sec3ΔN is synthetic lethal with the exo70-38 mutant
We have previously shown that the N terminus of Sec3 (Sec3N, aa 1–320) interacts with the Rho GTPases (Guo et al., 2001
; Zhang et al., 2001
). Surprisingly, cells expressing N terminus–deleted Sec3 (sec3ΔN
) as the only copy of Sec3 grew normally at all temperatures tested, and these cells did not have any defects in secretion (Guo et al., 2001
). A recent study showed that Exo70, like Sec3, resides on the bud tip membrane and that the other subunits, arriving via secretory vesicles, interact with Sec3 and Exo70 at the bud tip for exocyst assembly (Boyd et al., 2004
). Furthermore, it was shown that Exo70 binds directly to PIP2
(He et al., 2007b
; Liu et al., 2007
). It is thus possible that Sec3 and Exo70 function together in exocyst targeting in yeast. In that case, mutations in Sec3 or Exo70 that alone cause little or no phenotype may have a synthetic defect when combined. We recently obtained Exo70 mutants (He et al., 2007a
,b) that allowed us to test this hypothesis. Both sec3ΔN
were expressed under their endogenous promoters in CEN
has no growth defect. exo70-38
can survive at 25 but not 37°C (He et al., 2007a
is synthetic lethal with exo70-38
, as at all temperatures tested the sec3ΔN
double mutant sec3ΔN exo70-38
cannot survive on 5–fluoroorotic acid (5-FOA) plates on which the URA3
-based wild-type SEC3
balancer is eliminated (). The genetic interaction between sec3ΔN
suggests that the N terminus of Sec3 becomes indispensable in the exo70
mutant background. Besides exo70-38
also has synthetic defects with other exocyst mutants (Roumanie et al., 2005
; unpublished data).
Figure 1. The gic2-sec3 chimera is able to rescue synthetic lethality between sec3ΔN and exo70-38. (A) sec3ΔN is synthetic lethal with exo70-38. sec3ΔN and exo70-38 were expressed under SEC3 and EXO70 promoters in CEN plasmids. The sec3ΔN (more ...)
Functional replacement of the Sec3ΔN terminus with Gic2 N terminus
We have previously shown that the N terminus of Sec3 directly interacts with Cdc42, but we were unable to assess the functional importance of this interaction (Zhang et al., 2001
). Now, taking advantage of the synthetic lethality assay, we tested whether the lethality of the sec3ΔN exo70-38
double mutant could be rescued by adding the N terminus of Gic2 (Gic2N, aa 1–155) to sec3ΔN
(). Gic2 is a well-characterized effector of Cdc42 (Brown et al., 1997
, Chen et al., 1997
). Gic2N contains a Cdc42/Rac interactive binding (CRIB) domain that interacts with the GTP-bound form of Cdc42. We tested whether this gic2-sec3
chimera, when expressed under the SEC3
promoter, can function as well as the wild-type SEC3
in the exo70-38
background. We found that although exo70-38
were synthetic lethal, growth of the exo70-38 gic2-sec3
strain was similar to that of the exo70-38 SEC3
strain on plates at 25°C () and all other temperatures tested (not depicted).
Next, we performed experiments to identify the sequences of Gic2N that are crucial for its functional replacement of the Sec3 N terminus. First, we mutated or deleted the CRIB domain in Gic2N and tested the synthetic effects of these mutants with exo70-38. As shown in , the gic2-sec3 chimeras with the CRIB domain mutated or deleted were synthetic lethal with exo70-38, whereas the mutant proteins were expressed at similar levels to the wild-type protein (not depicted). Gic2 also contains a cluster of positively charged residues (aa 109–121) adjacent to the CRIB domain that are implicated in membrane association. As shown in , replacing these basic residues with alanine (K109A, K110A, K119A, K120A, and K121A) led to synthetic lethality between gic2-sec3 and exo70-38. These genetic analyses indicate that both the CRIB domain and the polybasic region of Gic2 are essential for the ability of the gic2-sec3 chimera to functionally replace SEC3 in yeast cells.
Localization of Sec3 in tropomyosin mutants and cells treated with latrunculin
It was previously reported by Finger et al. (1998)
that polarized localization of Sec3-GFP to the bud tip is independent of actin. In addition, fluorescence recovery after photobleaching (FRAP) of Sec3-GFP and Exo70-GFP at the bud was found to take place properly in the presence of latrunculin, which disrupted actin cables in cells (Boyd et al., 2004
). Consistent with these observations, Sec3-GFP was found to remain polarized in the tropomyosin mutant, tpm1-2 tpm2Δ
(Zajac et al., 2005
), in which actin cables were disrupted upon shifting to the restrictive temperature (Pruyne et al., 1998
). However, using an anti-Sec3 antibody, Roumanie et al. (2005)
reported that Sec3 immunofluorescence signals at the bud were lost in the tropomyosin mutant. In this study, we performed the immunostaining experiment using a Sec3 antibody (generated against a fusion protein containing aa 445–711 of Sec3) in parallel with GFP-tagged Sec3. This antibody detects Sec3 in wild-type (SEC3
) but not sec3
) cells by Western blotting or immunofluorescence (). The endogenous Sec3 was polarized in the TPM1 tpm2Δ
cells shifted to 34.5°C for 10 min (72% of the cells; n
= 300) or 60 min (58% of the cells; n
= 300; ). Sec3 remained polarized in many of the tpm1-2 tpm2Δ
cells shifted to 34.5°C for 10 min (53% of the cells; n
= 300) or 60 min (36% of the cells; n
= 300). However, in many cases, the polarized signals in these mutant cells were not concentrated as tight patches like those observed in the control cells. We also observed flat disc-shaped signals near the mother–daughter connections in the cells after zymolyase treatment (not counted as polarized signals in the experiments). In contrast, the Rab protein Sec4 was completely depolarized in the mutant cells. Sec3-GFP was well polarized in the tpm1-2 tpm2Δ
mutant cells. We noticed that some of the tpm1-2 tpm2Δ
mutant cells were easily lysed during the immunofluorescence procedure. The zymolyase and SDS treatments during yeast cell wall removal and membrane permeabilization steps may have led to the partial dispersion of the Sec3 signals in tpm
mutant cells. This is supported by a side-by-side comparison of the procedures for direct GFP observation and immunofluorescence in cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200704128/DC1
). In addition, the antibodies used in Roumanie et al. (2005)
and in this study were generated against different regions of Sec3, which may also contribute to the discrepancy. In fact, different antibodies and staining methods have been successfully used to reveal different pools of exocyst in mammalian cells (Yeaman et al., 2001
; Oztan et al., 2007
Figure 2. Localization of Sec3 in the tropomyosin mutant and cells treated with latrunculin. (A) The affinity-purified anti-Sec3 antibody recognizes Sec3 in wild-type (SEC3) but not sec3 deletion (sec3Δ) cells by Western blotting (left; molecular masses (more ...)
To avoid the cell lysis problems, we took another approach commonly used to study the role of actin cables in the localization of proteins involved in yeast cell polarity (Ayscough et al., 1997
). Cells were first arrested at G0
phase and then released to fresh medium for growth in the presence of latrunculin B (Lat B), which disrupts actin in yeast cells. Immunostaining was then performed in these cells to detect Sec3 localization. We found that although actin cables were clearly disrupted in the Lat B–treated cells, Sec3 remained polarized (66%; n
= 300; ).
Polarization of sec3ΔN-GFP is sensitive to latrunculin
The sec3ΔN protein, when expressed as the only copy of Sec3 in the cell, was well polarized to the bud tip (Guo et al., 2001
). We compared the targeting of sec3ΔN-GFP and Sec3-GFP to the emerging bud after G0
release in the presence of latrunculin. As shown in , in cells treated with Lat B, sec3ΔN-GFP was dispersed throughout the cells. However, the full-length Sec3 still formed a patch in the presumptive buds. As a control, both Sec3-GFP and sec3ΔN-GFP were well polarized in cells treated with DMSO. These results suggest that, unlike full-length Sec3, the delivery of sec3ΔN to the bud requires intact actin cables in a fashion similar to the other exocyst components that associate with secretory vesicles (Boyd et al., 2004
). Because the gic2-sec3
chimera is able to functionally replace SEC3
in yeast, we tested whether the initial targeting of gic2-sec3 to the bud is, as with the full-length Sec3, independent of actin cables. As shown in , similar to Sec3-GFP, gic2-sec3–GFP was localized to the presumptive bud sites in cells treated with Lat B. We conclude that the N terminus of Sec3 confers its actin-independent localization at the bud tip and that the interaction of Cdc42 with Sec3 is important for Sec3 targeting.
Figure 3. The actin-independent localization of Sec3 is conferred by its N terminus. (A) The targeting of sec3ΔN to the bud tip is dependent on actin. Yeast cells expressing Sec3-GFP or sec3ΔN-GFP under the SEC3 promoter as the sole copy of Sec3 (more ...)
Previous FRAP analyses demonstrated that Sec3-GFP fluorescence recovery at the bud can take place even in the presence of latrunculin (Boyd et al., 2004
). We asked whether sec3ΔN-GFP fluorescence could recover after photobleaching if actin is disrupted. Cells expressing Sec3-GFP and sec3ΔN-GFP under the SEC3
promoter were grown at 25°C. Small buds of these cells were bleached with laser, and pre-and postbleach images were recorded over time. As shown in , the full-length Sec3-GFP was able to recover its fluorescence at the bud tip in the presence of latrunculin, which is consistent with the previous observation (Boyd et al., 2004
). On the contrary, sec3ΔN-GFP could not recover. As controls, both Sec3-GFP and sec3ΔN-GFP recovered in the presence of DMSO. We also compared the recovery time of Sec3-GFP and sec3ΔN-GFP (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200704128/DC1
). sec3ΔN-GFP, like Sec8-GFP, reached nearly full recovery by 30 s, whereas Sec3-GFP did not fully recover until ~90 s. The data suggest that sec3ΔN may localize to the bud tip through its association with the other exocyst components.
Figure 4. FRAP of sec3ΔN-GFP at the bud relies on actin cables. (A) Recovery of Sec3-GFP or sec3ΔN-GFP fluorescence in cells treated with DMSO or 100 μM Lat B. Bar, 5 μm. (B) Fluorescence recovery graphs of cells expressing either (more ...)
The N terminus of Sec3 directly interacts with PIP2
Sequence analysis reveals that Sec3, like Gic2, also has a cluster of basic residues, including K134, K135, K136, and R137, located at its N terminus. In many cases, clusters of basic residues are implicated in direct interaction with the negatively charged phospholipids, including PIP2 and phosphatidylserine (PS), distributed in the inner leaflet of the plasma membrane. Because Sec3 is stably localized to the bud tip membrane, it is likely that Sec3 directly interacts with phospholipids via its N-terminal basic residues. To test this, we examined the binding of recombinant Sec3N to large unilamellar vesicles (LUVs) containing various phospholipids. As shown in , Sec3N bound to LUVs containing PIP2 but not to LUVs containing phosphatidylcholine (PC). Sec3N also bound to PS; however, the binding was indistinct unless the molar ratio of PS in the LUVs was raised to 60%. Noticeably, although Sec3N only bound diminutively to 20% PS LUVs, combining PIP2 and PS (5% PIP2 + 20% PS) significantly increased the affinity of Sec3N for the LUVs. As a control, GST did not bind to LUVs of any lipid composition. To measure the affinity of these interactions, we examined the binding of Sec3N to LUVs with increasing lipid concentrations. As shown in , although Sec3N barely bound to 20% PS, the affinity of Sec3N for LUVs containing 5% PIP2 was greatly enhanced when 20% PS was added (dissociation constant [Kd] = 14.4 ± 4 μM). This result suggests that membrane association of Sec3 involves multiple Sec3–phospholipid interactions. It is possible that PS, though binding to Sec3 with low affinity, synergistically contributes to Sec3 binding to PIP2 in physiological membranes.
Figure 5. The N terminus of Sec3 directly binds to phospholipids. (A) 0.3 μm GST-Sec3N (aa 1–320) purified from bacteria was incubated with liposomes containing 100% PC, 5% PIP2, 20% PS, 60% PS, or a combination of 5% PIP2 and 20% PS. After ultracentrifugation, (more ...)
We next tested whether mutating the polybasic region of Sec3 affects its binding to phospholipids. Mutagenesis was performed to change these residues to alanine (K134A, K135A, K136A, and R137A). As shown in , mutating theseresidues impaired the ability of Sec3 to bind to LUVs containing PIP2.
Identification of residues on Sec3 that are crucial for Cdc42 binding
After finding the interaction between Sec3 and Cdc42 (Zhang et al., 2001
), we performed several domain-mapping experiments to narrow down the region in Sec3 that mediates its interaction with Cdc42. We found that aa 140–155 were important for the binding (). We mutated four residues (I140A, L141A, S142A, and P145A) in this region and tested the mutant (named sec3-201
) for Cdc42 binding. The wild-type and mutant forms of the Sec3 N-terminal sequence (Sec3N and sec3-201N; aa 1–320) were expressed as GST fusion proteins, and Cdc42 was expressed as a Hisx6-tagged fusion protein. These recombinant proteins were purified from bacteria and used in an in vitro binding experiment. As shown in , the wild-type Sec3N bound strongly to Cdc42 in the presence of GTPγS, which is consistent with our previous observation (Zhang et al., 2001
). However, the sec3-201
mutant had almost no detectable binding to Cdc42. We also mutated four residues in the polybasic region (K134, K135, K136, and R137) into alanine (sec3-202
) or negatively charged glutamic acid (sec3-203
). The sec3-202
mutant (K/R→A) was able to bind to Cdc42 at a level comparable to wild-type Sec3. The sec3-203
mutant (K/R→E) had reduced binding to Cdc42. The dramatic charge reversion probably led to a certain degree of perturbation of the Sec3N structure. The sec3-201
mutant that failed in Cdc42 binding remains capable of binding to PIP2
-containing lipids in the LUV sedimentation assay ().
Figure 6. The Cdc42 binding domain and the polybasic region of Sec3 are important for Sec3 function. (A) Diagram of the Sec3 sequence containing the potential Cdc42 binding site and the polybasic region. Residues before R137 may be involved in lipid binding and (more ...)
The polybasic region and the Cdc42-binding region of Sec3 are critical for Sec3 function
Taking advantage of the synthetic lethality assay (), we assessed the functional implications of the Sec3 interaction with phospholipids and Cdc42. First, SEC3 was replaced with the Cdc42-binding–deficient mutant sec3-201 in exo70-38 cells. As shown in , the double mutant exo70-38 sec3-201 was inviable at 32°C. Next, we examined the synthetic genetic interaction between exo70-38 and sec3-202 or sec3-203. Both sec3-202 and sec3-203 had clear synthetic growth defects with exo70-38 at 32°C. The K/R→E mutation in sec3-203 led to a more severe growth defect than the K/R→A mutation in sec3-202. When mutations in the Cdc42 binding domain (sec3-201) were combined with the mutations in the polybasic region (sec3-202 and sec3-203), the resulting sec3 mutants sec3-204 and sec3-205 became synthetic lethal with exo70-38, even at 25°C (). Combining these results, we conclude that the dual interactions of Sec3 with Cdc42 and phospholipids are important for Sec3 function in cells.
Cdc42 and PIP2 interactions are important for Sec3 targeting to the bud tip
Using these sec3 mutants, we asked whether the interactions of Sec3 with Cdc42 and phospholipids are important for its polarization to the bud tip. The sec3 mutants were integrated into the SEC3 locus in the yeast chromosome to replace the endogenous SEC3. These sec3 mutants were then C-terminally tagged with GFP by chromosomal integration. The cells were arrested in G0 phase and then released into fresh medium in the presence of Lat B. Polarization of sec3-201, sec3-202, and sec3-203 to the presumptive bud emergence site were all affected to various extents in the presence of Lat B (). The sec3-204 and sec3-205 mutants that combine the Cdc42-binding and phospholipid-binding mutations failed to polarize to the bud tip. Quantification of the cells that polarized, partially polarized, or depolarized Sec3-GFP is presented in . These results suggest that the Cdc42 and phospholipid interactions synergistically control the actin-independent targeting of Sec3 to the bud tip during budding.
Figure 7. Mutations in the Cdc42 binding region or the polybasic region of Sec3 affect its targeting to the bud tip in the presence of Lat B. (A) Yeast cells expressing SEC3 or various sec3 mutants under the SEC3 promoter as the only copy of Sec3 were GFP tagged (more ...)
Using the same method, we also examined the polarization of the exo70-38 protein in cells treated with Lat B. In cells treated with Lat B, the wild-type Exo70 still formed a patch in the presumptive budding site; however, exo70-38–GFP was dispersed throughout the cells. As controls, both Exo70-GFP and exo70-38–GFP were well polarized in cells treated with DMSO ().
The sec3 exo70-38 double mutants display severe secretion defects
We examined the secretion of the periplasmic enzyme invertase in the exo70-38 sec3-201 and exo70-38 sec3-203 double mutants. As shown in , after a 2-h shift to 35°C, the exo70-38 or sec3ΔN single mutant cells secreted >90% of the total invertase, whereas the exo70-38 sec3-201 cells and the exo70-38 sec3-203 cells only secreted 34.1 and 38.8% of the total invertase, respectively. We also examined the secretion of the cell wall modification enzyme Bgl2. As shown in , no Bgl2 accumulation was detected in the exo70-38 or exo70-38 sec3-201 cells at 25°C, and only a small amount of Bgl2 was accumulated in the exo70-38 sec3-203 cells. After a 2-h shift to 35°C, although the exo70-38 cells accumulated only a moderate amount of Bgl2, the exo70-38 sec3-201 and exo70-38 sec3-203 cells showed a much more pronounced amount of Bgl2 accumulation. As a control, the sec3ΔN cells did not accumulate Bgl2 at either 25 or 35°C. These results indicate that combining sec3-201 or sec3-203 with exo70-38 greatly aggravates the Bgl2 secretion defects. Overall, these results suggest that disruption of the Cdc42 or lipid interaction of Sec3 in the exo70-38 background blocks secretion.
Figure 8. The exo70-38 sec3 double mutants display severe secretion defect. (A) The exo70-38 sec3 double mutants are defective in invertase secretion. The exo70-38 sec3-201 and exo70-38 sec3-203 mutants were tested for the secretion of the invertase after being (more ...)
We also performed thin-section electron microscopy on these mutants. As shown in , the exo70-38 cells began to accumulate vesicles (100 ± 38 vesicles/section) at 35°C, whereas the exo70-38 sec3-201 and exo70-38 sec3-203 double mutants accumulated much larger amounts of vesicles (366 ± 84 and 330 ± 69 vesicles/section, respectively). The double mutants accumulated vesicles even at 25°C. The sec3ΔN cells did not accumulate vesicles at any temperature tested. These results clearly show that the secretion defects are aggravated when the Cdc42–Sec3 or lipid–Sec3 interaction is disrupted in exo70-38 cells.
Exocyst localization and morphologies of the exo70-38 sec3 double mutants
To examine the localization of exocyst components in the mutant cells, we GFP tagged the exocyst components Sec3, 5, and 8 and Exo84 by chromosomal integration in the single or double mutants of exo70
. As shown in , at 35°C, in the sec3
single mutants, the exocyst components were polarized to the bud tip. However, in the exo70-38 sec3-201
and exo70-38 sec3-203
double mutants, the exocyst proteins were either completely depolarized or diffused inside or in the vicinity of the daughter cells. The assembly of the exocyst complex was mostly unaffected in the exo70-38 sec3ΔN
double mutants (Fig. S3, available at http://www.jcb.org/cgi/content/full/jcb.200704128/DC1
). In addition, Sec4 and actin are mostly polarized in these mutants, whereas Sec4 is less concentrated in the bud tip (Fig. S4).
Figure 9. Localization of the exocyst components in the exo70-38 sec3 double mutants. The exocyst components in exo70-38, sec3-201, sec3-203, and exo70-38 sec3 double mutant cells were GFP tagged by chromosomal integration. The cells were shifted from 25 to 35°C (more ...)
Because the correct targeting of the exocytic vesicles to the bud tip is required for polarized growth of the yeast cells, disrupting the interaction of Sec3 with Cdc42 or phospholipids in the exo70 mutant background would affect cell morphogenesis. As shown in , at 25°C, in contrast to the ellipsoidal shapes of the single mutants, the exo70-38 sec3 double mutants are significantly rounder. The double mutants have smaller axial ratios (length/width) compared with the single mutants (). The rounder morphology in the mutant cells suggest that although some vesicles are delivered to the daughter cells, their tethering is not restricted to the bud tip. Instead, vesicles are diffusely tethered to the daughter cell plasma membrane resulting in isotropic daughter cell expansion. When these round daughter cells reach the mother stage, the mother cells also appear round in shape. In addition to the rounder shape, the double mutant cells are also clearly larger in size (approximately twice as large as either the exo70-38 or the sec3 single mutants). This morphology suggests that some of the secretion occurs even in mother cells, leading to isotropic mother cell surface expansion. The cell biological characterization of the various sec3 mutant alleles generated in this study is summarized in Table I.
Figure 10. Morphological defects of the exo70-38 sec3 double mutants. (A) Morphology of sec3ΔN, exo70-38, exo70-38 sec3-201, and exo70-38 sec3-203. The exo70 sec3 double mutants were significantly larger and rounder than each single mutant, even at 25°C. (more ...)
Summary of the phenotypic characterization of the sec3 mutants