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Keywords:

  • actin cable;
  • cell morphogenesis;
  • cell polarity;
  • exocyst;
  • exocytosis;
  • fission yeast;
  • formin;
  • small GTPase

Abstract

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Proper cell morphogenesis requires the co-ordination of cell polarity, cytoskeletal organization and vesicle trafficking. The Schizosaccharomyces pombe mutant pob1-664 has a curious lemon-like shape, the basis of which is not understood. Here, we found abundant vesicle accumulation in these cells, suggesting that Pob1 plays a role in vesicle trafficking. We identified Rho3 as a multicopy suppressor of this phenotype. Because Rho3 function is related to For3, an actin-polymerizing protein, and Sec8, a component of the exocyst complex, we analyzed their functional relationship with Pob1. Pob1 was essential for the formation of actin cables (by interacting with For3) and for the polarized localization of Sec8. Although neither For3 nor Sec8 is essential for polarized growth, their simultaneous disruption prevented tip growth and yielded a lemon-like cell morphology similar to pob1-664. Thus, Pob1 may ensure cylindrical cell shape of S. pombe by coupling actin-mediated vesicle transport and exocyst-mediated vesicle tethering during secretory vesicle targeting.

Cell morphology and polarity have profound effects on cellular function. In mammals, neurite cells form a bipolar elongated shape containing two distinct ends, and epithelial cells form a sheet with surfaces that have different characteristics. Leukocytes move toward their target via the formation of a leading edge. In higher plants, polarized cell expansion is essential for morphogenesis. Several steps of cell morphogenesis must be co-ordinated and controlled, including the distribution of landmarks for cell polarity, cytoskeletal organization and the trafficking of membranes and cortical materials to appropriate sites (1).

The fission yeast Schizosaccharomyces pombe is an attractive organism for studying how cell morphogenesis is controlled at the molecular level. Various mutations that deform the normal cylindrical shape of this organism have been isolated and studied in detail (2,3). The primary system that establishes cell polarity during cell morphogenesis is a signaling pathway mediated by the small GTPases Ras1 and Cdc42 (4–7). Deregulation of the molecules involved in this pathway causes loss of cell polarity and produces round cells. Meanwhile, Tea1 and its associated proteins reach the cell tip via the elongation of the plus ends of microtubules and become anchored there as a landmark for polarized growth (8–11). Perturbation of the functions of these proteins disrupts the maintenance of cell polarity and induces the formation of extra cell tips, resulting in ‘T’-shaped cells or the distortion of the cell body into a ‘banana’ shape (2). We previously identified Pob1 (S.pombeboi-like) protein that is essential for cell elongation and cell separation after cytokinesis (12). Loss of Pob1 function causes a characteristic lemon-like cell shape, suggesting that the polarized cell axis remains although polarized cell growth is impaired (Figure S1). Despite the analysis of this mutant phenotype, the mechanisms underlying the formation of the lemon-like cell shape remain unclear.

Here, we studied the phenotype of a temperature-sensitive mutant, pob1-664, by transmission electron microscopy (TEM) using the high-pressure freezing and freeze-substitution method (13–15). We found that Pob1 is required for polarized vesicle trafficking during cell elongation. We also showed that Pob1 couples two distinct molecular pathways in vesicular trafficking, one involving actin cable-mediated vesicle transport and the other involving vesicle tethering to the plasma membrane, under the control of the Rho GTPase-mediated signaling pathway.

Results

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Mutation in the PH domain of Pob1 affects its localization and stability

Pob1 localizes to the growing tips of interphase cells and along the division plane during cytokinesis (12). Consistent with this localization, pob1-664 mutant cells show defects in cell elongation and cell separation at the restrictive temperature of 37°C. To probe the molecular mechanism underlying the phenotypes of pob1-664 mutant cells at the restrictive temperature, the mutation site in pob1-664 was identified. The pob1 locus in JX584 (pob1-664 cells) was amplified by polymerase chain reaction (PCR). Subsequent sequence analysis revealed a single-point mutation, substituting Tyr719 to Cys, near the N-terminus of the pleckstrin homology (PH) domain. The corresponding region is well conserved among PH domains and has been reported to bind phosphoinositides (16). Hereafter, we call the mutant protein Pob1Y719C.

If Pob1 uses its PH domain to localize at the growing site of the cell membrane, then Pob1Y719C may not localize properly, resulting in its apparent loss of function. To compare the intracellular localization of Pob1Y719C with that of wild-type Pob1, we prepared strains in which a hemagglutinin (HA) tag was introduced into the chromosomal locus before the stop codon of the open reading frame of pob1+ in the wild-type strain and that of pob1-664 in the mutant strain. In the control cells (JW100), wild-type Pob1-HA localized to the cell tip during interphase and along the dividing region during cytokinesis at both 25°C and 37°C, consistent with findings using green fluorescent protein (GFP)-fused Pob1 (12) (Figure 1A). By contrast, in the mutant cells (JW631), Pob1Y719C-HA was dispersed throughout the cytoplasm within 2 h of the cells being shifted to 37°C (Figure 1A). Pob1Y719C-HA and Pob1 were expressed equivalently in the two cell lines based on western blot analysis of cell homogenates prepared at the 2-h time-point (Figure 1B). Prolonged incubation for 4 h at 37°C, however, reduced the amount of Pob1Y719C-HA, whereas the level of the wild-type protein was not affected (Figure 1B), suggesting that Pob1Y719C-HA undergoes proteolysis after detachment from the cell cortex. Consistent with the idea, Pob1Y719C-mCherry was accumulated to the cytoplasmic compartments, possibly vacuole-related structure at 36°C (Figure S2A). Thus, the PH domain of Pob1 is involved in its proper subcellular localization and may contribute to the stability of the protein. Cortical localization of Pob1-HA was significantly reduced in its3-1, a phosphatidylinositol-4-phosphate 5-kinase mutant (17), at 36°C (Figure 1C). In the its3-1 mutant, the cellular amount of phosphatidylinositol 4, 5-bisphosphate (PIP2) reduces significantly (17). The interaction between PIP2 and Pob1 via its PH domain could be crucial for the cortical localization of Pob1.

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Figure 1. Cortical localization of Pob1 is impaired in the pob1-664 mutant. A) Intracellular localization of Pob1. Wild-type (WT) and pob1-664 cells expressing the triple HA-tagged protein and growing exponentially in YEA at 25°C were shifted to 37°C for the indicated time. The cells were fixed and stained with anti-HA (12CA5) to detect Pob1 (top) and with Hoechst 33342 to detect DNA (bottom). B) Protein level in cells cultured in (A). Antibodies 12CA5 and TAT-1 were used to detect Pob1 and tubulin (loading control), respectively. C) Localization of Pob1 in its3-1, a mutant defective of phosphatidylinositol-4-phosphate 5-kinase activity. its3-1 cells expressing Pob1-HA growing exponentially in YEA at 25°C were shifted to 36°C for 2 h. The cells were fixed and stained with 12CA5 to detect Pob1 (left), with Bodipy-phallacidin to detect F-actin (middle) and with DAPI to detect DNA (right). Bars: 10 µm.

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We noticed that Pob1Y719C-HA migrated faster than the wild-type protein during SDS–PAGE (Figure 1B). Saccharomyces cerevisiae Boi1p, a functional homolog of Pob1, is phosphorylated at multiple sites, and these modifications are closely related to its subcellular localization (18). The substitution Y719C in the mutant protein may thus affect its post-translational modification, possibly including phosphorylation, resulting in improper subcellular localization.

Accumulation of secretory vesicles in pob1-664

To investigate why pob1-664 has a characteristic lemon-like structure at the restrictive temperature, we observed the ultrastructure of JX584 cells by TEM using the high-pressure freezing and freeze-substitution method (13,15). Compared with chemical fixation, this method uses conditions that are more conducive to preserving cellular ultrastructure, especially that of the cytoskeleton. Electron micrographs revealed a markedly increased number of small vesicles in JX584 cells compared with wild-type cells (JY333) at 4 h after the shift to 37°C (Figure 2A). The accumulated vesicles in JX584 could be categorized as transparent (Figure 2A, arrows) or electron dense (Figure 2A, arrowheads). Both vesicle types may have been associated with cellular structures other than ribosomes because ribosomes were partly excluded from their surfaces (Figure 2B, a and b). Notably, we often observed several transparent vesicles near the Golgi and having almost the same electron density as the Golgi (Figure 2A, b). Therefore, those transparent vesicles were probably derived from the Golgi. To assess the population of vesicles according to their diameter, we counted the number of vesicles in 50 sections (summarized in Figure 2C). The diameter of the transparent vesicles was distributed in the range of 50–90 nm, which agrees well with a previous report of presumed secretory vesicles with a diameter of 50–80 nm derived from the Golgi apparatus and moving to the growing zones of wild-type S. pombe cells, which were found by three-dimensional reconstitution of serial sections visualized by TEM using the freeze-substitution method (19). The diameters of the electron-dense vesicles in both wild-type and pob1-664 cells showed two peaks at 20–30 and 50–60 nm (Figure 2C). Thus, three types of vesicles, namely transparent vesicles of diameter 50–90 nm and electron-dense vesicles of diameter 20–30 or 50–60 nm, were observed in both the wild-type and pob1-664 cells, suggesting that the vesicles that had accumulated in JX584 were a result of a defect in vesicle trafficking rather than an abnormality in membrane metabolism. To our knowledge, this is the first description defining that three types of vesicles with different sizes and densities were present in S. pombe.

image

Figure 2. Vesicles accumulate in the pob1-664 mutant cells. A) Electron micrographs of a wild-type (WT) cell (a) and a pob1-664 cell (b). Cells were grown in YEA at 25°C to mid-log phase and then shifted to the restrictive temperature, 37°C, for 4 h. Transparent vesicles (arrows) and electron-dense vesicles (arrowheads) were observed. AF, actin filament; G, Golgi; M, mitochondrion; N, nucleus; V, vacuole; Ves, vesicle. B) High-magnification images of pob1-664 cells incubated as in (A). A transparent vesicle (a, arrow) and an electron-dense vesicle (b, arrowhead) are shown. (c) Vesicles stained with PATAg which selectively visualizes polysaccharides as electron-dense deposits. (d) Vesicles associated with a bundle of microfilaments. C) Size distribution of small vesicles in WT (JY333) and pob1-664 (JX584) cells. The vesicles were counted and measured in 50 cell sections of electron micrographs. The white bars indicate transparent vesicles and solid bars indicate electron-dense vesicles.

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Cell growth in S. pombe is dependent on cell wall biosynthesis, and electron microscopy (EM) of S. pombe cell wall shows a three-layered structure with two electron-dense layers separated by a non-dense layer mainly consisted of the polysaccharides (1,3)β-d-glucan and (1,3)α-d-glucan (13,20). Secretion of highly glycosylated proteins is particularly relevant to the synthesis of the cell wall structure (21). They connect the cell wall components including glucans. To determine if the accumulated vesicles in JX584 contained highly glycosylated proteins, we examined the contents of the vesicles using the periodic acid-thiocarbohydrazide-silver (PATAg) staining method to selectively visualize polysaccharides as electron-dense deposits (22). PATAg stained all the accumulated vesicles, particularly the large vesicles (50–90 nm in diameter) that were probably transparent vesicles (Figure 2B, c). Therefore, the large vesicles seemed to contain more polysaccharides than the small/electron-dense vesicles. These results suggested that the accumulated vesicles, especially large transparent vesicles, might be involved in the cell wall biosynthesis.

We often observed that both the transparent vesicles and electron-dense vesicles localized along a bundled filamentous structure in the cytoplasm of JX584 cells incubated at 37°C (Figure 2B, d). The bundle contained ∼8-nm-diameter filaments, suggesting that it was composed of F-actin. No aligned F-actin filaments or accumulated vesicles along F-actin bundles were observed in wild-type cells (N. K., A. Y. and M. O., unpublished data). Because the formation of longitudinal actin cables that is observed in wild-type cells is impaired in pob1-664 mutant cells (12; see also Figure 6A), we considered that the vesicle-associated filamentous structures in JX584 cells were not intact actin cables. These results suggest that Pob1 may play an important role in vesicle transport by organizing the actin cytoskeleton.

image

Figure 6. S. pombe exocyst localized to cell tips without F-actin. A) Localization of the exocyst complex is maintained after Lat-A treatment. Wild-type cells expressing indicated GFP-fused proteins were treated with 10 µm Lat-A in YEA at 25°C. Bar: 10 µm. B) Actin-mediated vesicle transport is probably not essential for the recruitment of the exocyst complex to cell tips. Δfor3 null cells expressing GFP-fused subunits of the exocyst complex growing exponentially in YEA at 25°C were observed. Arrowheads indicate the localization of cell tips. Bar: 10 µm. C) Cells expressing Pob1-HA and Sec8-GFP were grown in YEA at 25°C to mid-log phase and then processed for immunostaining to detect Pob1-HA. DNA stained with DAPI is shown in blue in the merged image. Arrowheads indicate overlapping signals of both proteins. Bar: 5 µm.

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To know in which step of vesicle transport that Pob1 is involved, we further investigated the distribution of organelle and related structures engaging vesicle traffic by using GFP marker proteins (23–26) (Figure 3A). Among them, the distribution of Syb1, v-SNARE that is associated with secretory vesicles and is shuttling between the Golgi and plasma membrane, and Myo4/Myo52, type V myosin that engages in the transport of secretory vesicles to the plasma membrane, was perturbed in pob1-664 cells at 36°C (Figure 3A). Moreover, in pob1-664 cells, extracellular secretion of signal peptide-conjugated GFP was reduced and the protein was abnormally accumulated (Figure 3B). Acid phosphatase-secretion activity was significantly reduced in pob1-664 cells (Figure 3C). Taken together with EM data, Pob1 is likely to be involved in post-Golgi secretory pathway.

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Figure 3. Late secretory pathway is deteriorated in pob1-664. A) Live images of the cells growing at 25°C in mid-log phase (25°C), and shifted to 36°C for 4 h (36°C). Indicated GFP-fused proteins were expressed. Arrows indicate mislocalization of GFP-Syb1 and Myo4-YFP in the pob1-664 mutant cells and Δfor3 null cells. Note that the distribution of Sec13 and Sec24 [components of coat protein (COP)II-coated vesicle budding from the endoplasmic reticulum (ER) and transported to the Golgi apparatus], Erp2 (a presumed component of COPI-coated vesicle traveling from the Golgi to ER), Sec72 [a guanine-nucleotide exchange factor for ADP ribosylation factor (ARF) localized to trans Golgi] and Anp1 (a subunit of mannosyltransferase complex localized to cis Golgi) in pob1-664 cells was almost identical to wild-type cells at 36°C. Bars: 10 µm. B) SPL-GFP is abnormally accumulated in pob1-664 cells. After the removal of thiamine from media, pREP1SPL-GFP containing cells were incubated at 25°C for 16 h. Then the cells were transferred to fresh EMM and further incubated at 25°C or 36°C for 4 h. C) Measurement of acid phosphatase secretion to medium in pob1-664 mutant cells. See details for Materials and Methods.

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Rho3 is involved in the secretory pathway in which Pob1 is engaged

To clarify the molecular mechanism of Pob1-mediated vesicle transport, we screened for genes that were multicopy suppressors of temperature sensitivity in pob1-664. Among the ∼83 000 colonies transformed with an S. pombe genomic library, 7 of 18 suppressor clones contained the rho3+ locus (9 were pob1+ and the other 2 were uncharacterized because of weak suppression). Rho3 belongs to the Rho family of small GTPases that play important roles in eukaryotic cell polarization (27,28). Temperature-sensitive growth defect of pob1-664 cells was suppressed by overexpression of rho3Q71L, a GTP-locked mutant (Figure 4A), as well as wild-type rho3+. In contrast, no suppressing activity was seen by overexpression of rho3C202S, a mutant lacking a post-translational isoprenylation in the C-terminus of Rho3. Thus, Rho3 probably functions with Pob1 when it is active and membrane-associated. Although cells lacking rho3+ grow normally at 25°C, they show impaired growth and a lemon-like cell shape very close to the terminal phenotype of pob1 mutant cells at 37°C (28). In addition, large numbers of presumed secretory vesicles accumulate in Δrho3 null cells at the restrictive temperature (29).

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Figure 4. Rho3 may function with Pob1. A) Overexpression of either Rho3 or Rho3Q71L can compensate for the growth defect of pob1-664 cells. JX584 was transformed with the empty vector (pREP41), pREP41rho3, pREP41rho3Q71L or pREP1rho3C202S. The transformed cells were then streaked onto EMM plates and incubated at the indicated temperature for 5 days. B) Electron micrographs of pob1-664 (JX584) cells overexpressing the empty vector (a) or rho3+ (b). JX584 cells transformed with pREP41 or pREP41rho3 were grown in EMM at 25°C to mid-log phase and then shifted to the restrictive temperature, 37°C, for 4 h. Note that the abnormal accumulation of transparent (arrows) and electron-dense vesicles (arrowheads) was increased by Rho3 overexpression. CW, cell wall. Other abbreviations are defined in Figure 2. C) A rate of acid phosphatase secretion in pob1-664 mutant cells was improved by Rho3 overexpression. D) Overexpression of Rho3 restores the localization of Pob1Y719C to its correct site. JX584 cells transformed with pREP41 or pR41rho3 were grown in EMM at 25°C to mid-log phase and then shifted to the restrictive temperature, 37°C, for 4 h. Magnified images of the cells indicated by arrows in the F-actin images were shown on the right side. Note that actin cables (arrowheads) are seen in the cells overexpressing Rho3. Bar: 10 µm. E) For3 is not required for Rho3-dependent suppression of cell growth of pob1-664 mutant. Indicated transformants were streaked onto EMM plates and incubated at the indicated temperature for 5 days. WT, wild type.

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Given that the temperature-sensitive growth defect of the pob1-664 mutant strain was suppressed upon Rho3 overexpression, we examined whether Rho3 overexpression could also suppress the vesicle accumulation phenotype caused by pob1-664 mutation. At the restrictive temperature for pob1-664 mutant cells, JX584 control cells that were transformed with an empty vector accumulated a large number of presumed secretory vesicles (2.1 vesicles/µm2), indicating that the presence of the empty vector did not affect the vesicle accumulation phenotype of JX584 cells (Figure 4B, a). In contrast, JX584 cells overexpressing Rho3 showed very few secretory vesicles at the restrictive temperature (0.2 vesicles/µm2) (Figure 4B, b), similar to wild-type cells (Figure 2A). Consistent with the EM observations, Rho3 overexpression improved acid phosphatase secretion in pob1-664 cells at the restrictive temperature (Figure 4C). Thus, Rho3 overexpression was able to suppress the vesicle trafficking defect caused by the pob1-664 mutation.

How does Rho3 suppress the defect of vesicle trafficking in the pob1 mutant? In terms of the genetical relationship between Pob1 and Rho3, overexpression of Rho3 was unable to rescue the lethality of Δpob1 null cells, and Pob1 overexpression did not suppress the growth defect of Δrho3 null cells at 37°C (our unpublished data). Thus, Rho3 does not seem to function in a redundant pathway that parallels the Pob1 pathway, or perhaps the functions of these two proteins cannot be explained by a simple epistatic and hypostatic relationship. We found that Rho3 overexpression restored the localization of Pob1Y719C to the growing tips in interphase cells and to the dividing regions in mitotic cells at the restrictive temperature (Figure 4D). In Δrho3 null cells, Pob1 localization to the cell ends and septation site was weakened at 25°C (Figure S2B). After prolonged incubation at 36°C, Pob1 was gradually delocalized then almost disappeared from the cell cortex. Thus, Rho3 may be involved in the subcellular localization of Pob1. Consistent with this idea, Rho3 localizes to the cell cortex, especially to the growing tips and the septation site where Pob1 localizes (28,29). At this moment, only weak interaction between Rho3 and Pob1 was detected by two-hybrid assay (Figure S3A). Rho3 possibly acts downstream of Pob1 and controls cortical localization of Pob1 via the feedback signal from Rho3 signaling pathway. We cannot exclude other possibilities though.

Besides rho3+, S. pombe has five additional Rho GTPases, rho1+, rho2+, rho4+, rho5+ and cdc42+, which are involved in cell morphogenesis and septation (30). We found that, among them, not only rho3+ but also cdc42+ suppressed the temperature sensitivity of JX584 when cdc42+ was expressed under the strongest nmt1 promoter (Figure S3B). Nevertheless, Cdc42 overexpression could not restore, rather deteriorated, acid phosphatase-secretion activity of pob1-664 (Figure 4C). An independent study reported that Pob1 functions downstream of Cdc42 probably through direct interaction (31). pob1+ may interact with cdc42+ in a different way of that rho3+ does.

Loss of functional actin cables in pob1-664

Perturbation of the actin cytoskeleton impairs proper transport of secretory vesicles in S. pombe(26) as well as in other organisms. We previously reported that the pob1-664 mutation causes disorganization of the actin cytoskeleton during interphase at the restrictive temperature (12). When Rho3 was overexpressed in pob1-664 mutant cells, this phenotype was suppressed, and in these cells, actin patches were located in cell tips and actin cables were longitudinally elongated from cell tips (Figure 4D) like those in wild-type cells (32,33). Because Rho3 binds to For3, which is the S. pombe ortholog of formin, an actin-polymerizing protein that controls polarized cell growth (28), we hypothesized that Rho3 overexpression would suppress the pob1-664 mutant phenotype by interacting with and activating For3. To test this hypothesis, we examined whether For3-mediated actin cable formation was required for Rho3-mediated suppression of the pob1-664 mutation. Indeed, Rho3 was still able to suppress the pob1-664 mutation in the absence of for3+ (Figure 4E). In addition, For3 overexpression did not reverse the cell growth phenotype of the pob1-664 mutant. Therefore, For3-mediated actin cable formation was unlikely to be the primary cause of the growth defect of pob1-664 mutant cells at the restrictive temperature. It is possible that Rho3 and Pob1 have another cellular function(s) besides their roles in controlling actin cable formation. Recently, Rincón et al. reported a functional interaction between Pob1 and For3 in the formation of actin cables (31), and we have reached the same conclusion independently (Figure S4). Pob1 may control the actin cytoskeleton, at least in part, independently of the Rho3–For3 pathway.

Pob1 is required for polarized localization of the exocyst complex

The exocyst is a protein complex that tethers secretory vesicles to the target membrane prior to SNARE-dependent membrane fusion (34–36). Eight subunits of the exocyst, Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70 and Exo84, are conserved among budding yeast, higher plants and humans, and their conformational conversion induces docking of secretory vesicles to the plasma membrane. Exocyst activity is controlled by small GTPases. In S. pombe, a vesicle accumulation phenotype similar to that of pob1-664 is observed in sec8-1, a conditional exocyst mutant, at the restrictive temperature (37). The sec8-1 mutant shows an abnormal elongated multicellular morphology, and Rho3 overexpression can compensate for this phenotype (29). Given the similarity in the vesicle accumulation phenotype and Rho3-mediated suppression in sec8-1 or pob1-664 mutant cells, we speculated that these two genes interact functionally and that Rho3 may play a key role in the interaction. Thus, we examined the phenotype of the double mutant by crossing pob1-664 (JX584) and sec8-1 (KNS808) cells. Sporulation was induced in the pob1-664 sec8+/pob1+sec8-1 strain (KNS812), and the progeny asci were examined. The genotype of each progeny was judged by their growth and morphology at a common restrictive temperature for pob1-664 or sec8-1 (37°C). The pob1-664 sec8-1 double mutant cells showed a severe synthetic growth defect (Figure 5A); they ceased to grow after several rounds of cell division and did not form colonies detectable by the naked eye, even at 25°C (a cell growth-permissive temperature for each single mutant). Thus, Pob1 and Sec8 interact genetically to promote cell growth. The synthetic growth defect of the pob1-664 sec8-1 double mutant was suppressed by Rho3 overexpression (Figure 5B), indicating that Rho3 interacts functionally with both Pob1 and Sec8. In addition, a genetic interaction was found between pob1+ and exo70+, a gene encoding non-essential subunit of the exocyst complex in S. pombe (Figure S5A). Similar genetic interaction is found between rho3+ and exo70+(29). Therefore, Pob1 and Rho3 may be involved in controlling the cellular functions of the exocyst complex.

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Figure 5. Pob1 is required for polarized localization of Sec8. A) Tetrad analysis of the pob1+sec8-1/pob1-664 sec8+ diploid cells. The left panel shows five sets of dissected asci incubated at 25°C. The right panel shows the replicated colonies incubated at 37°C. The genotypes of the descendants were judged by their growth and terminal morphology at 37°C. D, pob1-664 sec8-1 (pob1 sec8); P, pob1-664; S, sec8-1; W, wild type. B) Overexpression of Rho3 can complement the synthetic lethality of the pob1 sec8 double mutant. Cells containing pR41rho3 were streaked onto EMM plates with or without 5 µm thiamine and incubated at 37°C for 5 days. C) Mislocalization of Sec8-GFP in the pob1-664 mutant cells. WT cells and pob1-664 cells expressing Sec8-GFP were incubated at 37°C for 4 h and then processed for F-actin staining. Arrowheads indicate the correct localization of Sec8-GFP to the tips of interphase cells and the centers of mitotic cells. D) Exocyst function is not necessary for the cortical localization of Pob1. WT cells and sec8-1 cells expressing Pob1-HA were incubated at 37°C for 4 h and then processed for F-actin staining. Arrowheads indicate the correct localization of Pob1-HA. Bars: 5 µm.

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In wild-type cells, the exocyst complex localizes to regions of the cell surface that are expanding, namely at the growing tips of interphase cells and in the septated region of dividing cells (37). This cortical localization of the exocyst complex was maintained after disrupting F-actin structure (Figure 6A), and so was Pob1 (Figure S2C). Actin cables were dispensable for recruiting Pob1 as well as the exocyst complex to the polarized region throughout the cell cycle (Figures 6B and S2B). We compared the cellular localization of Pob1 with that of Sec8 by observing Pob1-HA Sec8-GFP cells (KNS855). Fluorescence microscopy showed that Pob1 and Sec8 localized within the same regions of cell tips and septation sites (Figure 6C). Moreover, detailed observation revealed that the signals of several Pob1 dots overlapped with those of Sec8. Thus, Pob1 was likely to work together with the exocyst in growth zones.

Next, we examined the localization dependency of Pob1 and Sec8. The polarized localization of Sec8 was impaired in pob1-664 mutant cells shifted to 37°C (Figure 5C). Concomitantly, GFP-Sec6, GFP-Sec8 and GFP-Sec10 were depolarized in pob1-664 mutant cells at a restrictive temperature 36°C (Figure S5B). In contrast, Pob1 remained in the cell tips and division sites under a genetic background of sec8-1, even at 37°C, the restrictive temperature of sec8-1 (Figure 5D). Thus, the cellular localization of Sec8 requires Pob1 activity, and the reciprocal relationship is not feasible. These results suggested that Pob1 plays a crucial role in targeting of secretory vesicles to the appropriate site by recruiting the exocyst complex.

Simultaneous impairment of two distinct systems in vesicle trafficking may occur in pob1-664

Although cells lacking exocyst activity accumulated an abundance of presumed secretory vesicles, they were able to continue polarized growth but without cell separation for several cell cycles (37). Meanwhile, cells lacking Pob1 activity, such as the pob1-664 mutant at the restrictive temperature, accumulated secretory vesicles and ceased polarized elongation and swelled from their middle. What underlies the difference in cell shape between these two vesicle trafficking-defective strains? As mentioned earlier, Pob1 may play an important role in actin cable formation in addition to recruiting the exocyst. Because actin cables are considered to be ‘rails’ for type V myosin that is used in vesicle transport (26,38), we speculated that, in pob1-664 cells, both the vesicle transport system and vesicle tethering system would be impaired, whereas only the latter system would be impaired in sec8-1 cells. To examine this possibility, Myo4/Myo52 or For3 was deleted from the sec8-1 mutant, and the phenotype of the resultant double mutants was assessed. A severe synthetic cell growth defect was observed in both sec8-1Δfor3 and sec8-1Δmyo4 mutants (Figure 7A). Moreover, the double mutant cells frequently showed a lemon-like shape similar to pob1-664 cells (Figure 7A, arrows) that were obviously distinct from the abnormal elongated shape of cells defective in exocyst function (37). Both Δfor3 null cells and Δmyo4 null cells can continue polarized growth although they become somewhat enlarged (26,28,39). Therefore, the two molecular systems in vesicle trafficking, namely vesicle transport and vesicle tethering, may support each other during the polarized growth of S. pombe cells.

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Figure 7. The disruption of vesicle transport system in sec8-1 mutant cells resembles the terminal phenotype of the pob1-664 mutant. A) Tetrad analysis of diploid cells derived from crossing sec8-1 with Δfor3 or Δmyo4. The left panel shows three sets of dissected asci incubated at 25°C with uracil. The middle and right panels show the replicated colonies incubated at 25°C without uracil or at 37°C with uracil, respectively. The genotypes of the descendants were judged by their requirement for uracil as a marker of Δfor3 or Δmyo4 and their growth and terminal morphology at 37°C as a marker of sec8-1. D, double mutant of sec8-1 and Δfor3 or Δmyo4; F, Δfor3; M, Δmyo4; S, sec8-1; W, wild type. B) Model of the functions of Pob1 in polarized growth of S. pombe cells. See the text for details.

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Discussion

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Pob1 is required for vesicle trafficking into growth zones

Here, we showed that, in pob1-664 mutant cells, presumed secretory vesicles markedly accumulated in the cytoplasm and along F-actin filaments at the restrictive temperature. In addition, we found that Pob1 may control not only the organization of actin cables—presumably through interaction with For3—but also the localization of the exocyst complex at growth sites cell tips. It has been shown that neither the process of vesicle transport along actin cables nor exocyst-dependent vesicle tethering is essential for polarized elongation in S. pombe(26,28,37,39). Our present results show, however, that when both processes are impaired cells do not elongate (and later die) and have pob1-664-like morphology. This finding suggests that the two processes, i.e. vesicle transport along actin cables and exocyst-dependent vesicle tethering, are likely to be closely involved in achieving polarized vesicle trafficking for cell elongation, and thus Pob1 may couple these two events. Our hypothesis is illustrated in Figure 7B.

In the budding yeast S. cerevisiae, most exocyst subunits are transported to the bud site along actin cables (40). This may not be the case in S. pombe, however, because Sec6, Sec8 and Sec10 localize in growing tips in Δfor3 null cells (Figure 6B) that lack actin cables. Moreover, a mutant Pob1 lacking For3-binding ability normally localizes to cell tips (31). Saccharomyces cerevisiae and S. pombe, evolutionarily distant two yeasts, may employ different molecular systems recruiting exocyst complex to the polarized region hence achieving different types of cell growth. Meanwhile, actin cable organization appeared normal and Myo4 tightly localized cell ends in sec8-1 incubated at the restrictive temperature (Figures 3A and 5D). Therefore, although the two events that are required for proper cell elongation, namely vesicle transport along actin cables and vesicle tethering to the target membrane, are ordinarily considered as sequential processes, in fact these processes appear to work independently in S. pombe.

In the absence of actin cable-mediated vesicle transport in Δmyo4 null cells, although marked accumulation of presumed secretory vesicles is seen, polarized elongation progresses slowly (26). Under these conditions, some secretory vesicles may drift to the plasma membrane of growing tips because the distance from the Golgi apparatus to the target zone is quite short in S. pombe. When a cylindrical cell is regenerated from an S. pombe protoplast, the number of Golgi that are near the nucleus at the beginning increases dramatically and Golgi become redistributed throughout the cytoplasm near the cell cortex (22). Active transport of secretory vesicles to the cell tips as a means of producing a high concentration of such vesicles at growth sites may be important for relieving defects caused by an impairment of exocyst-mediated vesicle tethering. This hypothesis persuasively explains why For3 or Myo4, but not both, is essential for cell growth in an sec8-1 genetic background. In the absence of Pob1 function, secretory vesicles have difficulty reaching the cell tips because actin cables are absent. Moreover, in these cells, the exocyst is delocalized from the cell tips, and thus vesicle targeting to the cell surface may occur randomly in pob1-defective cells, although membrane fusion efficiency is probably reduced. Because cell surface expansion occurs randomly in the cortex, pob1-defective cells may gradually swell. The pob1-defective cells show lemon-like cell shape suggesting that another cell polarity cue that marks the cell tip remains intact and defines the cell axis.

Pob1 may induce the formation of actin cables by interacting with For3

For3 is required for the formation of actin cables by interacting with the Rho GTPases Rho3 and Cdc42 at its N-terminus (28,41). The N-terminal sequence of For3 is essential and sufficient for its localization to cell tips, and Rho GTPases play important roles in this localization. It is considered that For3 activity is restrained through interaction of its N-terminal diaphanous inhibitory domain (DID) with its C-terminal diaphanous autoregulatory domain (DAD), similar to other diaphanous/formin family proteins (41). Therefore, For3 seems to be activated by interacting with Rho GTPases at cell tips. Meanwhile, Tea4, a cell end marker protein, has also been reported as an interacting protein for For3 at its N-terminus (11). However, their interaction does not seem to be required for the activation of For3 because, in the absence of Tea4, actin cables are still well organized from one tip of the cell (11). Tea4 is probably involved in switching the localization pattern of For3 from monopolar to bipolar during cell elongation. We previously found that elongated actin cables are not present in the pob1-664 mutant at the restrictive temperature (12). Pob1 uses its central region, which contains the sterile alpha motif (SAM) domain, to bind to the N-terminus of For3 (Figure S4). Therefore, like Rho3 or Cdc42, but unlike Tea4, Pob1 may play an important role in For3 function. Rincón et al. recently proposed that Pob1 is required for the For3-mediated formation of actin cables, probably by directly interacting with For3 co-operatively with Cdc42 (31). Our data agree with those of Rincón et al. (Figure S4). Our TEM observation revealed that, in pob1-664, vesicles accumulated along a bundle of microfilaments (presumably F-actin). The bundle structure is unlikely to be composed of normal actin cables because, as described earlier, longitudinal actin cables are not observed in pob1-664. In Δfor3 null cells, short and curled abnormal F-actin structures are often observed (28). It remains unclear whether the bundle of microfilaments observed in pob1-664 corresponds to the abnormal F-actin structures observed in Δfor3 null cells. In wild-type cells, the normal/rapid transport of secretory vesicles may ensure that they do not accumulate along actin cables. Consistent with this speculation, a vesicle accumulation phenotype is observed in Δmyo4 null cells, i.e. in the absence of type V myosin (26). In pob1-664 cells, secretory vesicles might associate with abnormal actin bundles via type V myosin.

Pob1 enables exocyst localization to the growth region

In S. cerevisiae, the primary role of Sec3p is presumably to serve as a landmark for localization of other subunits in the bud tip by interacting (via its N-terminus) with the Rho GTPases Rho1p and Cdc42p as well as PIP2(42–44). The majority of the remaining components located on secretory vesicles subsequently arrive at the target membrane by vesicle transport along actin cables (40). Exo70p is able to localize in the bud tip independently of the actin cytoskeleton by binding to PIP2, and Exo70p function seems to be redundant with Sec3p in the spatial control of the exocyst complex (44,45). Exo70p can directly bind to Rho3p (46), although this interaction is not necessarily required for Exo70p localization to the bud tip (47,48).

Curiously, no obvious Sec3p homolog was found in S. pombe by our genome database search. Other molecular mechanisms may be used to define the cellular localization of the exocyst in this organism. In S. pombe, Exo70 is involved in vesicle trafficking as a component of the exocyst, but its function is dispensable at 25°C (29). It is possible that Pob1 primarily helps control exocyst localization. In S. cerevisiae, Sec15p, an exocyst complex subunit, directly interacts with the cell polarity protein Bem1p (49) that associates with Boi1p and Boi2p (functional homologs of Pob1) and contributes to bud formation (50,51). Perhaps, Pob1 controls exocyst localization by interacting with Scd2 (Ral3), a protein structurally related to Bem1p in S. pombe. Consistent with this idea, cells lacking Scd2 are round, indicating the loss of cell polarity (4,52).

Boi family proteins and Rho GTPases

Proteins related to Pob1 are widely conserved among yeast and fungi, and S. cerevisiae Boi1 and Boi2 were the first such proteins identified (12,50,51,53). We showed that the temperature sensitivity of pob1-664 was suppressed by overexpression of Rho3 or Cdc42. Conversely, pob1+ was isolated as a multicopy suppressor of a cdc42 temperature-sensitive mutant (31). These investigators also showed that Pob1 interacts with Cdc42. In S. cerevisiae, two-hybrid experiments have shown that Boi proteins interact with Cdc42p, a Cdc42p-effector protein, and the GTPase-activating protein for the Rho GTPase Bem2p (50,54). Moreover, under regulation of the cyclin-dependent kinase 1, both Boi1p and Boi2p form a protein complex containing the Cdc42p-regulatory proteins Cdc24p (a guanine nucleotide exchange factor) and Rga2p (a GTPase-activating protein), together with the scaffolding protein Bem1p in S. cerevisiae(18). This protein complex seems to act as the cell polarity cue that induces bud formation by controlling actin-based membrane transport and activating the GTPase cycle of Cdc42p as a positive feedback system (55–57). Rho3p may functionally interact with this protein complex because several components have been identified as multicopy suppressors of the rho3-deficient mutant (58). In S. pombe, Rho3 may stabilize cortical localization of Pob1 probably in an indirect manner. Thus, all members of this Boi family seem to function closely with the Rho GTPase signaling network that is involved in actin-related polarity control. Our results implicate Pob1 in both vesicle transport through the control of actin cable formation and vesicle tethering through the exocyst in S. pombe. It remains to be clarified if all the Boi family proteins use a similar network of Rho GTPases or function in the same way as Pob1.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Genetic techniques and DNA manipulation

Standard genetic procedures for S. pombe were followed according to Alfa et al. (59) and Moreno et al. (60). Complete medium yeast extract plus adenine (YEA) and minimum medium Edinburgh minimum medium (EMM) were used to grow the S. pombe strains. DNA manipulation was carried out according to Sambrook et al. (61). The S. pombe strains and plasmids used in this study are listed in Tables S1 and S2, respectively.

Gene expression in fission yeast

The fission yeast expression vectors pREP1 (62), pREP41 and pREP81 (63) were used in this study. Expression of exogenous genes was induced by removing thiamine from the medium.

Screening

Multicopy suppressors of the temperature-sensitive pob1-664 mutant (JX584) were screened as follows. Cells of JX584 were transformed with an S. pombe genomic library that was constructed with the expression vector pDB248, which is based on pBR322. Transformants were incubated on EMM plates at 26°C for the first 2 days and then transferred to 37°C. From the colonies that grew at 37°C, plasmid DNA was recovered and transformed into Escherichia coli HB101 and sequenced.

Fluorescence microscopy

The cell staining process used to prepare specimens for fluorescence microscopy was as described (33,59). The cells were stained with 4′, 6-diamino-2-phenylindole (DAPI) (Molecular Probes) for DNA and rhodamine-phalloidin (Molecular Probes) or Bodipy-phallacidin (Molecular Probes) for F-actin. Anti-HA (Roche Diagnostics) and Alexa Fluor 594-conjugated anti-rat immunoglobulin G (IgG) (Molecular Probes) were used for immunostaining of HA-Pob1. The stained cells were viewed using an ORCA-3CCD camera (Hamamatsu Photonics) attached to an IX71 or BX51 microscope (Olympus) equipped with a 100×/1.40 numerical aperture (NA) UPlanSApo objective lens (Olympus), and the images were processed in Adobe Photoshop. Three-dimensional reconstitution was accomplished with the Delta Vision system (Applied Precision) as described (28).

Electron microscopy

Schizosaccharomyces pombe cells were cryofixed using a hat-shaped gold specimen carrier (BTBU 012 126-T; BAL-TEC/Leica) covered with a dome-shaped gold specimen carrier (BTBU 012 125-T; BAL-TEC/Leica) that had been coated with lecithin (1, 2-dipalmitoyl-sn-glycero-3-phosphocholine; Fluka) to allow the easy removal of the cover later on. The cells were then frozen under 210 MPa using a high-pressure freezing instrument, the HPM 010 (BAL-TEC/Leica). The cover of the cryopreparation chamber was then removed, and the hat-shaped specimen carrier was soaked in acetone anhydride containing 2% OsO4 in a microtube cooled with liquid nitrogen for more than 80 h. Following a series of incubations at −20°C for 2 h, at 4°C for 1 h and at room temperature for 1 h, the specimen was removed from the specimen carrier and rinsed with acetone anhydride 3× and dehydrated with a 1:1 mixture of QY1 (n-butyl glycidyl ether; Nissin EM) and acetone anhydride and embedded in Quetol 812 (Nissin EM). Ultrathin sections were prepared and stained with 6% uranyl acetate for 10 min and 0.4% lead citrate for 5 min. EM observation was performed using a JEM 1200EXS (JEOL) at 100 kV.

The PATAg staining method for polysaccharides was performed as described (22) with slight modification. Briefly, ultrathin sections on formvar-coated gold grids were pretreated by floating the grid on a drop of 1% periodic acid solution for 20 min and then washed by floating the grid on a drop of deionized water, twice. The grid was then incubated with 0.2% thiocarbohydrazide in 20% acetic acid for 2 h, washed with drops of 10% acetic acid, 5% acetic acid, and deionized water twice, with 5-min incubation each. The sections on the grid were then developed in 1% silver protein solution in the dark for 30 min and then washed in deionized water for 5 min twice.

Expression and detection of SPL-GFP

Experiment using GFP fused with the signal peptide of pho1 acid phosphatase (SPL-GFP) was performed according to Cheng et al. (64) with some modifications. Cells containing pREP1SPL-GFP were grown to early log phase in EMM. After 16 h, cells were collected by a brief centrifugation and transferred to fresh EMM. Then, cells were incubated at 25°C or 36°C for 4 h. Fluorescence signal of the SPL-GFP-expressing cells was visualized without fixation. For western blotting, proteins secreted to the growth media were precipitated with 0.015% deoxycholic acid and 10% trichloroacetic acid, then washed with acetone 2×. To prepare the cell lysate, cells were broken mechanically by vortexing with glass beads in the homogenizing buffer [20 mm Tris–HCl (pH 7.5), 100 mm NaCl, 5 mm ethylenediaminetetraacetic acid (EDTA), 0.5% Triton-X-100], then sample buffer [the final concentrations; 125 mm Tris–HCl (pH 6.8), 5% 2-mercaptoethanol, 4% SDS, 10% glycerol, 0.004% bromophenol blue] was added. After incubation at 95°C for 5 min, proteins were subjected to SDS–15% PAGE and immunoblotted with anti-GFP antibodies.

Measurement of acid phosphatase-secretion activity

Acid phosphatase secretion was assayed according to Craighead et al. (65) and Tanaka and Okayama (66) with some modifications. Cells were grown in EMM at 25°C, pelleted, washed with EMM without phosphate (EMMP) (66) and suspended in fresh EMMP at 25°C or 36°C. Samples were taken at 0 h and at hourly intervals thereafter. For each sample, 500 µL of culture was centrifuged, and 400 µL of the supernatant was added to 400 µL of substrate solution (2 mmp-nitrophenyl phosphate, 0.1 m sodium acetate, pH 4.0; prewarmed to 30°C), then incubated at 30°C for 5 min. Reactions were stopped by the addition of 400 µL of 1 m NaOH. The absorbance at 405 nm was measured by using the 0-h sample as a blank control.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

We are grateful to Mohan K. Balasubramanian (National University of Singapore), Masaki Edamatsu (University of Tokyo), Masak Takaine (University of Tsukuba), Snezhana Oliferenko (Temasek Life Sciences Laboratory), Takayoshi Kuno (Kobe University School of Medicine) and Pilar Perez (Universidad de Salamanca) for providing us strains and plasmids. We also thank Mami Konomi, Yuka Kondo and Mamiko Sato for preparation of electron micrographs. Several strains are provided from the Yeast Genetic Resource Center of the National Bio-Resource Project in Japan. This work was supported by a Grant-in-Aid for Scientific Research to K. N. from the Ministry of Education, Culture, Sports, Sciences, and Technology (MEXT) (Nos. 16044205, 20770150 and 22770184). M. T. was supported by the Special Postdoctoral Researchers program of RIKEN.

References

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Results
  4. Discussion
  5. Materials and Methods
  6. Acknowledgments
  7. References
  8. Supporting Information

Supporting Information

Additional Supporting Information may be found in the online version of this article:

Figure S1: Pob1 function is required for polarized elongation of growing S. pombe cells. Differential interference contrast images of typical phenotypes of the indicated strains are shown. pob1-664 mutant cells have a lemon-like shape at the restrictive temperature, perhaps caused by defective growth while the polarity cue remains functional. WT, wild type.

Figure S2: Cortical localization of Pob1. A) Pob1Y719C is detached from the cell cortex upon high temperature shift. Cells expressing red fluorescent protein mCherry-fused Pob1 and Pob1Y719C instead of Pob1 growing exponentially in YEA at 25°C were shifted to 36°C for the indicated time (min). Wild-type protein (left column) partially strayed away from cell ends to other cortical area upon temperature shift from 25°C to 36°C (arrowheads). This perturbation of Pob1-mCherry localization was repaired within 50 min. On the other hand, mutant protein Pob1Y719C-mCherry, displaying fainter signal of cortical localization even at 25°C, was detached from the cell cortex upon the temperature shift (right column). In additional incubation, polarized localization of Pob1Y719C-mCherry was never returned, and many spots of its fluorescence signal were detected in a cytoplasm (small arrows). Size and intensity of spots were gradually increased (medium arrows). Finally, signal was detected in a vacuole-like structure after around 140 min (large arrows). B) Localization of Pob1 in Δfor3 and Δrho3 null cells. Strains expressing Pob1-mCherry growing exponentially in YEA at 25°C were shifted to 36°C for the indicated time. C) Localization of Pob1 is maintained after Lat-A treatment. Fluorescence microscopy images of F-actin and Pob1-HA are shown. Wild-type cells expressing the triple HA-tagged protein and growing exponentially in YEA at 25°C were treated with 10 µM Lat-A for 10 min. The cells were fixed and stained with anti-HA to detect Pob1 (right) and with Bodipy-phallacidin to detect F-actin (left). Bars: 10 µm.

Figure S3: Pob1 genetically and physically interacts with Cdc42. A) Two-hybrid assay between Pob1 and Rho3. A two-hybrid experiment was carried out using the indicated combination of genes in Y190 strain. Colony formation on a plate lacking histidine, in the presence of 20 mM 3-aminotriazole for suppressing false-positive growth, was diagnostic of a positive interaction between the proteins indicated to the bottom. Cdc42 is used as a positive control (31). Unlike Cdc42, Rho3 seems not to strongly bind to Pob1. B) Cdc42 may also function with Pob1. Overexpression of either Rho3 or Cdc42 can compensate for the growth defect of pob1-664 cells. JX584 was transformed with the empty vector (pREP41), pR81pob1, pR41rho1, pR41rho2, pR41rho3, pR41rho4, pR41rho5 or pR1cdc42. The transformed cells were then streaked onto EMM plates and incubated at the indicated temperature for 5 days.

Figure S4: Pob1 controls the subcellular localization of For3. A) Delocalization of For3 in pob1-664 mutant cells. The expression of YFP-For3 is shown in wild-type (WT) and pob1-664 cells. Bar: 5 µm. B) Pob1 binds to the N-terminus of For3. A two-hybrid experiment was carried out using the indicated combination of gene fragments in HF7c. Colony formation on a plate lacking histidine was diagnostic of a positive interaction between the proteins indicated to the right.

Figure S5: Pob1 functionally interacts with exocyst. A) Genetic interaction between Pob1 and Exo70. The indicated cells were streaked onto YEA plates and incubated at 25°C or 30°C for 4 days. B) Mislocalization of the exocyst complex in pob1-664 mutant. Indicated GFP-fused proteins were expressed in wild-type cells and pob1-664 cells. Cells growing at 25°C in mid-log phase were incubated at 36°C for 2 h and then observed. Bar: 10 µm.

Table S1: List of strains used in this study.

Table S2: Plasmids used in this study.

FilenameFormatSizeDescription
TRA_1190_sm_FigS1.tif786KSupporting info item
TRA_1190_sm_FigS2.tif759KSupporting info item
TRA_1190_sm_FigS3.tif619KSupporting info item
TRA_1190_sm_FigS4.tif964KSupporting info item
TRA_1190_sm_FigS5.tif880KSupporting info item
TRA_1190_sm_TableS1.doc71KSupporting info item
TRA_1190_sm_TableS2.doc46KSupporting info item

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