Taken together, our data provide a model by which the timely export of Chs2 from the ER to the bud neck for septum formation can be explained (). CHS2
that is expressed during metaphase (Pammer et al., 1992
; Choi et al., 1994
; Spellman et al., 1998
) is retained in the ER prior to sister chromatid separation by Clb2-Cdc28 phosphorylation at the N-terminus (VerPlank and Li, 2005
; Zhang et al., 2006
; Teh et al., 2009
). On APCCdc20
activation, sister chromatids separate and the destruction of the mitotic cyclin Clb2 occurs, leading to the initiation of the first phase of mitotic exit (Yeong et al., 2000
; Wasch and Cross, 2002
). At this point, Cdc14 is released throughout the nucleus by the FEAR pathway, and Chs2 that is phosphorylated continues to be retained in the ER. This is consistent with previous observations that MEN mutants defective in reducing Cdk1 activity are unable to export Chs2 from the ER (VerPlank and Li, 2005
; Zhang et al., 2006
; Teh et al., 2009
; Meitinger et al., 2010
) as Chs2 remains phosphorylated at telophase (Supplemental Figure S2).
Schematic showing Cdc14 (green) and Chs2 (red) distribution during mitotic exit. See the text for details.
As sister chromatids separate and the spindles elongate, the migration of one spindle pole body into the daughter cell results in the activation of the MEN that promotes the complete inactivation of Cdk1 (Stegmeier and Amon, 2004
). At the same time, Cdc14 released from the nucleolus to the cytoplasm () during MEN activation now has access to the N-terminal of Chs2 and acts efficiently to relieve the inhibitory phosphorylation on Chs2 (). In the absence of competing Cdk1 activity, dephosphorylated Chs2 is exported from the ER via the secretory pathway to the bud neck (Chuang and Schekman, 1996
; Zhang et al., 2006
) in time to deposit the primary septum and stabilize the actomyosin ring as it undergoes constriction (VerPlank and Li, 2005
The dispersal of Cdc14 to the nucleoplasm in early anaphase by FEAR and to the cytoplasm in late mitosis by MEN has been suggested to provide a means by which cells regulate the dephosphorylation of distinct groups of substrates of Cdc14 at different times in mitosis (Sullivan and Morgan, 2007
). However, in addition to Cdc14 dispersal, the Cdk1 activity could also contribute to the differential dephosphorylation of Cdc14 substrates. For instance, in early anaphase, substrates including Ase1 (Khmelinskii et al., 2007
), Sli15 (Pereira and Schiebel, 2003
), and Ask1 (Higuchi and Uhlmann, 2005
) are dephosphorylated by the FEAR-released Cdc14 in the nucleoplasm even though the Cdk1 activity is still relatively high. This could reflect the higher efficiency that Cdc14 has in dephosphorylating these substrates. Substrates such as Cdh1 (Zachariae et al., 1998
; Jaspersen et al., 1999
), Swi5 (Moll et al., 1991
; Visintin et al., 1998
). and Sic1 (Visintin et al., 1998
) are dephosphorylated in late anaphase by Cdc14 released into the cytoplasm when APCCdc20
is active and the Cdk1 activity likely has decreased to 50% of its peak level. In line with our data, Chs2 likely represents a member of the group of late Cdc14 substrates that are dephosphorylated in telophase when Cdk1 activity has been reduced to a very low level (). As such, the different levels of Cdk1 activity at early anaphase, late anaphase, and telophase, coupled to the nucleoplasmic or cytoplasmic localization of Cdc14, together contribute toward ordering mitotic events.
Consistent with this notion, we observed that the export of Chs2 was effectively triggered when Cdk1 was inhibited by overexpression of SIC1 in metaphase-arrested cells (). Presumably, the Cdc14 that is forced out of the nucleolus under these conditions acts unopposed in the absence of Cdk1 and efficiently dephosphorylates its substrates, including Chs2. These observations are in line with the findings that cdc55Δ, leading to FEAR activation, did not cause premature Chs2 ER export in metaphase (), but, rather, bub2Δ that leads to activation of the MEN and Cdc14 cytoplasmic localization caused Chs2 ER export ().
Besides promoting mitotic exit, the MEN components such as Tem1p, Cdc15, Dbf2, Dbf20, and Cdc14 are known to localize to the neck at the end of mitosis to promote events needed for cytokinesis and septation (Yeong et al., 2002
; Balasubramanian et al., 2004
). The exact function(s) of the MEN components in cytokinesis and septum formation is unclear. It was recently suggested that the MEN plays a role in the neck localization of cytokinesis components such as Inn1, Cyk3, and Chs2 (Meitinger et al., 2010
). In dbf2-2 dbf20Δ
cells in which SIC1
was overexpressed to inhibit the mitotic Cdk1 activity, Chs2 translocation to the neck was inefficient. Consequently, the authors proposed that the MEN is needed for targeting Chs2 to the neck. However, these observations could be due to the fact that Dbf2 is needed for promoting Cdc14 cytoplasmic retention during exit from mitosis (Mohl et al., 2009
) and that in the dbf2-2 dbf20Δ GAL-SIC1
cells, Cdc14 cytoplasmic localization was not efficient even in the presence of Sic1. As a result, Chs2 remained at the ER and failed to translocate to the neck.
It should also be noted that the cdc14-NES
mutant shows a cytokinesis defect (Bembenek et al., 2005
). Observation of the GFP-tagged Cdc14-NES protein revealed that the mutant protein is defective in neck localization, suggesting that Cdc14 neck localization might be important for septum formation and/or cytokinesis. However, in the cdc14-NES
mutant, phosphorylation-deficient Chs2(4S-to-4A)-YFP was localized to the neck even in metaphase (). This indicates that Chs2 localization at the neck per se might not normally depend upon Cdc14 function at the neck, but, rather, that Cdc14 acts on Chs2 at the ER to promote its export and translocation to the neck. The cytokinesis defect in the cdc14-NES
mutant is therefore likely to be a failure of Chs2 ER export.
It remains to be seen how the dephosphorylation of Chs2 by Cdc14 during mitotic exit allows for its incorporation into COP II vesicles (Lee and Miller, 2007
; Sato and Nakano, 2007
) for its transport to the neck. It would be of interest to determine whether the COP II coat protein Sec24 interacts with the dephosphorylated form of Chs2 and, if so, what the underlying basis is that allows for the association. Furthermore, it is unclear how the interaction with COP II complex enables the selection of Chs2 into ER exit sites (Budnik and Stephens, 2009
) in a timely manner for targeting to the bud neck to aid in actomyosin ring constriction, and this requires further study. Given that several other cargoes of the secretory pathway studied are constitutively exported out of the ER (Makarow, 1988
; Nevalainen et al., 1989
) unlike Chs2, understanding the differences in the mechanisms by which the COP II coat proteins and other components of the early secretory pathway interacts with distinct cargoes is an important future goal.
Cdc14 belongs to a family of highly conserved dual-specificity phosphatases (Mocciaro and Schiebel, 2010
), with several novel substrates in mitosis and cytokinesis identified recently (Bloom et al., 2011
). Our finding that Chs2, a cargo of the secretory pathway (Chuang and Schekman, 1996
; VerPlank and Li, 2005
; Zhang et al., 2006
), is a novel Cdc14 substrate highlights an important link between the cell division machinery and the protein-trafficking pathway in the coordination of cell cycle events. More than transporting Chs2, the secretory vesicles carrying Chs2 that are targeted to the neck might also be required for the delivery of other proteins and, more critically, membranes that are needed during cytokinesis (McKay and Burgess, 2011
). It is intriguing that Cdc14A in mammalian cells (Lanzetti et al., 2007
) has been shown to interact with Rab5, a small GTPase implicated in the secretory and the endocytic pathways (Zerial and McBride, 2001
), whereas Cdc14C has been found to localize to the ER (Rosso et al., 2008
). The implications of these findings are unknown, although the findings point to possible roles of Cdc14 in higher eukaryotes in membrane trafficking.
Although questions remain regarding how dephosphorylation of Chs2 affects its selection into COP II vesicles and how Cdc14 might further contribute to cytokinesis through its role in promoting vesicular transport, our data nonetheless highlight the significance of the combined effects of low Cdk1 activity and cytoplasmic localization of Cdc14 in promoting the dephosphorylation of telophase substrates such as Chs2 (). The constitutive neck localization of Chs2p(4S-to-4A)-YFP in cycling cells even when mitotic exit has not occurred (Supplemental Figure S4) further suggests that the presence of phosphorylation at the N-terminal Cdk1 sites normally prevents untimely localization of Chs2 during a regular cell division. Conversely, it also implies that the dephosphorylation of Chs2 is needed to alleviate the restraint on its ER export. More important, the untimely dephosphorylation of Chs2 can result in premature transport to the neck in an unperturbed cell division cycle.
The tight regulation of Chs2 ER export at the end of mitosis is critical, as premature localization of Chs2 to the neck in mitosis can result in aberrant septation (Zhang et al., 2006
; Meitinger et al., 2010
), whereas the phosphorylation-deficient Chs2(4S-to-4A) kills cells when overexpressed (Teh et al., 2009
). Moreover, a transient release of Cdc14 from the nucleolus in metaphase upon SIC1
induction () was sufficient to cause Chs2 ER export, indicating that the sequestration of Cdc14 from the cytoplasm normally ensures Chs2 ER retention until after the activation of the MEN. The observation further raises the possibility that the lowering of Cdk1 during mitotic exit could contribute to Cdc14 cytoplasmic localization. Such an interdependence of chromosome segregation, MEN activation, decrease in mitotic Cdk1 activity, and Cdc14 dispersal is likely to play a critical role in the execution of late mitotic events. Indeed, the coupling of these events in promoting late mitotic processes such as the export of Chs2 from the ER provides a simple yet effective mechanism for cells to order sister chromatid separation, mitotic exit, and cytokinesis in the proper temporal sequence.