Physical interactions between the Rrd proteins and the catalytic subunits of PP2A and 2A-like phosphatases have been demonstrated previously (Mitchell and Sprague, 2001
; Fellner et al., 2003
; Van Hoof et al., 2004
). However, because these phosphatases exist in both Tap42-dependent and -independent forms (Di Como and Arndt, 1996
), it is not clear which form of the phosphatases is the direct target of the Rrd proteins. As a first proof that the Rrd proteins were part of the Tap42–phosphatase complexes, we reexamined the interactions between the Rrd proteins and the phosphatases in our strain background. Accordingly, we immunoprecipitated myc epitope-tagged Rrd1 and Rrd2 from cell extracts and determined the presence of Sit4 and PP2Ac in the precipitates by Western blot analysis. As shown in , we found that both phosphatases copurified with the Rrd proteins (, lane 2 and 3). Interestingly, the interaction between the Rrd proteins and the phosphatases seems to be highly selective, because we found that Sit4 coprecipitated almost exclusively with Rrd1 (, middle, compare lanes 2 and 3) and Pph21 mainly with Rrd2 (, bottom, compare lanes 2 and 3). These findings demonstrate a distinct feature for Rrd1 and Rrd2 in phosphatase interaction. Nevertheless, a small amount of Sit4 was found to be copurified with Rrd2 (, lane 3). A slight increase in the level also was observed when RRD2
was overexpressed or RRD1
was deleted from the cells (Zheng and Jiang, unpublished observation). These findings suggest a partially overlapped binding activity between Rrd1 and Rrd2. Similarly, a small portion of Pph21 was found to be associated with Rrd1 (, lane 3). However, in this case, some of Pph21 was believed to be from the Tpd3–Pph21–Rrd1 complex (see below). The drastic difference in the amount of the phosphatases copurified with Rrd1 and Rrd2 was not due to their expression levels, because we found that the levels of the phosphatases were the same in all the cells used in this experiment ().
Figure 1. Interaction of the Rrd proteins with Sit4 and Pph21. Yeast cells (Y162) expressing HA-PPH21 were transformed with pRS314 (lane 1), pRS314-RRD1(myc)13 (lane 2), or pRS314-RRD2(myc)13 (lane 3). (A) Interaction of the myc-tagged Rrd proteins (Rrd-myc) with (more ...)
On determination of the specific interactions between the Rrd proteins and the two different phosphatases, we assessed the association between the Rrd proteins and Tap42 by coimmunoprecipitation. As shown in , when Tap42 antibody was used to precipitate Tap42 from cell extracts, both Rrd1 and Rrd2 were found in the precipitates (, lanes 2 and 3). Conversely, when myc antibody was used to purify the myc epitope-tagged Rrd1 and Rrd2, Tap42 was found to be copurified with both Rrd1 and Rrd2 (, lanes 2 and 3). These results demonstrate that Rrd1 and Rrd2 physically interact with Tap42.
Figure 2. Rrd1 and Rrd2 physically associate with Tap42. The interaction of Tap42 with either Rrd1-myc or Rrd2-myc was examined by coimmunoprecipitation. (A) Anti-Tap42 antibody was used to precipitate extracts from cells expressing a control vector (Y847, lane1), (more ...)
To confirm that the Rrd proteins were part of the Tap42–phosphatase complexes, we examined the existence of the Tap42–Sit4–Rrd1 ternary complexes by using two sequential pull-down assays. In the first pull-down, GST-Sit4, along with proteins associated with it, was precipitated with glutathione beads. In the second pull-down, the proteins eluted from the glutathione beads were precipitated with anti-Tap42 antibody. As shown in , in the first pull-down both Tap42 and Rrd1 were found to be copurified with GST-Sit4 (lane 4) but not with GST alone (lane 3). In the second pull-down, both GST-Sit4 and Rrd1 were found in the Tap42 precipitates (lane 6). Should the three proteins form independent dimers between them, only GST-Sit4 or Rrd1 but not both would occur in the Tap42 precipitate. The occurrence of both GST-Sit4 and Rrd1 in the precipitate indicated that the three proteins were in the same complex. As a control, GST was not found in the Tap42 precipitate (lane 5), indicating that Tap42 bound only to Sit4 but not GST.
Figure 3. Rrd1 forms a ternary complex with Tap42 and Sit4. Extracts from cells expressing the RRD1(myc)13 gene (Y850) together with either a GST (lane 1) or GST-SIT4 gene (lane 2) were precipitated with glutathione beads. The presence of Rrd1-myc and Tap42 in (more ...)
Previously, it has been reported that Rrd1 interacts with Sit4 by binding to its C-terminal catalytic domain (Mitchell and Sprague, 2001
). On the other hand, our recent work shows that Tap42 binds to the N-terminal region of Sit4 (Wang et al., 2003
). It is thus possible that phosphatases may act as a bridge to bring Tap42 and the Rrd proteins into the same complex. To test this notion, we examined the interaction of Tap42 with Rrd1 and Rrd2 in cells lacking of either Sit4 or Pph21 and Pph22. As shown in , a significantly decreased amount of Tap42 was found to be copurified with Rrd1 in cells lacking Sit4 compared with wild-type cells (, compare lanes 1 and 2). Similar reduction in the amount of Tap42 coprecipitated with Rrd2 also was observed in the pph21 pph22
cells in comparison with that in the wild-type cells (, compare lanes 3 and 4). The diminished interaction between Tap42 and the Rrd proteins in the absence of the phosphatases suggests that the phosphatases play a significant role in facilitating the interaction. Nevertheless, these results also reveal that Tap42 is able to interact with the Rrd proteins independently of the phosphatases. To rule out the possibility that the reduced interaction between Tap42 and Rrd1 in the absence of Sit4 was mediated by other 2A-like phosphatases, we examined the interaction by using an in vitro GST pull-down assay. As shown in , we found that bacterially expressed recombinant Tap42 was copurified with a GST-Rrd1 fusion protein but not with GST (, compare lanes 4 and 5), suggesting that Tap42 possessed an intrinsic binding activity toward Rrd1.
Figure 4. Tap42 is able to interact with the Rrd proteins independent of the phosphatases. Plasmids pRS314-RRD1(myc)13 or pRS314-RRD2(myc)13 were introduced into the sit4 (Y397), pph21 pph22 (Y531) mutants as well as their isogenic wild-type alleles (Y062 and Y661). (more ...)
The mammalian PTPA was shown to act on the AC dimeric core of the PP2A holoenzyme (Cayla et al
). We thus determined whether the AC dimer of PP2A in yeast was also a target of the Rrd proteins. Accordingly, we immunopurified myc epitope-tagged Rrd1 and Rrd2 from cell extracts and examined the precipitates for the presence of Tpd3, the only A subunit of PP2A in yeast. As shown in , Tpd3 was found to be coprecipitated with both Rrd1 and Rrd2, suggesting that the A subunit was able to interact with both proteins. However, Cdc55, one of the B subunits of PP2A, was not found in the precipitates. This result indicates that the Rrd proteins interact with the AC dimeric core but not the holoenzyme. In addition, the finding that both Rrd1 and Rrd2 interact with Tpd3 indicates that at least some of the Pph21 copurified with Rrd1 shown in was from the Tpd3-containing complex.
Figure 5. The Rrd proteins interact with the A subunit of PP2A. Extracts from cells expressing either RRD1(myc)13 (Y929) or RRD2(myc)13 (Y930) were precipitated with anti-myc antibody. The presence of Tpd3 (middle) and Cdc55-HA (bottom) in the cell extracts (Ext) (more ...)
Interestingly, the amount of Tpd3 copurified with Rrd1 was found to be similar to that copurified with Rrd2, indicating that Rrd1 and Rrd2 were equally effective in their interaction with the AC dimer. This is in contrast to their interaction with Pph21 shown in , in which case Pph21 was mainly associated with Rrd2, and only a small fraction was in complex with Rrd1. This discrepancy suggests that the most of Pph21 copurified with Rrd2 shown in was in the Tap42-containing complex. To confirm this notion, we compared the amount of Rrd2 associated with Tap42 with that associated with Tpd3. This was done by immunoprecipitating Tap42 and Tpd3 by using excessive amount of antibodies so that nearly all the antigens were precipitated from cell extracts (). As shown in , the amount of Rrd2 copurified with Tpd3 was significantly less than that copurified with Tap42 (). Similarly, significantly less Rrd1 was found to be associated with Tpd3 than with Tap42 (). These findings suggest that the Tap42-containing complexes are the major targets of the Rrd proteins.
Figure 6. The Tap42–phosphatase complexes are the major targets of the Rrd proteins. Extracts from cells expressing RRD2(myc)13 (Y852) were precipitated with either anti-Tap42 or anti-Tpd3 antibody. (A) Levels of Tap42 (top) and Tpd3 (bottom) in the extracts (more ...)
The interaction of Tap42 with Sit4 and PP2Ac has been shown to be rapamycin sensitive (Di Como and Arndt, 1996
). It is believed that rapamycin induces the release of the phosphatases from Tap42, and consequently their activation (Jacinto et al., 2001
). Therefore, upon demonstration that the Rrd proteins were part of the Tap42–phosphatase complexes, we sought to determine whether the interaction of Tap42 with the Rrd proteins also was rapamycin sensitive. As shown in , rapamycin treatment significantly reduced the amount of Rrd1 (, middle) and Rrd2 (, middle) copurified with Tap42. Interestingly, the extent of reduction was similar to the decrease in the amount of Tap42 associated with the phosphatases (, bottom), indicating that rapamycin induced the release of both the Rrd proteins and the phosphatases from Tap42. Conversely, rapamycin did not seem to affect the interaction between the Rrd proteins and the phosphatases, because the amounts of Sit4 and Pph21 copurified with Rrd1 and Rrd2, respectively, from the treated and untreated cells were similar (, compare lanes 1 and 2). These findings indicate that although the interaction between Tap42 and the Rrd proteins is rapamycin sensitive, the interaction between the Rrd proteins and phosphatases is not. It is thus evident that rapamycin treatment causes the release of the Rrd-phosphatase dimer from Tap42.
Figure 7. Interaction between Tap42 and the Rrd proteins is rapamycin sensitive. Yeast cells (Y162) expressing either RRD1(myc)13 or RRD2(myc)13 were treated with rapamycin (200 nM) or drug vehicle for 1 h. Tap42, Sit4 and HA-Pph21 were precipitated from extracts (more ...)
Release of Sit4 from Tap42 accompanies activation of the phosphatase, which is essential for rapamycin-induced dephosphorylation of many factors downstream of the Tor proteins, including Gln3, a GATA transcription factor that is involved in nitrogen catabolism in yeast (Beck and Hall, 1999
; Cardenas et al., 1999
; Bertram et al., 2000
). On finding that the Rrd proteins were released together with the phosphatases, we asked whether these proteins were required for the rapamycin-induced activity of the phosphatases. Accordingly, we examined the dephosphorylation of Gln3 in response to rapamycin treatment in cells lacking either Rrd1 or Rrd2 or both. As published previously (Cardenas et al., 1999
; Bertram et al., 2000
) and as shown in , rapamycin induced dephosphorylation of Gln3, which was characterized by the appearance of a faster migrating band on SDS-PAGE (compare lanes 1 and 2). The dephosphorylation of Gln3 was absent in cells lacking Sit4 (compare lanes 9 and 10). Rapamycin caused a full dephosphorylation of Gln3 in cells without Rrd2 (compare lanes 2, 5, and 6) and a partial dephosphorylation in cells lacking Rrd1 (compare lanes 2, 3, and 4). However, rapamycin failed to induce Gln3 dephosphorylation in cells lacking both Rrd1 and Rrd2 (compare lanes 7 and 8). These results indicate that the Rrd proteins are required for rapamycin-induced activation of Sit4. The absence of Sit4 activation in the rrd1 rrd2
double mutant and the partial activation of it in the rrd1
single mutant seem to indicate a partially overlapped function between Rrd2 and Rrd1 in supporting Sit4 activation.
Figure 8. Rrd proteins are required for rapamycin-induced activation of the Sit4 phosphatase. Wild-type (Y661), rrd1 (842), rrd2 (Y843), rrd1 rrd2 (Y874), and sit4 (Y397) cells expressing a myctagged GLN3 gene were treated with or without rapamycin (200 nM) for (more ...)
As mentioned in the introduction, the RRD1
genes are characterized by the rapamycin resistance associated with cells lacking either gene (Rempola et al., 2000
). Because the rrd1
and rrd1 rrd2
mutants displayed defect in Sit4 activation in the presence of rapamycin (), we asked whether Sit4 activity was required for the drug resistance trait associated with the rrd1
cells. We thus examined the rapamycin sensitivity of the sit4 rrd1
and sit4 rrd2
double mutants. As published previously (Rempola et al., 2000
) and as shown in , the rrd1, rrd2
and rrd1 rrd2
double deletions were found to be resistant to rapamycin. The drug resistance of the mutants seemed to correlate reversely with the Sit4 activity, with the rrd1 rrd2
double mutant showing the least activity () and the most resistance (, compare the growth of the treated and untreated cells). Surprisingly, the sit4 rrd1
and sit4 rrd2
double mutants were found to be as sensitive to rapamycin as the sit4
single mutant. This finding indicates that the sit4
deletion is epistatic to both the rrd1
mutants. On the other hand, we found that the sit4
deletion barely affected the resistance of the gln3
deletion to rapamycin. The drug treatment reduced the growth of both the gln3
and gln3 sit4
cells by 1 order (, compare the growth of the cells in the presence and absence of the drug). The epistatic interaction of gln3
is in accordance with the fact that Gln3 acts downstream of Sit4. Together, our findings suggest that the rapamycin resistant trait of the rrd
mutants requires Sit4.
Figure 9. Rapamycin resistance of the rrd1 and rrd2 mutants is dependent upon Sit4. (A) Mid-log phase cells of wild-type (Y062), rrd1 (Y884), rrd2 (Y885), rrd1 rrd2 (Y871), sit4 (Y397), rrd1 sit4 (Y886), and rrd2 sit4 (Y887) strains were subjected to a series of (more ...)