Differential stability of Arabidopsis D-type cyclins: CYCD3;1 is a highly unstable protein degraded by a proteasome-dependent mechanism.
In Arabidopsis, the D-type cyclin CYCD3 is rate-limiting for transition of the G(1)/S boundary, and is transcriptionally upregulated at this point in cells re-entering the cell cycle in response to plant hormones and sucrose. However, little is known about the regulation of plant cell-cycle regulators at the protein level. We show here that CYCD3;1 is a phosphoprotein highly regulated at the level of protein abundance, whereas another D-type cyclin CYCD2;1 is not. The level of CYCD3;1 protein falls rapidly on sucrose depletion, correlated with the arrest of cells in G(1) phase, suggesting a rapid turnover of CYCD3;1. Treatment of exponentially growing cells with the protein synthesis inhibitor cycloheximide (CHX) confirms that CYCD3;1 is normally a highly unstable protein, with a half-life of approximately 7 min on CHX treatment. In both sucrose-starved and exponentially growing cells, CYCD3;1 protein abundance increases in response to treatment with MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), a reversible proteasome inhibitor, but not in response to the cysteine protease inhibitor E-64 or the calpain inhibitor ALLN (N-acetyl-leucyl-leucyl-norleucinal). The increase on MG132 treatment is because of de novo protein synthesis coupled with the blocking of CYCD3;1 degradation. Longer MG132 treatment leads to C-terminal cleavage of CYCD3;1, accumulation of a hyperphosphorylated form and its subsequent disappearance. We conclude that CYCD3;1 is a highly unstable protein whose proteolysis is mediated by a proteasome-dependent pathway, and whose levels are highly dependent on the rate of CYCD3;1 protein synthesis.
Planchais
Séverine
S
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK.
Samland
Anne K
AK
Murray
James A H
JA
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002478:Cells, Cultured; D016213:Cyclins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003513:Cycloheximide / Q000494:pharmacology; D017931:DNA Primers; D004355:Drug Stability; D016193:G1 Phase; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D010750:Phosphoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013395:Sucrose / Q000378:metabolism
D029681:Arabidopsis Proteins; D016213:Cyclins; D017931:DNA Primers; D010446:Peptide Fragments; D010750:Phosphoproteins; D011500:Protein Synthesis Inhibitors; D013395:Sucrose; D003513:Cycloheximide