Plant 21D7 Protein, a Nuclear Antigen Associated with Cell Division, Is a Component of the 26S Proteasome

Abstract

Previously, we cloned a carrot (Daucus carota L.) cDNA encoding a 45-kD protein, 21D7, located in the nuclei of proliferating cells. The 21D7 protein is similar to the partia1 sequence of a regulatory subunit of the bovine 26s proteasome, p58 (C. DeMartino, C.R. Moomaw, O.P. Zagnitko, R.J. Proske, M. Chu-Ping, S.J. Afendis, J.C. Swaffield, C.A. Slaughter [1994] J Biol Chem 269:20878-20884) and to the deduced sequence encoded by the Saccharomyces cerevisiae gene SUN2 (M. Kawamura, K. Kominami, J. Takeuchi, A. Toh-e [1996] MOI Cen Cenet 251 : 11 46-1 521). I n our work, the expression of plant 21 D7 cDNA rescued the yeast sun2 mutant. Fractionation of carrot and spinach (Spinacia oleracea L.) crude extracts showed that the 21 D7 protein sedimented with the active 26s proteasomes. The cessation of cell proliferation in carrot suspensions at the stationary phase caused 26s proteasome dissociation and, correspondingly, the 21 D7 protein sedimented together with the free regulatory complexes of the 26s proteasomes. Large-scale purification of carrot 26s proteasomes resulted i n coisolation of the 21 D 7 protein. Polyacrylamide gel electrophoresis under nondenaturing conditions showed that the 21 D7 protein had the same mobility as the 26s proteasome and that proteasome dissociation changed the mobility of the 21 D7 protein accordingly. We conclude that the 21D7 protein is a subunit of the plant 26s proteasome and that it probably belongs to the proteasome regulatory complex.Most molecular studies of the cell division cycle in higher plants are based on the cloning of plant homologs of yeast and animal genes that control the cell cycle progression. However, in a number of cases the association of a gene with cell division has been shown first in a plant system (eg. Kodama et al., 1991;Ito and Komamine, 1993). In one of these cases, immunochemical studies of somatic embryogenesis resulted in the identification of a 45-kD carrot (Daucus carota L.) antigen, named 21D7 after the

Homologues of 26S proteasome subunits are regulators of transcription and translation

“…This suggests proteasomal functions of Sun2 orthologues (Kawamura et al, 1996) rather than previously proposed diphenol oxidase activities (Pentz & Wright, 1991). In addition, a plant Sun2 homologue is localized to the nucleus and shows a cell-cycle dependent variation in levels (Smith et al, 1997). This suggests that these homologues are involved in cell cycle stage specific regulation of proteasome function.…”

N α-Acetylation and Proteolytic Activity of the Yeast 20 S Proteasome

“…The 20 S proteasome from yeast extracts was purified by sequential chromatographs on octyl-Sepharose CL-4B, BioGel A-1.5 m, DEAE-Sepharose CL-6B, PD-10, and FPLC Mono-Q columns. The purification was performed according to the method reported previously (23), except that Bio-Gel A-1.5m gel filtration was used instead of dialysis (24) and FPLC with a Mono-Q column was used as the final purification step (25).…”

Anopheles stephensi Dox-A2 Shares Common Ancestry with Genes from Distant Groups of Eukaryotes Encoding a 26S Proteasome Subunit and Is in a Conserved Gene Cluster

“…Evidence that human p58 is also an ortholog of SUN2 came from direct sequencing of subunits of the purified PA700 (DeMartino et al 1994). With more recent studies on mammals, plants, and yeast, the role of these Dox-A2-related genes in 26S proteasome is without dispute (Kominami et al 1997;Smith et al 1997;Ito et al 1997). An alternative role for insect Dox-A2 genes is therefore highly improbable, and an additional role which is not shared by at least the mammalian genes is also unlikely.…”

“…Kawamura et al (1996) and Smith et al (1997) speculate that Dox-A2 is involved in the activation of phenol oxidase. This was originally discounted by on the grounds that mutations at the Dox-A2 locus only affected the A2 band on polyacrylamide gels.…”

“…Subsequent studies confirmed this and showed that the SUN2 product is a non-ATPase component of the PA700 complex. An identical role has been found for the mammalian and plant genes related to Dox-A2 (Kominami et al 1997;Smith et al 1993Smith et al , 1997. This has lead to speculation that Dox-A2 is not the structural locus for diphenol oxidase A2, but instead is involved in the activation of the proenzyme (Kawamura et al 1996;Smith et al 1997).…”