Cell Cycle–Dependent Proteolysis in Plants: Identification of the Destruction Box Pathway and Metaphase Arrest Produced by the Proteasome Inhibitor MG132

Abstract

It is widely assumed that mitotic cyclins are rapidly degraded during anaphase, leading to the inactivation of the cell cycle-dependent protein kinase Cdc2 and allowing exit from mitosis. The proteolysis of mitotic cyclins is ubiquitin/26S proteasome mediated and requires the presence of the destruction box motif at the N terminus of the proteins. As a first attempt to study cyclin proteolysis during the plant cell cycle, we investigated the stability of fusion proteins in which the N-terminal domains of an A-type and a B-type tobacco mitotic cyclin were fused in frame with the chloramphenicol acetyltransferase ( CAT ) reporter gene and constitutively expressed in transformed tobacco BY2 cells. For both cyclin types, the N-terminal domains led the chimeric cyclin-CAT fusion proteins to oscillate in a cell cycle-specific manner. Mutations within the destruction box abolished cell cycle-specific proteolysis. Although both fusion proteins were degraded after metaphase, cyclin A-CAT proteolysis was turned off during S phase, whereas that of cyclin B-CAT was turned off only during the late G 2 phase. Thus, we demonstrated that mitotic cyclins in plants are subjected to post-translational control (e.g., proteolysis). Moreover, we showed that the proteasome inhibitor MG132 blocks BY2 cells during metaphase in a reversible way. During this mitotic arrest, both cyclin-CAT fusion proteins remained stable. INTRODUCTIONThe cell division cycle is a sequential process that permits the replication of the genome, the segregation of chromosomes to two daughter nuclei, and finally cytokinesis. Progression through the cell cycle involves many proteins, which are regulated both at the transcriptional and posttranslational levels. Today, it has become clear that the timed destruction of key proteins plays an essential role in cell cycle progression. The first protein shown to be subjected to cell cycle-specific proteolysis was cyclin B (Evans et al., 1983). For many other proteins, cell cycle-specific proteolysis has been reported: among them are the G 1 cyclins and the mitotic cyclins, the cyclin-dependent kinase inhibitors (CKIs), proteins involved in sister chromatid separation, and spindle components (reviewed in Peter and Herskowitz, 1994;Murray, 1995; King et al., 1996; Hoyt, 1997). The degradation of cell cycle regulator proteins is not just a way to break down a protein whose function is achieved at a specific step of the cell cycle, but it is also a way to directly control the cell cycle. Thus, the highly regulated proteolysis of CKIs allows DNA replication to begin, and degradation of proteins involved in sister chromatid separation is required at the onset of anaphase.Proteins subjected to degradation are marked with ubiquitin tags and subsequently are targeted to the degradative action of the 26S proteasome. The ubiquitin/26S proteasome proteolytic pathway is highly conserved in eukaryotes and is involved in many other important cellular functions aside from cell cycle progression (reviewed in Hochstrasser, 1995). Deg...

Cyclin dependent kinases and their role in regulation of plant cell cycle

“…Proteolysis of securin is ensured by APC/CCDC20 before anaphase onset, but degradation is maintained until the end of G1 by APC/CCDH1 (Nasmyth 2001). Although plant orthologues of securin proteins have not yet been reported (these proteins are poorly conserved) inhibition of the proteasome during prophase blocks the cells in metaphase, indicating that securing orthologues do exist in plants (Genschik et al 1998). The degradation of securins is especially significant in a mechanism called the spindle assembly checkpoint (Hoyt et al 1991).…”

Involvement of the ubiquitin/proteasome pathway in the organisation and polarised growth of kiwifruit pollen tubes

“…In plants, this proteolytic pathway has been shown to control a large number of events such as cell cycle progression (Genschik et al 1998), stress response and senescence (Belknap and Garbarino 1996;Stephenson and Rubinstein 1998), hormone signaling (Callis and Vierstra 2000;Worley et al 2000), vascular tissue differentiation (Woffenden et al 1998), and pollen maturation and germination (Callis and Bedinger 1994;Scoccianti et al 1999;Speranza et al 2001). Some of these studies have been carried out by using cellpermeable proteasome inhibitors such as the reversible, competitive synthetic tripeptide aldehyde inhibitor carbobenzoxyl-leucinyl-leucinal (MG132) and the irreversible inhibitors clasto-lactacystin b-lactone and epoxomicin (Dick et al 1997;Meng et al 1999;Tsubuki et al 1996).…”

Insights into the G1/S transition in plants

“…At the moment, only data concerning proteolytic control of mitotic cyclins have been reported in plants (Genschik et al 1998). However, an increasing body of evidence indicates that the ubiquitinmediated proteolysis pathway is a common component in plant hormone signaling (Boniotti and Griffith 2002).…”