Article |
Address correspondence to Akio Toh-e, Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 7-3-1 Hongo, Tokyo 113-0033, Japan. Tel.: 81-3-5684-9420. Fax: 81-3-5841-4465. E-mail: toh-e{at}biol.s.u-tokyo.ac.jp
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Abstract |
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Key Words: cell cycle; mitotic exit; Lte1; Ras; budding yeast
* Abbreviations used in this paper: CHD, Cdc25 homology domain; GEF, guanine nucleotide exchange factor; PKA, protein kinase A.
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Introduction |
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Lte1 (low temperature essential) is a protein with homology to Ras guanine nucleotide exchange factor (GEF) protein Cdc25 and is required for mitotic exit at a lowered temperature (Shirayama et al., 1994a). Lte1 was identified as a multicopy suppressor of the heat shock sensitivity of the ira1-1 and RAS2Val19 mutants (Shirayama et al., 1994a). As Lte1 contains the Cdc25 homology domain (CHD), Lte1 was supposed to interfere with the RascAMP pathway by direct interaction with Ras, not downstream of PKA. On the other hand, LTE1 was also identified as a multicopy suppressor of a late mitotic defect of the ras1
ras2 cyr1 mutant (Morishita et al., 1995). How the overexpression of LTE1 suppressed both dominant and deletion mutants of yeast RAS is an important question to understand the role of Lte1.
A possible role of Lte1 was suggested by the identification of Tem1 GTPase, which is essential for mitotic exit, as a multicopy suppressor of the lte1 mutant (Shirayama et al., 1994b). Because LTE1 encodes a Ras GEF homology protein, Lte1 would be a GEF for Tem1, and assuming this, an attractive model, in that the interaction of Lte1 at the bud cortex with Tem1GTPase at the spindle pole determines the timing of mitotic exit, has been proposed (Bardin et al., 2000). However, it was reported that Lte1 is not involved in the timing of mitotic exit at 30°C or above (Adames et al., 2001), and there is no positive biochemical evidence indicating that the Ras GEF domain of Lte1 possesses GEF activity toward Tem1 (Geymonat et al., 2002).
Recent studies have revealed that the Lte1 localization at the bud cortex is dependent on the Cdc42 GTPase and its effector Cla4 kinase, the phosphorylation state of Lte1, cell polarity protein Kel1, and septin, but is independent of the microtubule or actin cytoskeleton (Hofken and Schiebel, 2002; Jensen et al., 2002; Seshan et al., 2002). Hofken and Schiebel (2002) reported that Cdc42 and Cla4 are essential for priming Lte1 at the bud cortex; however, it remains unknown what anchors Lte1 there. We report here that active Ras2-GTP anchors Lte1 at the bud cortex via the direct interaction through the CHD of Lte1. We also found that the Ras GEF domain of Lte1 is essential for localization but not essential for mitotic exit, indicating that the CHD of Lte1 does not act as a GEF of Tem1.
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Results |
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RAS functions upstream of LTE1 in mitotic exit
The physical interaction between Ras2 and Lte1 raised the possibility that Ras regulates mitotic exit in concert with Lte1. One of the reasons why ras1
ras2
bcy1 cells, in which BCY1 is deleted to keep a
ras1
ras2 double mutant alive by bypassing its defect in the RascAMP pathway, are defective in mitotic exit is the failure of Cdc14 liberation, a critical step of mitotic exit, because Cdc145GFP release from the nucleolus is partially defective in
ras1
ras2
bcy1 cells compared with that in
bcy1 cells cultured at 10°C (Fig. 2 a). It has been reported that Cdc14 liberation is governed by at least two steps; one is by the Cdc14 early anaphase release (FEAR) and the other is by the mitotic exit network (MEN) (Pereira et al., 2002; Stegmeier et al., 2002; Yoshida et al., 2002). Lte1 is known to act at the top of the MEN and shows synthetic lethality in combination with a null mutation in a FEAR component SPO12 (Stegmeier et al., 2002). We found by tetrad dissection of SAY510 (RAS1/ras1, ras2/ras2, BCY1/bcy1, SPO12/spo12) cells that
ras1
ras2
bcy1
spo12 segregants were inviable whereas
ras1
ras2
bcy1
lte1 segregants were viable (Fig. 2 c). We also found that the low temperature sensitivity of
ras1
ras2
bcy1 as well as that of
lte1 was partially suppressed by the deletion of the BUB2 gene (Fig. S1 b). These genetic interactions indicate that Ras and Lte1 function in the same pathway.
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Ras is essential for Lte1 localization at the bud cortex
To investigate the role of Ras in the regulation of Lte1, we examined the bud cortex localization of Lte1 in the ras1 ras2 mutants because Ras proteins exist at the plasma membrane and Lte1 and Ras proteins interact with each other. In the ras1,
ras2, or
bcy1 cells, Lte12HA protein localized properly to the bud cortex as in wild-type cells (Fig. 3 a), but in
ras1
ras2
bcy1 cells, although LTE12HA was expressed at a same level, Lte12HA localization at the bud cortex was abolished and Lte12HA was diffused to the cytoplasm (Fig. 3 a) (unpublished data). This observation indicates that Ras proteins regulate Lte1 localization to the bud cortex, and Ras1 and Ras2 share a redundant role in Lte1 localization.
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Ras and Cdc42Cla4 pathways have a separate role in Lte1 localization
The recent finding that Cdc42 and its effector Cla4 kinase are essential for the hyperphosphorylation and for the bud cortex localization of Lte1 (Hofken and Schiebel, 2002; Jensen et al., 2002; Seshan et al., 2002) raises a possibility that Ras regulates Lte1 localization indirectly via activation of the Cdc42Cla4 pathway because Ras2 has recently been reported to regulate actin polarity and Cdc42 localization at a higher temperature (Ho and Bretscher, 2001).
To examine the phosphorylation level of Ras-bound Lte1, Lte12HA was pulled down with GFPRas (Fig. 4 a). By analyzing cell extracts, we found in ras1 ras2 bcy1 cells that the hyperphosphorylated form of Lte12HA was not so obvious as in the cla4 cells (Fig. 4 b), suggesting that the Cla4-dependent hyperphosphorylation of Lte1 is partially impaired in the absence of Ras. There are two possibilities that can explain the reason why Lte1 hyperphosphorylation is impaired in the absence of Ras: (1) the Cdc42Cla4 pathway (or Cla4 kinase activity) is not active in the ras mutant, and (2) Cla4 cannot interact with Lte1 simply because Lte1 is diffused in the cytoplasm in the absence of Ras. To examine whether Lte1 mislocalization in the absence of Ras is due to a failure in Cdc42Cla4 activation, we introduced an extra copy of CDC42 or dominantly active CDC42Val12 under an inducible promoter in the SAY627 (ras1 ras2 bcy1 pLTE12HA) cells and found that the induction of neither CDC42 nor CDC42Val12 recovered the Lte1 localization at the bud cortex at all (Fig. 4 c). This observation indicates that the Lte1 mislocalization in the ras mutant was not due to the lack of Cdc42 activation. We also found that Cla4GFP was localized at the bud cortex in the ras1
ras2
bcy1 cells as well as in
bcy1 cells (Fig. 4 d). Judging from the normal morphology of
ras cells and normal localization of Cdc12 septin shown in Fig. 4 d, Cla4 seems functional in the absence of Ras. Thus, we think that impaired hyperphosphorylation of Lte1 is not due to the misregulation of the Cdc42Cla4 pathway but due to the absence of Lte1 around Cla4 residing at the bud cortex. We also found that the hyperphosphorylation of Lte1 is not required for the Lte1Ras2 interaction because the Lte1 protein immunoprecipitated with GFPRas2 was not hyperphosphorylated (Fig. 4, a and b). Assuming that Lte1 and Ras2 interact at the plasma membrane, the hyperphosphorylation of Lte1 is not required for its localization.
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Ras GEF homology domain of Lte1 is essential for the interaction of Lte1 with Ras2
Lte1 contains the Ras GEFN (GEF at the NH2 terminus) domain at its NH2 terminus and the Ras GEF CHD at its COOH terminus, but the role of each domain is not yet clear. We constructed various truncated mutants of LTE1 to determine the domains of Lte1 that are essential for the interaction of Lte1 with Ras2 in vivo (Fig. 5 a). We found that deletion of 103 amino acids from the COOH terminus or of 500 amino acids from the NH2 terminus of Lte1 abolished the Lte1Ras2 interaction, as judged by immunoprecipitation, even when these truncated versions of Lte1 were overproduced (Fig. 5 b). In contrast to the bacterial two-hybrid system (Fig. 1 a), the CHD alone is not sufficient for the interaction with Ras2 in vivo (Fig. 5 b). These results indicate that both NH2- and COOH-terminal GEF homology domains of Lte1 are essential for in vivo association of Lte1 with Ras2.
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Ras GEF domain of Lte1 is not essential for mitotic exit
If Lte1 is the GEF of Tem1 GTPase, the CHD of Lte1 is functioning both for the activation of Tem1 and for anchoring itself to the bud cortex via Ras binding. We attempted to determine an essential domain of Lte1 for mitotic exit. If the CHD of Lte1 acts as a Tem1 GEF, a mutant protein lacking this domain would not fulfill the function of Lte1 in mitotic exit. We found that overexpression of the CHD domain of Lte1 could not suppress the low temperature sensitivity of lte1, but to our surprise, overexpression of the internal region of Lte1 (LTE1-mini) that lacks both NH2- and COOH-terminal Ras GEF homology domains cured the cold sensitivity shown by lte1 cells (Fig. 5 e). This was further substantiated by following the length of the spindle during synchronized cultures of lte1 GFPTUB1 cells; the cell cycle of the cells containing Lte1-mini progresses more or less like those containing genuine Lte1 (Fig. 5 f). We also found that overexpression of LTE1-mini, as well as full-length LTE1, rescued the temperature sensitivity of the ras1 ras2 mutant (Fig. 5 a). Lte1d10 lost activities expressed by the Lte1mini (Fig. 5, a and e), indicating the importance of the amino acid sequence 868926 for Lte1mini activity. These results indicate that the LTE1-mini domain is sufficient for transmitting the signal of mitotic exit to its downstream target, and the Ras GEF homology domain of Lte1 is essential only for its proper localization by association with Ras and is dispensable when the mini domain of Lte1 is overproduced.
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Discussion |
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We think there is another target of Ras involved in mitotic exit because the ras1 ras2 bcy1 cells are defective in mitotic exit not only at a higher but also at a lower temperature, whereas lte1 cells are defective in mitotic exit only at a lower temperature. Although it is also possible that active Ras inhibits, and at the same time promotes, mitotic exit because the active RascAMP pathway does some harm on mitotic exit by inhibiting the anaphase-promoting complex (APC) (Anghileri et al., 1999; Irniger et al., 2000). It requires further study to elucidate the role of Ras in mitotic exit.
The role of Lte1
Our findings strongly support the idea that Lte1 functions as an effector of Ras because (a) Lte1 binds to Ras2-GTP, and this interaction is essential for Lte1 localization, and (b) overexpression of Lte1 rescues the mitotic exit defect of a ras1
ras2 mutant whereas active RAS2Val19 mutation does not rescue the cold sensitivity shown by the
lte1 mutant. Lte1 has long been regarded as a GEF for Tem1 because Lte1 contains a putative Ras GEF domain and Lte1 functions upstream of Tem1. But our finding that overexpression of the LTE1-mini (659926) domain, which lacks both NH2- and COOH-terminal Ras GEF homology domains, fulfills the LTE1 function (Fig. 5, e and f) suggests that Lte1 is not necessarily a GEF for Tem1.
Our finding that Lte1 physically associated with active Ras2 strongly supports the idea that overexpressed Lte1 blocks the RascAMP pathway by titrating out the excess amount of active Ras. The facts that overexpression of LTE1 suppressed the heat shock sensitivity of the active Ras mutation RAS2Val19 and ira1-1, but not that of the bcy1 mutation (Shirayama et al., 1994a), and that truncated Lte1 mutants that failed to interact with Ras2 also failed to suppress the heat shock sensitivity of RAS2Val19 (Fig. 5 d) further support the idea that Lte1 acts directly with Ras. However, it is unlikely that Lte1 functions as an inhibitor of the RascAMP pathway in a physiological state because lte1 cells did not show the heat shocksensitive phenotype (unpublished data).
Genetic data indicate that Lte1 possibly functions upstream of Tem1 (Shirayama et al., 1994b), but it remains unclear how Lte1 regulates the Tem1 pathway. Recent studies have revealed that Lte1 physically associates with Kel1/Kel2, and the deletion of either protein rescues the mitotic defect of lte1 cells (Hofken and Schiebel, 2002). These interactions suggest that the function of Lte1 is to inactivate Kel1/Kel2.
Finally, we would like to emphasize that the CHD of Lte1 has a unique property that prefers to bind to GTP-bound Ras2. It is of interest whether there are other proteins whose exchange factor domain does not act as an exchange factor but acts as if it is an effector.
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Materials and methods |
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Purification of flagLte1
SAY623 cells overexpressing flagLte1 under the GAL1 promoter in YPGalactose medium were lysed in lysis buffer LBI (50 mM Tris-HCl, 150 mM NaCl, 0.5% Triton X-100) and incubated with anti-flag M2 beads. The beads were subsequently washed with LBII (50 mM Tris-HCl, 500 mM NaCl, 1.5% Triton X-100) and LBIII (50 mM Tris-HCl, 1.5 M NaCl, 0.1% Triton X-100), and then flagLte1 was eluted from the beads with 0.1 M glycine (pH 3.4). Eluted flagLte1 was neutralized and separated by anion exchange mono-Q column (Amersham Biosciences) and used for the Ras2 binding assay.
In vitro binding of Ras2Lte1
A two-hybrid test using the BacterioMatch system (Stratagene) was performed per the manufacturer's instructions. XL1-Blue MRF' Kan (Stratagene) was used as the host for the bacterial two-hybrid assay. The ampr gene was used as a reporter gene whose expression was detected by resistance to carbenicillin. Purified flagLte1 was precipitated with M2 beads, and 10 µl of the aliquots (containing 30 pmol flagLte1) was incubated with 6 pmol each of various forms of purified Ras2 protein, which had been loaded with GTPS or GDP, in 30 µl of buffer A (20 mM Tris-HCl, pH 7.4, 40 mM NaCl, 5 mM MgCl2, 1 mM EDTA, 1 mM dithiothreitol, 0.1% Lubrol PX) (Shima et al., 2000) for 30 min with continuous mixing at 30°C and subsequently washed three times with buffer A. The bound Ras2 was separated by SDS-PAGE and detected by Western immunoblotting with the anti-Ras2 antibody (Santa Cruz Biotechnology, Inc.).
Online supplemental material
The supplemental figure and tables are available at http://www.jcb.org/cgi/content/full/jcb.200301128/DC1. The localization of Ras2 and its derivatives, the suppression of the ras1 ras2 bcy1 low temperaturesensitive phenotype by bub2, and a detailed domain analysis of Lte1 are included in the figure. The yeast strains and plasmids used in this study are listed in the tables.
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Acknowledgments |
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This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology. S. Yoshida is a recipient of a Japan Society for the Promotion of Science fellowship for young scientists.
Submitted: 29 January 2003
Revised: 5 March 2003
Accepted: 7 March 2003
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