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Chaperoning the Cln3 Cyclin Prevents Promiscuous Activation of Start
Molecular Cell
Preview Chaperoning the Cln3 Cyclin Prevents Promiscuous Activation of Start Noel F. Lowndes1,* 1
Genome Stability Laboratory, Department of Biochemistry, National University of Ireland, Galway, University Road, Galway, Ireland *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.06.018
In a recent issue of Molecular Cell, Verge´s et al. (2007) described a new mechanism of cell-cycle control. Nuclear translocation of the G1 cyclin Cln3 is prevented by its retention at the endoplasmic reticulum (ER), and its release requires growth-associated increases in chaperone activity. In eukaryotic cells, a wave of late G1 periodic transcription is required to complete cell division. Once the transcription factors involved have been activated, in regulatory processes termed ‘‘Start’’ in yeast, or the ‘‘restriction point’’ in metazoans, the cell is committed to completion of that round of the cell cycle. This regulation is intimately linked to cell growth, and thus cells must reach a critical size before they commit to completing the cell cycle (Hartwell et al., 1974). In budding yeast, the most upstream activator of Start is the G1 cyclin Cln3, which partners the cyclin-dependent kinase Cdc28. Cln3 is an unstable and therefore low abundance protein present throughout the cell cycle. Although Cln3 levels can change in response to nutritional and other external signals, when these signals are kept constant posttranslational mechanisms are required to prevent activation of Cdc28-Cln3 until the critical cell size is reached. In a recent study from Martı´ Aldea and colleagues in Molecular Cell, the translocation of Cln3 into the nucleus, an event critical to the execution of Start, is mechanistically dissected (Verge´s et al., 2007). Their data indicate that Cln3 associates with the ER (cosedimentation with the microsomal fraction and punctate cytoplasmic localization) throughout the cell cycle except in late G1, when a significant fraction of Cln3 becomes nuclear and triggers Start. Bioinformatic analysis revealed a novel ‘‘J-like domain’’ within Cln3. J domains are so termed due to homology with a motif found in
the prototypical E. coli cochaperone, DnaJ, which has been implicated in the regulation of ‘‘DnaK-like’’ (or ‘‘Hsp70-like’’) chaperones. Together these chaperones and cochaperones are known to modulate the assembly and disassembly of protein complexes (Walsh et al., 2004). Crucially, Verge´s et al. noticed that the J domain of Cln3 is missing the HPDK tetrapeptide involved in activating the ATPase activity of Hsp70s. The authors hypothesized that this would make the Cln3 J domain inactive or inhibitory, and hence they term this a Ji domain. Consistent with this hypothesis, deletion or structural disruption of the Ji domain resulted in release of Cln3 from the ER, allowing its transport to the nucleus via its nuclear localization domain. Furthermore, it is the intact Cdc28-Cln3 complex that interacts with the ER, as when this complex is disrupted (using a Cln3 cyclin box mutant that cannot associate with Cdc28), Cln3 cannot associate with the ER. Previous studies have implicated the yeast Ydj1 cochaperone in both positive and negative regulation of Cln3 (mutation of YDJ1 results in delayed execution of Start, whereas Ydj1 binds the C terminus of Cln3, facilitating its phosphorylation by Cdc28 as a signal for degradation). Therefore, the authors examined ER retention of Cln3 and found that Ydj1, as well as the Hsp70-related Ssa1 and 2 chaperones, is required for release of Cdc28Cln3 from the ER and its consequent nuclear import in late G1. Furthermore, CLN3 overexpression suppresses the large size and G1 accumulation of
ydj1D cells. Thus, Ydj1 is an activator of Cln3 function, being required for its release from the ER and consequent nuclear accumulation. In nice domain swap experiments, the authors demonstrate that when the J domain of Ydj1 was replaced with the Ji domain of Cln3, although the chimeric protein (Ydj1DJ::JiCln3) was still capable of binding the Hsp70 chaperone, Ssa1, it failed to activate the ATPase activity of Ssa1, suggesting that the Ji domain of Cln3 could function as a competitive inhibitor of Ssa1 activity. Consistent with this possibility, when the missing HPDK tetrapeptide was inserted into the Cln3 Ji domain (Cln3Ji::HPDK), Cln3 was converted into an efficient activator of Ssa1 ATPase activity. Thus, the inhibitory Ji domain of Cln3 and the activating J domain of Ydj1 play interdependent and opposing roles. Consistent with this, overexpression of Ydj1 results in an earlier execution of Start, as judged by smaller size at budding, fewer unbudded cells, and advanced S phase. In a cell-free system, addition of recombinant cochaperone (Ydj1) and chaperone (Ssa1) together with an ATP regenerating system resulted in appreciable release of Cln3 from the microsomal (ER) fraction and a concomitant increase in soluble Cdc28Cln3 complexes. There was no significant change in the overall level of the more abundant Cdc28 protein in the microsomal fraction, indicating that most ER-bound Cdc28 remains bound to the ER. By mixing extracts with nearly all Cln3 bound to the ER with
Molecular Cell 27, July 6, 2007 ª2007 Elsevier Inc. 1
Molecular Cell
Preview soluble extracts with epitope-tagged Cdc28 in the absence of any G1 cyclins, Cln3 released from the ER upon chaperone addition was determined to be associated with the soluble epitope-tagged Cdc28. This suggests that chaperones mediate the transfer of Cln3 from Cdc28-Cln3 complexes bound at the ER to non-ER-bound, soluble Cdc28, leaving the ER-bound Cdc28 behind. This study nicely complements the authors’ previous studies demonstrating that CLN3 mRNA, as well as the Cdc28 protein, is also retained at the ER in a Whi3-dependent fashion (Gari et al., 2001; Wang et al., 2004). The new mechanism is consistent with translated Cln3 being immediately retained at the ER, where it interacts with Cdc28. This docking of the Cdc28-Cln3 complex onto the ER is mediated by interaction between the Ssa1/2 chaperones and the Cln3 Ji domain. The inhibitory Ji domain prevents the Hsp70 chaperone cycle, effectively locking Cdc28-Cln3 com-
plexes onto the ER, where they remain until Ydj1, and possibly other cochaperones, attain levels sufficient to activate the Ssa1/2 ATPase activity and release the bound Cln3. Thus, CLN3 mRNA and Cln3 protein are prevented from inappropriate activation of the Start transcriptional program by their retention at the ER. The authors’ model predicts that as chaperone and cochaperone levels increase linearly at a constant rate during early G1, eventually sufficient surplus is produced to induce release of Cln3 from the ER and trigger Start. Later, as cells proceed into S phase there is a change in the rate of protein synthesis (presumably as a direct consequence of the wave of late G1 transcription), which would once more reduce chaperone availability and result in Cln3 being retained at the ER again. The authors have presented a fascinating study that very significantly advances our understanding of cell-cycle control in budding yeast. It will be interesting to extrapolate their model
2 Molecular Cell 27, July 6, 2007 ª2007 Elsevier Inc.
to human cells and test whether any G1-specific cyclins are similarly retained at the ER in early G1 phase. A recent study indicating that the mouse Hsc70 chaperone associates with cyclin D1 suggests that similar mechanisms may well be conserved in higher eukaryotes (Diehl et al., 2003).
REFERENCES Diehl, J.A., Yang, W., Rimerman, R.A., Xiao, H., and Emili, A. (2003). Mol. Cell. Biol. 23, 1764– 1774. Garı´, E., Volpe, T., Wang, H., Gallego, C., Futcher, B., and Aldea, M. (2001). Genes Dev. 15, 2803–2808. Hartwell, L.H., Culotti, J., Pringle, J.R., and Reid, B.J. (1974). Science 183, 46–51. Verge´s, E., Colomina, N., Garı´, E., Gallego, C., and Aldea, M. (2007). Mol. Cell 26, 649–662. Walsh, P., Bursac, D., Law, Y.C., Cyr, D., and Lithgow, T. (2004). EMBO Rep. 5, 567–571. Wang, H., Garı´, E., Verge´s, E., Gallego, C., and Aldea, M. (2004). EMBO J. 23, 180–190.
Preview Chaperoning the Cln3 Cyclin Prevents Promiscuous Activation of Start Noel F. Lowndes1,* 1
Genome Stability Laboratory, Department of Biochemistry, National University of Ireland, Galway, University Road, Galway, Ireland *Correspondence: [email protected] DOI 10.1016/j.molcel.2007.06.018
In a recent issue of Molecular Cell, Verge´s et al. (2007) described a new mechanism of cell-cycle control. Nuclear translocation of the G1 cyclin Cln3 is prevented by its retention at the endoplasmic reticulum (ER), and its release requires growth-associated increases in chaperone activity. In eukaryotic cells, a wave of late G1 periodic transcription is required to complete cell division. Once the transcription factors involved have been activated, in regulatory processes termed ‘‘Start’’ in yeast, or the ‘‘restriction point’’ in metazoans, the cell is committed to completion of that round of the cell cycle. This regulation is intimately linked to cell growth, and thus cells must reach a critical size before they commit to completing the cell cycle (Hartwell et al., 1974). In budding yeast, the most upstream activator of Start is the G1 cyclin Cln3, which partners the cyclin-dependent kinase Cdc28. Cln3 is an unstable and therefore low abundance protein present throughout the cell cycle. Although Cln3 levels can change in response to nutritional and other external signals, when these signals are kept constant posttranslational mechanisms are required to prevent activation of Cdc28-Cln3 until the critical cell size is reached. In a recent study from Martı´ Aldea and colleagues in Molecular Cell, the translocation of Cln3 into the nucleus, an event critical to the execution of Start, is mechanistically dissected (Verge´s et al., 2007). Their data indicate that Cln3 associates with the ER (cosedimentation with the microsomal fraction and punctate cytoplasmic localization) throughout the cell cycle except in late G1, when a significant fraction of Cln3 becomes nuclear and triggers Start. Bioinformatic analysis revealed a novel ‘‘J-like domain’’ within Cln3. J domains are so termed due to homology with a motif found in
the prototypical E. coli cochaperone, DnaJ, which has been implicated in the regulation of ‘‘DnaK-like’’ (or ‘‘Hsp70-like’’) chaperones. Together these chaperones and cochaperones are known to modulate the assembly and disassembly of protein complexes (Walsh et al., 2004). Crucially, Verge´s et al. noticed that the J domain of Cln3 is missing the HPDK tetrapeptide involved in activating the ATPase activity of Hsp70s. The authors hypothesized that this would make the Cln3 J domain inactive or inhibitory, and hence they term this a Ji domain. Consistent with this hypothesis, deletion or structural disruption of the Ji domain resulted in release of Cln3 from the ER, allowing its transport to the nucleus via its nuclear localization domain. Furthermore, it is the intact Cdc28-Cln3 complex that interacts with the ER, as when this complex is disrupted (using a Cln3 cyclin box mutant that cannot associate with Cdc28), Cln3 cannot associate with the ER. Previous studies have implicated the yeast Ydj1 cochaperone in both positive and negative regulation of Cln3 (mutation of YDJ1 results in delayed execution of Start, whereas Ydj1 binds the C terminus of Cln3, facilitating its phosphorylation by Cdc28 as a signal for degradation). Therefore, the authors examined ER retention of Cln3 and found that Ydj1, as well as the Hsp70-related Ssa1 and 2 chaperones, is required for release of Cdc28Cln3 from the ER and its consequent nuclear import in late G1. Furthermore, CLN3 overexpression suppresses the large size and G1 accumulation of
ydj1D cells. Thus, Ydj1 is an activator of Cln3 function, being required for its release from the ER and consequent nuclear accumulation. In nice domain swap experiments, the authors demonstrate that when the J domain of Ydj1 was replaced with the Ji domain of Cln3, although the chimeric protein (Ydj1DJ::JiCln3) was still capable of binding the Hsp70 chaperone, Ssa1, it failed to activate the ATPase activity of Ssa1, suggesting that the Ji domain of Cln3 could function as a competitive inhibitor of Ssa1 activity. Consistent with this possibility, when the missing HPDK tetrapeptide was inserted into the Cln3 Ji domain (Cln3Ji::HPDK), Cln3 was converted into an efficient activator of Ssa1 ATPase activity. Thus, the inhibitory Ji domain of Cln3 and the activating J domain of Ydj1 play interdependent and opposing roles. Consistent with this, overexpression of Ydj1 results in an earlier execution of Start, as judged by smaller size at budding, fewer unbudded cells, and advanced S phase. In a cell-free system, addition of recombinant cochaperone (Ydj1) and chaperone (Ssa1) together with an ATP regenerating system resulted in appreciable release of Cln3 from the microsomal (ER) fraction and a concomitant increase in soluble Cdc28Cln3 complexes. There was no significant change in the overall level of the more abundant Cdc28 protein in the microsomal fraction, indicating that most ER-bound Cdc28 remains bound to the ER. By mixing extracts with nearly all Cln3 bound to the ER with
Molecular Cell 27, July 6, 2007 ª2007 Elsevier Inc. 1
Molecular Cell
Preview soluble extracts with epitope-tagged Cdc28 in the absence of any G1 cyclins, Cln3 released from the ER upon chaperone addition was determined to be associated with the soluble epitope-tagged Cdc28. This suggests that chaperones mediate the transfer of Cln3 from Cdc28-Cln3 complexes bound at the ER to non-ER-bound, soluble Cdc28, leaving the ER-bound Cdc28 behind. This study nicely complements the authors’ previous studies demonstrating that CLN3 mRNA, as well as the Cdc28 protein, is also retained at the ER in a Whi3-dependent fashion (Gari et al., 2001; Wang et al., 2004). The new mechanism is consistent with translated Cln3 being immediately retained at the ER, where it interacts with Cdc28. This docking of the Cdc28-Cln3 complex onto the ER is mediated by interaction between the Ssa1/2 chaperones and the Cln3 Ji domain. The inhibitory Ji domain prevents the Hsp70 chaperone cycle, effectively locking Cdc28-Cln3 com-
plexes onto the ER, where they remain until Ydj1, and possibly other cochaperones, attain levels sufficient to activate the Ssa1/2 ATPase activity and release the bound Cln3. Thus, CLN3 mRNA and Cln3 protein are prevented from inappropriate activation of the Start transcriptional program by their retention at the ER. The authors’ model predicts that as chaperone and cochaperone levels increase linearly at a constant rate during early G1, eventually sufficient surplus is produced to induce release of Cln3 from the ER and trigger Start. Later, as cells proceed into S phase there is a change in the rate of protein synthesis (presumably as a direct consequence of the wave of late G1 transcription), which would once more reduce chaperone availability and result in Cln3 being retained at the ER again. The authors have presented a fascinating study that very significantly advances our understanding of cell-cycle control in budding yeast. It will be interesting to extrapolate their model
2 Molecular Cell 27, July 6, 2007 ª2007 Elsevier Inc.
to human cells and test whether any G1-specific cyclins are similarly retained at the ER in early G1 phase. A recent study indicating that the mouse Hsc70 chaperone associates with cyclin D1 suggests that similar mechanisms may well be conserved in higher eukaryotes (Diehl et al., 2003).
REFERENCES Diehl, J.A., Yang, W., Rimerman, R.A., Xiao, H., and Emili, A. (2003). Mol. Cell. Biol. 23, 1764– 1774. Garı´, E., Volpe, T., Wang, H., Gallego, C., Futcher, B., and Aldea, M. (2001). Genes Dev. 15, 2803–2808. Hartwell, L.H., Culotti, J., Pringle, J.R., and Reid, B.J. (1974). Science 183, 46–51. Verge´s, E., Colomina, N., Garı´, E., Gallego, C., and Aldea, M. (2007). Mol. Cell 26, 649–662. Walsh, P., Bursac, D., Law, Y.C., Cyr, D., and Lithgow, T. (2004). EMBO Rep. 5, 567–571. Wang, H., Garı´, E., Verge´s, E., Gallego, C., and Aldea, M. (2004). EMBO J. 23, 180–190.