Ssd1 localizes to bud cortex and bud necks
Current data suggest that Cbk1 kinase and Ssd1 are functionally linked to cell growth and maintenance of cell wall integrity (Jorgensen et al., 2002
; Kurischko et al., 2005
; Jansen et al., 2009
); however, the molecular function of Ssd1 is poorly understood. The majority of Cbk1 kinase localizes to the bud cortex during bud growth and to the bud neck during cytokinesis, consistent with a role in polarized growth (Racki et al., 2000
; Colman-Lerner et al., 2001
; Weiss et al., 2002
; Nelson et al., 2003
). In contrast, Ssd1 was reported to localize uniformly throughout the cytoplasm (Uesono et al., 1997
; Jansen et al., 2009
Given the cooperative roles of Cbk1 and Ssd1 in polarized growth and cell wall biogenesis, we postulated that some Ssd1 must localize similarly to Cbk1. Thus, we reinvestigated the subcellular distribution of Ssd1 using cells expressing integrated Ssd1-GFP under the control of its physiological promoter. We observed a complex pattern of Ssd1-GFP localization in logarithmically growing cells, indicating that Ssd1 is spatially regulated. Most Ssd1 localized diffusely to the cytoplasm, as previously reported (Uesono et al., 1997
; Jansen et al., 2009
). In addition, Ssd1 concentrated to the bud cortex in some (13–40%, depending on the culture) small budded cells (). Ssd1 also localized to the bud neck in 4–9% of large budded cells (). These data indicate that a fraction of Ssd1 localizes similarly to Cbk1, which is consistent with a Cbk1-dependent function for Ssd1 during polarized growth.
Figure 1. Ssd1 localizes to sites of polarized growth. Logarithmically growing cells expressing Ssd1-GFP and Pap1-RFP (a nuclear marker) (FLY3433) were observed by fluorescence microscopy. Ssd1-GFP was enriched in small buds (13–40% of cells, n = 250 cells (more ...)
Cbk1 influences Ssd1 localization
To determine if Cbk1 kinase influences Ssd1 localization we monitored Ssd1-GFP in cells expressing an analogue-sensitive cbk1-as
allele, which encodes a derivative of Cbk1 that is specifically inhibited by the drug 1NA-PP1 (Weiss et al., 2002
). When cbk1-as
cells were treated with 1NA-PP1 for 45 min, some Ssd1-GFP redistributed from the predominantly diffuse cytoplasmic localization to randomly distributed cytoplasmic puncta (). We rarely (<1% cells) observed Ssd1-GFP at the bud cortex or bud neck after Cbk1 inhibition, suggesting that the establishment or maintenance of Ssd1 at sites of polarized growth is Cbk1 dependent. Ssd1 localization was not affected in mock-treated cbk1-as
cells or in 1NA-PP1–treated wild type (unpublished data). These data suggest that Ssd1 localization is modulated by Cbk1-dependent phosphorylation and support the hypothesis that Cbk1 kinase promotes the cortical localization of Ssd1 and/or represses the association of Ssd1 with cytoplasmic puncta.
Figure 2. Ssd1 localizes to P-bodies. (A) Ssd1-GFP localizes to cytoplasmic puncta upon Cbk1 kinase inhibition. cbk1-as cells expressing Ssd1-GFP (FLY3249) were treated with 1NA-PP1 for 45 min and analyzed by fluorescence microscopy. Approximately 70% of 1NA-PP1–treated (more ...)
Modest Ssd1 overexpression promotes an association with P-bodies in mother cells
We also monitored Ssd1 localization after its modest overexpression. We introduced low copy plasmids of SSD1-GFP
under the control of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GPD) promoter and monitored Ssd1 localization in asynchronous ssd1Δ
cells. Modest Ssd1-GFP overexpression did not cause any obvious growth or cell integrity defects. However, up to 60% of the cells containing GPD-driven Ssd1-GFP plasmids contained prominent cytoplasmic foci that were usually excluded from buds (). Considering that most Cbk1 kinase localizes to buds during polarized growth (Racki et al., 2000
; Colman-Lerner et al., 2001
; Weiss et al., 2002
; Nelson et al., 2003
), these data are consistent with the model that moderate Ssd1 overexpression overwhelms regulatory Cbk1 phosphorylations in mother cells where Cbk1 concentrations are low, but not in buds where Cbk1 concentrations are higher.
Ssd1 associates with P-bodies and stress granules
The Ssd1 cytoplasmic puncta caused by modest Ssd1 overexpression or by Cbk1 inhibition were reminiscent of cytoplasmic mRNA P-bodies and stress granules, which sequester untranslated mRNAs and contribute to translational repression during cellular stress (Parker and Sheth, 2007
; Anderson and Kedersha, 2008
; Buchan et al., 2008
). In support, Ssd1 was reported to partly colocalize with a P-body protein during stationary phase (Jansen et al., 2009
). To determine if Ssd1 associates with P-bodies or stress granules during logarithmic growth, we monitored Ssd1 localization in cells expressing GPD-driven Ssd1-GFP and RFP-tagged P-body and stress granule proteins. We observed that ~60% of the Ssd1-GFP cytoplasmic puncta colocalized with the core P-body protein Edc3 in logarithmically growing cells (). These data support the model that Ssd1 at least transiently associates with mRNA-sequestering protein complexes that mediate translational repression.
Cellular stresses, such as glucose depletion, hypertonic stress, and heat shock trigger P-body aggregation and stress granule formation (Parker and Sheth, 2007
; Anderson and Kedersha, 2008
; Buchan et al., 2008
). To test if cellular stress influences Ssd1 localization, we monitored Ssd1-GFP localization after glucose depletion. Ssd1 localized prominently to cytoplasmic puncta within 10 min of glucose depletion (; Fig. S1
). Most of the Ssd1 cytoplasmic puncta (~73–90%) colocalized with the P-body proteins Edc3, Lsm1, and Dcp2 (; Fig. S1). Ssd1 also colocalized with the stress granule proteins Pab1 and Pub1 upon glucose depletion. We observed similar results in salt-stressed cells (unpublished data). Collectively, these data suggest that Cbk1 kinase inhibition and cellular stress promote Ssd1 recruitment to both P-bodies and stress granules, which in turn may lead to the translational repression of Ssd1-associated mRNAs.
Figure 3. Ssd1 localizes to P-bodies and stress granules in response to cellular stress. ssd1Δ cells (FLY2184) containing Ssd1-GFP and RFP-tagged P-body proteins Edc3, Lsm1, Dcp2, or stress granule proteins Pab1 and Pub1 were transferred to glucose-depleted (more ...)
To biochemically corroborate data from the colocalization experiments, we conducted coprecipitation experiments with cells expressing Ssd1-GFP and representative TAP-tagged P-body and stress granule proteins. We discovered that Ssd1 specifically coprecipitated with the P-body or P-body–associated proteins Dcp2, Not1, and Xrn1 (). Ssd1 also coprecipitated with Pat1, a topoisomerase II–associated deadenylation-dependent mRNA-decapping factor necessary for both P-body and stress granule assembly (Buchan et al., 2008
). These data confirm our colocalization experiments and suggest that a key function for Ssd1 involves P-body and stress granule interactions.
Figure 4. Ssd1 coprecipitates with P-body and stress granule proteins. Ssd1-GFP was immunoprecipitated from extracts of logarithmically growing yeast expressing TAP-tagged P-body or stress granule proteins and Ssd1-GFP. Immunoblots were probed with anti-GFP or (more ...)
mRNA binding is not necessary for Ssd1–P-body association
Because P-bodies and stress granules are comprised of mRNA–protein complexes, Ssd1 recruitment or binding to P-bodies might occur via mRNA interactions. To determine if Ssd1 mRNA binding is a prerequisite for associating with P-bodies, we analyzed the localization of truncated derivatives of Ssd1, such as Ssd11-450
, which lack the mRNA-binding domain, as defined by Uesono et al. (1994)
. Upon stress induction (hypertonic stress, heat shock, and glucose depletion), Ssd11-450
prominently localized to P-bodies within several minutes of stress induction (unpublished data). These data indicate that the N-terminal 450–amino acid region of Ssd1 is sufficient for P-body recruitment and that establishment and maintenance of Ssd1–P-body interactions are independent of mRNA binding.
Phosphorylation-deficient Ssd1 is toxic and causes cellular lysis
Ssd1 contains multiple Cbk1/LATS kinase consensus sequences for phosphorylation in its N-terminal region, as defined by Hao et al. (2008)
and Mazanka et al. (2008)
. Significantly, in vivo expression of an Ssd1 mutant protein lacking multiple Cbk1 phosphorylation sites was reported to be toxic to yeast (Jansen et al., 2009
); however, the nature of its toxicity and the effect of Cbk1-dependent phosphorylations on Ssd1 localization were not established. To explore the physiological significance of Cbk1-mediated phosphorylations on Ssd1 function and localization, we analyzed the phenotypes of Ssd1 phosphorylation site mutants. We converted nine putative Cbk1 phosphorylation sites to nonphosphorylatable alanines to yield Ssd1-9A. We also converted putative Cbk1 phosphorylation sites to phosphomimetic aspartate residues to yield phosphomimetic Ssd1-9D. We introduced low copy plasmids encoding wild-type or mutant Ssd1 into wild-type, ssd1Δ,
and cbk1Δ ssd1Δ
yeast strains and analyzed cellular phenotypes.
We discovered that Ssd1-9A expression was dominantly toxic to wild-type and ssd1Δ cells at 22°C and lethal at 37°C (). The Ssd1-9A transformants of wild-type and ssd1Δ cells were very fragile and propagated poorly. Microscopic analysis revealed that Ssd1-9A expression caused severe cell morphology and lysis defects that were remarkably similar to the phenotypes of conditional cbk1 mutants (). Ssd1-9A expression was also lethal in cbk1Δ ssd1Δ double mutant cells, as is wild-type Ssd1 (unpublished data). These data support the model that Cbk1 negatively regulates Ssd1 and indicate that hypo-phosphorylated Ssd1 causes dominant and severe growth and cell integrity defects.
Figure 5. In vivo expression of Ssd1-9A and Ssd1-9D. (A) Transformants of ssd1Δ (FLY2184) cells carrying low copy plasmids encoding wild-type Ssd1, Ssd1-9A, or Ssd1-9D (FLE1079, FLE1080, FLE1081) under the control of the physiological SSD1 promoter. (B) (more ...)
Figure 6. Ssd1-9A causes lysis phenotypes that are suppressed by cbk1 dosage suppressors. (A) Top panels: wild-type cells and cbk1-8 mutants at 34°C. The lysis phenotypes of cbk1-8 cells resemble those of Ssd1-9A–expressing cells at 22°C. (more ...)
To test if the toxicity of Ssd1-9A expression is dependent on mRNA binding, we constructed a plasmid expressing Ssd1-9A protein that lacks the mRNA-binding domain (RBD 686–788), as defined by Uesono et al. (1997)
. Ssd1-9A-RBDΔ expression was not toxic to ssd1Δ
or wild-type cells and did not cause any obvious growth defects (
). Furthermore, Ssd1-RBDΔ is not toxic to cbk1Δ
cells, in contrast to wild-type Ssd1, indicating that the RNA-binding domain is essential for Ssd1 function (unpublished data). Ssd1-9A-RBDΔ expression also disrupted polarized Srl1 mRNA localization, similarly to ssd1Δ
(see Fig. S4 B
). Thus, the Ssd1 mRNA-binding domain is essential for Ssd1 function, in agreement with Uesono et al. (1997)
Ssd1-9A toxicity is diminished by cbk1 dosage suppressors
If Cbk1 phosphorylation prevents the toxicity of Ssd1, then the lysis phenotypes of cbk1
loss-of-function mutants and cells expressing Ssd1-9A must occur via similar mechanisms. If this hypothesis is correct, then some cbk1
dosage suppressors may abrogate or diminish the toxicity of Ssd1-9A expression. We previously established that high copy plasmids of the cell wall biogenesis proteins, Ccw12, Sim1, Srl1, and Uth1 suppress the lethality of cbk1
loss-of-function mutants (Kurischko et al., 2005
and Fig. S2
). Thus, we introduced Ssd1-9A plasmids into cells containing cbk1
dosage suppressor plasmids (pCCW12
and assayed cell morphology and cell integrity by microscopy. We discovered that the cellular lysis and aberrant morphology phenotypes of Ssd1-9A cells were partially rescued by these cbk1
dosage suppressors at 22°C, 34°C (), and 37°C (not depicted). These data support the hypothesis that the toxicity of Ssd1-9A expression and the cell integrity defects of cbk1
loss-of-function mutants occur via the same or similar mechanisms.
Ssd1-9A constitutively localizes to P-bodies
Because Ssd1 localizes to P-bodies in response to Cbk1 inhibition, we hypothesized that hypo-phosphorylated Ssd1-9A constitutively localizes to P-bodies and stress granules. Thus, we analyzed the localization of GFP-tagged Ssd1-9A in ssd1Δ cells. Because constitutive Ssd1-9A expression (via GPD promoter) was toxic for cell growth, we conducted experiments with cells containing galactose-inducible Ssd1-9A plasmids. When cells were grown in repressing conditions (glucose-containing medium), Ssd1-9A–GFP was not detectable by fluorescence microscopy and did not cause any obvious phenotype (unpublished data). Upon 2 h galactose induction, Ssd1-9A localized prominently to cytoplasmic puncta in nearly all cells (). Most of the Ssd1-9A cytoplasmic puncta colocalized with P-body markers. We never observed phospho-deficient Ssd1-9A at bud tips or bud necks. These results suggest that the lethality of ssd1-9A and conditional cbk1 mutants correlates with enhanced or constitutive Ssd1 localization to P-bodies and stress granules. Furthermore, these data support the model that hypo-phosphorylated Ssd1 is toxic to cells via P-body–dependent translational repression of Ssd1-associated mRNAs. In agreement, Ssd1 is no longer toxic when it cannot bind mRNAs ( and Fig. S4 B).
Figure 7. pGAL-Ssd1-9A-GFP localizes to P-bodies in otherwise unstressed cells and Ssd1-9D localizes to the cytoplasm, bud cortex, and bud neck. (A) ssd1Δ cells were transformed with plasmids encoding galactose-inducible Ssd1-9A-GFP and Dcp2-RFP or Lsm1-RFP (more ...)
Cbk1 promotes the cortical functions of Ssd1
To determine the role of Cbk1 phosphorylation on Ssd1 function, we analyzed the phenotypes of cells expressing phosphomimetic Ssd1-9D. In contrast to phospho-deficient Ssd1-9A, phosphomimetic Ssd1-9D was not toxic to wild-type, ssd1Δ, or cbk1Δ ssd1Δ cells when expressed on low copy plasmids under the control of its endogenous promoter or the GPD promoter ( and unpublished data). Moreover, the morphology and viability of cells expressing Ssd1-9D plasmids were indistinguishable from corresponding control cells containing empty vectors. These data support the model that Cbk1 negatively regulates Ssd1 by phosphorylation, and thereby prevents the dominant toxic/lethal effects of hypo-phosphorylated Ssd1.
To determine if Cbk1 phosphorylation directly influences Ssd1 localization, we analyzed Ssd1-9D-GFP localization in asynchronous cells. Like wild-type Ssd1, most Ssd1-9D localized diffusely to the cytoplasm. Ssd1-9D also localized prominently to the bud cortexes and bud necks of some small and large budded cells, similar to wild-type Ssd1 (). Significantly, and in contrast to wild-type Ssd1 or Ssd1-9A, Ssd1-9D rarely localized to bright cytoplasmic foci (P-bodies) when expressed via the constitutive GPD promoter. These data support the model that Cbk1-dependent phosphorylations prevent Ssd1 from associating with P-body aggregates, but allow Ssd1 to localize to sites of polarized growth.
Phosphorylated Ssd1 regulates the polarized distribution of associated mRNAs
Ssd1 binds a subset of mRNAs, many of which encode cell wall biogenesis proteins (Hogan et al., 2008
; Jansen et al., 2009
). Intriguingly, several Ssd1-associated mRNAs are encoded by cbk1
dosage suppressors, such as SRL1
, and UTH1
(Kurischko et al., 2005
; see Fig. S2). Moreover, two of these mRNAs (SRL1
) are known to polarize to the bud cortex in an actin- and myosin V–dependent fashion (Shepard et al., 2003
). In light of the polarized localizations of wild-type Ssd1 and phosphomimetic Ssd1-9D in some budded cells, it seemed plausible that Ssd1 influences asymmetric mRNA localization during polarized growth.
To determine whether Ssd1 influences mRNA localization, we monitored SRL1
mRNA localization in vivo using a GFP-tagging strategy described previously (Haim et al., 2007
; Haim-Vilmovsky and Gerst, 2009
). We compared the pattern of SRL1
mRNA localization in asynchronously growing ssd1Δ
cells containing Ssd1 plasmids or empty vector. In ~50% of the cells expressing wild-type SSD1
mRNA polarized to 1–3 prominent spots in the bud (; Shepard et al., 2003
). We also observed that SRL1
mRNA localized to multiple faint spots randomly distributed throughout the cytoplasm in ~40–50% of the cells. The overall distribution of SRL1
mRNA localization was similar in small, medium, and large budded cells, indicating that the different patterns of SRL1
mRNA localization were not obviously enhanced during cell cycle progression (unpublished data). We obtained similar results with wild-type cells (unpublished data).
Figure 8. Asymmetric SRL1 mRNA localization is significantly diminished in ssd1Δ cells and rescued by phosphomimetic Ssd1-9D.
SRL1 mRNA was GFP tagged and analyzed from budded cells, as described in Materials and methods. SRL1 mRNA localization was analyzed (more ...)
In ssd1Δ cells containing empty vector, SRL1 mRNA polarity was diminished by ~10-fold compared with SSD1 cells (). Moreover, the percentage of ssd1Δ cells with multiple faint SRL1 mRNA puncta or no spots was ~30% and severalfold greater, respectively, than corresponding SSD1 cells (). These data indicate that Ssd1 is critical for establishing or maintaining asymmetric SRL1 mRNA localization during polarized growth.
Ssd1-9D promotes the polarized localization of SRL1 mRNA
If Cbk1-mediated phosphorylations simply inactivate Ssd1, then constitutively phosphorylated Ssd1 should phenocopy ssd1 loss-of-function alleles, such as ssd1Δ. However, in light of the cortical localization of wild-type Ssd1 and Ssd1-9D, we thought it to be likely that Cbk1 stimulates some cortical Ssd1 functions and protein interactions. To test if Ssd1-9D can rescue the SRL1 mRNA localization defects in ssd1Δ cells, we monitored SRL1 mRNA localization in ssd1Δ cells expressing Ssd1-9D plasmids. Significantly, Ssd1-9D expression restored the diminished SRL1 mRNA polarity in ssd1Δ cells to a greater degree (~1.4-fold) than wild-type Ssd1 (). Moreover, Ssd1-9D expression reduced the percentage of cells with dispersed SRL1 mRNA puncta by approximately two- to threefold, in comparison to corresponding ssd1Δ cells and SSD1-expressing cells. These data indicate that Cbk1-phosphorylated Ssd1 promotes and enhances the polarity of SRL1 mRNA in asynchronously growing cells.
Dephosphorylated Ssd1 traps associated mRNAs in P-bodies
Because Ssd1-9A constitutively localizes to P-bodies, we predicted that Ssd1-9A expression also brings its associated mRNAs to P-bodies. Thus, we monitored SRL1 mRNA localization in cells expressing galactose-inducible RFP-tagged Ssd1-9A. Significantly, within 2 h of Ssd1-9A induction, SRL1 mRNA dispersed to multiple cytoplasmic puncta, all of which colocalized with Ssd1-9A (). We observed the same punctate SRL1 mRNA localization in cells expressing HA-tagged Ssd1-9A (Fig. S4). In contrast, SRL1 mRNA did not disperse or redistribute to P-bodies when wild-type Ssd1 or Ssd1-9D expression was induced for the same duration (unpublished data). Corresponding immunoblots confirmed that Ssd1-9A and wild-type Ssd1 were expressed to similar levels in these experiments (unpublished data). These results support the model that in the absence of Cbk1 phosphorylations, hypo-phosphorylated Ssd1 sequesters its associated mRNAs in P-bodies and stress granules, where the mRNAs are translationally repressed. These data further suggest that enhanced Ssd1–mRNA interactions with P-bodies and stress granules are the primary cause of Ssd1 toxicity in cbk1 mutants.
Figure 9. SRL1 mRNA colocalizes with Ssd1-9A. Cells expressing GAL-inducible Ssd1-9A-RFP and SRL1 mRNA-GFP (FLY3196 with plasmid FLE1244) were transferred to galactose medium for 1–2 h. SRL1 mRNA and Ssd1-9A localization were subsequently observed by spinning (more ...)