mut2-1 Identifies a Gene Product Important for Cell Separation and Is Defective in the Exocyst Component Sec8p
We performed a screen to identify mutants defective in cytokinesis in S. pombe. To visualize nuclei easily, a strain was constructed in which the coding region of the histone H3 gene (hht2) was fused to green fluorescent protein sequences (hht2-GFP). As expected, Hht2-GFP localized to the nucleus throughout the cell cycle (our unpublished results). This starting strain (MBY816) was mutagenized by UV irradiation, and the resulting ts− mutants were subjected to microscopic analysis to detect mutants that accumulated multiple nuclei (Tang, X., and Balasubramanian, M.K., unpublished results). The characterization of one such mutant, mut2-1, is described in this study. mut2-1 cells grew and formed colonies at 24°C (permissive temperature) but were unable to do so at 36°C (restrictive temperature). Although wild-type cells continued to grow and divide upon temperature shift from 24 to 36°C, the cell number of a mut2-1 strain did not increase after an identical temperature shift (Figure A), whereas the number of attached cell bodies increased, indicating failed cell separation. To better characterize the phenotype of mut2-1, we monitored changes in the subcellular distribution of F-actin and cell wall material after a shift from 24 to 36°C (Figure B). Under permissive conditions, F-actin rings and septa in the majority of mut2-1 cells resembled those found in wild-type cells (Figure B, 0 h). After 4 h at 36°C, >50% of mut2-1 cells contained four nuclei, indicative of the successful completion of two rounds of mitosis despite the aberrant cytokinesis (Figure B, 4 h). Interestingly, under these conditions, assembly and constriction of the actomyosin ring were not impaired in mut2-1 cells (Figure B, arrow). In addition, mut2-1 cells were also capable of assembling medial division septa (Figure B). However, the septa apparently could not be disassembled in mut2-1 cells, leading to the accumulation of elongated cells with one or three septa. Thus, mut2-1 identifies a protein important for cell separation after assembly of the division septum.
Figure 1 Phenotype of mut2-1 (sec8-1) cells. (A) Growth curves of wild-type and mut2-1 cells. Cell numbers of wild-type and mut2-1 cells were quantified by counting cells using a hemacytometer after temperature shift from 24 to 36°C. ●, wild-type; (more ...)
To identify the gene responsible for the mut2-1 phenotype, a plasmid rescuing the temperature-sensitive lethality of mut2-1 was identified (see MATERIALS AND METHODS). The rescuing DNA encoded a 1088-amino acid polypeptide. Database searches using the predicted protein sequence showed that it was related to S. cerevisiae Sec8p (16% amino acid identity), a component of the exocyst, as well as to Sec8p-like proteins from humans (13% identity, Figure ). Several lines of evidence established that mut2-1 is an allele of sec8+ (see MATERIALS AND METHODS).
Figure 2 S. pombe Sec8p is homologous to proteins from S. cerevisiae and human. Amino acid sequences were aligned using ClustalX and Boxshader programs. Identical amino acids are shaded in black, and conservative substitutions are shaded in gray. Hs, Homo sapiens (more ...)
The exocyst is required for polarized cell growth and cell surface expansion in S. cerevisiae
(TerBush et al., 1996
; Roth et al., 1998
). In contrast, the sec8-1
mutant described in this study appeared to be defective only in septum disassembly and cell separation. To test the role of Sec8p in cell elongation, wild-type and sec8-1
cells were synchronized by nitrogen starvation and monitored for the ability to undergo polarized cell growth and septum assembly. This protocol provides a convenient means to assess the function of a protein in cell elongation, because wild-type cells start at the length of 4 μm and elongate to 12–14 μm before division (Figure A). After release into rich medium, sec8-1
cells were able to elongate, enter mitosis, and assemble division septa with kinetics similar to that of wild-type cells (Figure B). However, unlike wild-type cells, sec8-1
cells failed to disassemble the division septa, leading to the accumulation of binucleate cells with a medial division septum. Even although septum cleavage and cell separation failed, sec8-1
cells reinitiated polarized growth and underwent a second round of mitosis and division septum assembly 7 h after release into the rich medium. Septum cleavage again failed in these cells resulting in the accumulation of tetranucleate cells with three septa. On prolonged incubation (12 h) sec8-1
cells lysed. Cell length and the percentage of septated cells at each time point were also quantified (Table ), which indicated that sec8-1
is not defective in cell elongation and is not delayed for septum assembly. These data suggest that sec8-1
cells are specifically defective in septum cleavage and cell separation.
Figure 3 sec8-1 cells are not defective in polarized cell growth. Wild-type cells (A) and sec8-1 cells (B) were synchronized in G1 by growth in nitrogen-free medium for 18 h at 24°C, and shifted to 36°C for 1 h to inactivate the Sec8-1p protein. (more ...)
Cell elongation and septum assembly in wild-type and Sec8-1 cells.
Identification of sec6+, sec10+, sec15+, and exo70+ Sequences from the S. pombe Genome Database
The exocyst in S. cerevisiae
is a mulitprotein complex comprised of Sec3p, Sec5p, Sec6p, Sec8p, Sec10p, Sec15p, Exo70p, and Exo84p (TerBush et al., 1996
). We have identified Sec8p in S. pombe
as a homologue of one component of the exocyst. We therefore searched the S. pombe
databases to determine whether other exocyst components were also present in fission yeast. Interestingly, homologues of S. cerevisiae
Sec6p, Sec10p, Sec15p, and Exo70p were also present in S. pombe
(see MATERIALS AND METHODS). We were unable to identify proteins related to Sec3p, Sec5p, or Exo84p. The alignments of the S. pombe
exocyst proteins (named like their S. cerevisiae
homologues) with their counterparts in other organisms are shown in Figure . These four exocyst proteins in S. pombe
share ~20% identities in sequences and align through their entire lengths with their homologues. Thus, several components of the exocyst complex are conserved in S. pombe
Figure 4 Alignment of S. pombe Sec6p, Sec10p, Sec15p, and Exo70p with related proteins from S. cerevisiae, rat, and human. Amino acid sequences are aligned using ClustalX and Boxshader programs. Identical amino acids are shaded in black, and conservative substitutions (more ...)
The Exocyst Components Interact In Vivo
Immunoprecipitation experiments were performed in order to determine whether S. pombe Sec6p, Sec8p, Sec10p, and Exo70p form a complex in vivo, as has been demonstrated with their counterparts in other organisms. A number of strains expressing either c-Myc– or GFP-tagged versions of Sec6p, Sec8p, Sec10p, and Exo70p were constructed. To test the interaction between Sec8p and Sec6p, protein extracts from strains expressing sec8-GFP alone, sec8-GFP and sec6-Myc, or sec6-Myc alone were immunoprecipitated using anti-GFP antibodies and analyzed using a Myc mAb. Sec6p-Myc was only detected in the immunoprecipitates from the sec8-GFP sec6-Myc strain (Figure A), suggesting that these two proteins associate in vivo. To test the interaction of Sec8p with Sec10p, similar immunoprecipitations were performed using extracts of strains expressing sec8-GFP alone, sec8-GFP and sec10-Myc, or sec10-Myc alone. Sec10-Myc was detected only in the immunoprecipitates from sec8-GFP sec10-Myc cells (Figure B). Thus, Sec8p also interacts with Sec10p. Finally, the interaction of Sec8p with Exo70p was demonstrated in similar experiments (Figure C). In addition, we observed interactions in the other pairwise combinations (Sec6p-Sec10p, Sec6p-Exo70p, and Sec10p-Exo70p; our unpublished results). Thus, the exocyst components Sec6p, Sec8p, Sec10p, and Exo70p physically interact with each other in S. pombe.
Figure 5 Sec6p, Sec8p, Sec10p, and Exo70p associate in vivo. (A) Protein extracts were prepared from cells expressing Sec8-GFP (lanes 1), Sec8-GFP and Sec6-Myc (lanes 2), or Sec6-Myc alone (lanes 3). Total lysates (right panel) and immunoprecipitates prepared (more ...)
Sec6p, Sec8p, Sec10p, and Exo70p Localize to the Division Site
The subcellular localization of Sec8p was determined by tagging the chromosomal copy of sec8+ with GFP sequences. In this strain, the expression of Sec8p-GFP was under the control of the sec8+ promoter. The sec8-GFP cells resembled wild-type cells in morphology and growth rates, establishing that the addition of GFP did not compromise the function of Sec8p. However, the Sec8p-GFP signal was prone to rapid photobleaching. Therefore, indirect immunofluorescence was performed to visualize Sec8p-GFP. In interphase cells, identified as uninucleate cells with uncondensed chromosomes, tip localization was observed in 55% (Figure A, marked with arrowheads). In early mitotic cells, tip localization was absent and Sec8-GFP was seen as a ring in the medial region of the cell that resembled the actomyosin ring (Figure A, cells marked with 1 and 4). However, in late mitotic cells, unlike the actomyosin ring, which undergoes constriction, medial staining of Sec8p-GFP was detected as double rings (Figure A, cells marked with 2 and 3). To examine whether these structures were real ring structures, confocal microscopy and 3D-projection software were used to determine the localization of Sec8-GFP. When Sec8-GFP double ring images were rotated, they appeared clearly as rings (Figure B; arrow marks the entire ring visualized upon rotation by 139°). Essentially identical localization patterns were observed for Sec6p-GFP, Sec10p-GFP, Sec6p-Myc, Sec10p-Myc, and Exo70p-Myc (Figure , A, C, and D). Thus, consistent with their coimmunoprecipitation, components of the exocyst also colocalized in S. pombe cells, supporting the hypothesis that the exocyst components interact in vivo.
Figure 6 Localization of exocyst components in S. pombe. (A) Exocyst components localize to the division site and cell tips. sec8-GFP sec6-Myc cells were stained with antibodies against GFP and Myc. In the merged micrograph, Sec8-GFP is in red, Sec6-Myc in green, (more ...)
To investigate the localization of exocyst components in relation to the actomyosin ring, we examined the localization of Sec10p-GFP and Myo2p (an actomyosin ring component) in the same cells. Both proteins assembled into ring structures at early mitosis and approximately colocalized (Figure E, top panel). However, in cells undergoing actomyosin ring constriction (Figure E, bottom panel), constriction of the Sec10-GFP rings was not observed. Instead, the Sec10-GFP rings split into a pair of rings on either side of the constricting actomyosin ring.
The Medial Localization of the Exocyst Is Dependent on the Actomyosin Ring but not on Exocytosis
Given that the S. pombe
exocyst components assembled as a medial ring that colocalized approximately with the actomyosin ring at early mitosis, we addressed the roles of the F-actin cytoskeleton and of proteins important for actomyosin ring formation in the assembly of the exocyst complex at the division site. First, we monitored the localization of Sec10-GFP after treatment of G2-synchronized cells with latrunculin A (LatA), a drug that prevents actin polymerization. Although DMSO alone did not affect assembly of medial Sec10-GFP rings, cells treated with LatA in DMSO were unable to assemble medial Sec10p-GFP rings (Figure F). Thus, the proper assembly of Sec10p and, by inference, the other exocyst components as a medial ring at the division site is F-actin dependent. We then examined Sec10p-GFP localization in cdc8-110
(Balasubramanian et al., 1992
(Chang et al., 1997
), and cdc15-140
(Fankhauser et al., 1995
) mutants. Although Sec10p-GFP was observed as a medial ring at 24°C in all these mutants, at 36°C none of the mutants was able to assemble Sec10p-GFP into ring structures (Figure , G and H, and our unpublished data). Thus, the assembly of the exocyst to the medial region appears to depend on the proteins essential for actomyosin ring assembly and actin patch mobilization.
Because the exocyst in S. pombe
is potentially involved in secretion, we wanted to ascertain whether the localization of the exocyst as a medial ring is dependent on the secretory pathway. Synchronous cells expressing Sec10-GFP were treated with brefeldin A (BFA), a drug blocking membrane trafficking of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi (Turi et al., 1994
). Gma12p, a Golgi marker protein that has been reported to relocate from Golgi to ER upon BFA treatment (Brazer et al., 2000
), was used to test the efficacy of BFA treatment. The ER in S. pombe
is distributed primarily around the nuclear membrane region, whereas Golgi is seen as patches throughout the cytoplasm (Brazer et al., 2000
). As expected, Gma12p-GFP relocated from Golgi to ER upon treatment with BFA (Figure I). In contrast, the localization of Sec10p was not affected by BFA (Figure I), indicating that the exocyst localization is independent of exocytosis. Thus, the exocyst complex in S. pombe
could serve as a landmark for the targeting of the exocytic machinery.
Phenotype of Exocyst Null Mutants
Although exocyst mutants in S. cerevisiae
are defective in polarized growth (Hsu et al., 1999
mutants in S. pombe
appear to be unaffected with respect to polarized growth. Given this dramatic difference in phenotype, it seemed possible that sec8-1
was not defective in a polarized growth function of Sec8p. In this case, a sec8
null mutant would be expected to show a stronger phenotypic defect with respect to cell growth. To test this, we replaced sec8+
in a diploid strain. By analysis of meiotic products from the heterozygous strain, we found that spores bearing the sec8-
null mutation were incapable of forming colonies. Thus, Sec8p is essential for cell viability. To characterize the terminal phenotype, the mutant spores were germinated and stained to visualize F-actin, DNA, and septa (Figure A). The mutant spores were capable of germination, cell elongation, mitosis, actomyosin ring assembly, and septum assembly. However, the septa assembled in the germinating mutant cells were not cleaved. Cell growth, cell elongation, and mitosis continued in the unseparated mutant cells, leading eventually to the accumulation of tetranucleate cells with septa placed between each pair of nuclei. Similar results were obtained with sec6
null mutants (Figure , D and E).
Figure 7 (A, D, and E) Null mutants of sec8 (A), sec6 (D), and sec10 (E) are defective in cleavage of the septum. Mutant spores were germinated in appropriate selective media and stained with DAPI, phalloidin, and Calcofluor to visualize DNA, F-actin, and septa, (more ...)
To ensure that the phenotype was not due to inherited maternal exocyst proteins, we tested whether the maternal Sec8p was present in sec8
-null cells using a diploid strain in which one sec8
locus was replaced with ura
and the other was tagged with Myc
. We examined whether the maternal Sec8-Myc protein was present in the germinated null mutant cells. Spores were germinated in medium selective for ura
and stained with antibodies against Myc and Mok1p to visualize Sec8-Myc and the α-glucan synthase Mok1p (Katayama et al., 1999
). Although Sec8-Myc localization was clearly observed in sec8
cells (our unpublished results), it was not observed in the sec8
null cells (Figure B), suggesting that there was no significant carry-over of maternal Sec8p in these cells. Mok1p, used as a control, was observed in both cases as expected.
To analyze the sec8 loss-of-function phenotype using a different approach, we constructed a sec8 shut-off strain in which sec8 transcription was under the control of the low-strength and thiamine-repressible 81nmt1 promoter. On growth under repressing conditions, sec8 shut-off cells again appeared defective only in the disassembly of division septa but not in polarized cell growth (Figure C). We conclude that the exocyst is essential for septum disassembly and cell separation, whereas cell elongation and division septum assembly might require reduced levels of exocyst function or might be independent of it.
sec8-1 Mutant Cells Are Defective in Exocytosis
The exocyst in S. cerevisiae
and mammals is involved in membrane trafficking from the Golgi apparatus to the plasma membrane (Hsu et al., 1999
). To test whether the exocyst in S. pombe
has a role in exocytosis, we used electron microscopy to ask if the targeting and fusion of secretory vesicles with the plasma membrane could occur normally in sec8
mutant cells. Presumed secretory vesicles (100 nm in diameter) were observed only rarely in wild-type cells (Figure A). In contrast, 60–100 such vesicles were detected in every section in the sec8-1
mutant (Figure B; average three vesicles/μm2
). These vesicles were stained intensely after permanganate fixation and most likely represent post-Golgi secretory vesicles (Armstrong et al., 1993
). In mutant cells undergoing septum assembly, most of the vesicles were clustered approximately in the vicinity of the septa. These observations suggested that targeting of secretory vesicles to the correct location occurs in sec8-1
cells but that the subsequent docking and/or fusion with the plasma membrane failed. During interphase, sec8-1
cells were also found to accumulate ~100 nm vesicles, indicating that Sec8p might also participate in exocytic events during interphase. Similarly, sec8
shut-off cells accumulated a large number of ~100 nm vesicles under repressing conditions (Figure D), whereas cells under nonrepressing conditions resembled wild-type cells (Figure C).
Figure 8 sec8 mutants are defective in exocytosis. (A-D) sec8-1 mutant and sec8 shut-off cells accumulate large numbers of secretory vesicles. (A and B) Wild-type (A1, A2) and sec8-1 (B1–B3) mutant cells were grown at 24°C and shifted to 36°C (more ...)
To test whether sec8 mutants are defective in exocytosis using a different approach, we monitored the transport of the enzyme acid phosphatase through the S. pombe secretory pathway in sec8-1 cells (Figure E). The activity of secreted acid phosphatase was assayed using the culture supernatant (see MATERIALS AND METHODS). Wild-type cells at 36°C secreted acid phosphatase about twice as fast as at 24°C. sec8-1 cells secreted much less acid phosphatase than wild-type cells at both temperatures. After 4 h, they secreted 67% of the level of activity of wild-type cells at 24°C and 42% of the activity at 36°C. Thus, sec8-1 is indeed defective in exocytosis.