immunoblotting were similar to those estimated in permeabilized cells. These experiments indicate that permeabilized cells provide a useful approach to the study of rapidly turning over ubiquitin conjugates in mammalian cells.

Ubiquitin in Chlamydomonas reinhardii

“…The proteins were transferred to nitrocellulose filters and immunoblotted as described previously [44]. The anti-(human ubiquitin) antibodies were used at a dilution of lj2000.…”

Presence of antibodies to ubiquitin during the autoimmune response associated with systemic lupus erythematosus.

“…"Ubiquitination" of histones is a postsynthetic and reversible modification that occurs preferentially in actively transcribed regions of chromatin after core particle assembly (20)(21)(22). The level of histone ubiquitination changes as cells progress through the cell cycle or respond to stress (21,23,24). Recently, ubiquitin was also found to be covalently attached to certain cell-surface glycoproteins, especially to lymphocyte-homing receptor (25), and it was suggested that ubiquitination could play an important role in receptor function (26).…”

Ubiquitin immunoreactivity shows several proteins varying with development and sporulation in the basidiomycete Coprinus cinereus

“…Ubiquitin conjugates with molecular masses similar to 28 and 31 kDa have also been observed in Chlamydomonas (Shimogawara and Muto 1989). The 23-and 29-kDa proteins may be ubiquitinated histone 2A and diubiquitinated histone 2A, respectively (Haas and Bright 1985;Raboy et al 1986). Ubiquitin conjugates with molecular masses up to 26 kDa have been observed in the ribosomal fraction (Raboy et al 1986).…”

“…The 15-kDa protein is probably a ubiquitin dimer (Haas and Bright 1985;Raboy et al 1986) or a 15-kDa protein of an ubiquitin moiety linked to the C-terminal extension as observed in both Dictyostelium d i s c o i h m and Saccharornyces cerevisiae (Miiller-Taubenberger et al 1988). Ubiquitin conjugates with molecular masses similar to 28 and 31 kDa have also been observed in Chlamydomonas (Shimogawara and Muto 1989).…”