Fig 1.
Spa2 interacts with IPCs during cytokinesis.
(A) INN1-TAP CHS2-9MYC (YMF38) and control (YMF79) strains were grown at 24°C in YPRaff medium and synchronised in G1 phase with mating pheromone and then released for 105 minutes. Cell extracts were prepared before the immunoprecipitation of Inn1-TAP (or TAP in control) on IgG-beads. The isolated material was released from the beads by cleavage with TEV protease. Purified material was subjected to immunoprecipitation of Chs2-9MYC before analysis by mass spectrometry [59]. The protein composition of purified fractions was analysed by mass spectrometry. Spectral count number (SpC), percentage sequence coverage (Cov.) and Fold-Change are shown (i). INN1-TAP MYO2-5FLAG (YMF969) and control (YMF914) strains were grown at 24°C in YPD medium and synchronised in G1 phase with mating pheromone and then released for 90 minutes before preparing the protein extracts. DNA content was monitored by flow cytometry. The asterisk denotes the time when the sample for making protein extracts was collected (ii) (note that the cytokinesis peak is slightly advanced because the carbon source is different from that in Fig 1A (i)). Cell extracts were prepared and analysed by SDS-PAGE and immunoblotting (iii). (B) TAP-SPA2 CHS2-9MYC (YMF1302) (i) and TAP-SPA2 MYO1-5FLAG IQG1-6HA (YMF1176) (ii), together with the corresponding control strains, were grown at 24°C in YPD medium and synchronised in G1 phase with mating pheromone and then released for 90 minutes. Cell extracts were prepared before immunoprecipitation of Spa2 and detection of the indicated proteins by immunoblotting. (C) TAP-SPA2 CHS2-9MYC (YMF1302) (ii) and TAP-SPA2 MYO1-FLAG CYK1-6HA (YMF1176) (iii) strains were grown at 24°C in YPD medium and synchronised in G1 phase with mating pheromone (G1 sample) or released from G1 block for 30 minutes (S phase sample) or 90 minutes (cytokinesis sample); DNA content was monitored by flow cytometry (i). Cell extracts were then prepared before immunoprecipitating Spa2 on IgG-beads and detecting the indicated proteins by immunoblotting (ii) and (iii). (D) MYO1-Tomato SPA2-GFP (YMF1256) strain was grown at 24°C in YPD and arrested in G1 phase with mating pheromone. Cells were washed and released from G1 arrest into fresh YPD for 30 minutes. Subsequently cells were shifted to Synthetic Complete (SC) medium before being placed on the time-lapse slide to examine the localisation of Myo1 and Spa2 every 2 minutes as cells completed cell division at 24°C (see Methods for details). A z-stack of images was gathered. A two-dimensional projection of the three dimensional data is shown. Scale bar: 2 μm. The grey and black circles denote the timing of the actomyosin ring contraction.
Fig 2.
Spa2 interacts directly with multiple domains of Cyk3.
(A) Diagram of protein structures and yeast two-hybrid interactions between different fragments of Spa2 and tested fragments of Cyk3 (i). Summary of yeast two-hybrid interactions between Spa2-1-145 fragment and different truncations of Cyk3 (ii). (B) The SH3 domain of Cyk3 interacts directly with Spa2-1-552. Pairs of E. coli cell cultures expressing Strep-tag-SH3-Cyk3 and 6His-Spa2-1-552 were mixed and used to purify putative protein complexes via Strep-Tactin Superflow resin. The final purified fractions were analysed by SDS-PAGE and the tagged proteins were detected with anti-Strep or anti-His antibodies. His-tag was only used for protein detection, but not for purification purposes. (C) Fragment of Cyk3 containing transglutaminase-like domain interacts directly with Spa2-1-552. Pairs of E. coli cell cultures expressing Strep-tag- Spa2-1-552 and 6His-Cyk3-475-885 were mixed and used to purify putative protein complexes via Strep-Tactin Superflow resin. The final purified fractions were analysed by SDS-PAGE and the tagged proteins were detected with anti-Strep or anti-His antibodies. His-tag was only used for protein detection, not for purification purposes.
Fig 3.
Spa2 interacts directly with multiple domains of Hof1.
(A) Summary of yeast two-hybrid interactions between the fragments of Spa2-1-145 containing SHD-I and the fragments of Hof1 containing SH3 or F-BAR domains. (B) SH3 domain of Hof1 interacts directly with Spa2-1-552. Pairs of E. coli cell cultures expressing Strep-tag-SH3-Hof1 and 6His-Spa2-1-552 were mixed and used to purify putative protein complexes as in Fig 2B. The final purified fractions were analysed as in Fig 2B. (C) The F-BAR domain of Hof1 interacts directly with Spa2-1-552. Pairs of E. coli cell cultures expressing Strep-tag-Hof1-1-300 and 6His-Spa2-1-552 were mixed and we proceeded as in Fig 2B. (D) Schematic alignment and protein sequence comparison of Spa2 (S. cerevisiae) and its orthologues from S. pombe, together with SHD domain of Caenorhabditis elegans (F14F3.2) and Homo sapiens (KIAA0148). The identical residues have been boxed. Conserved Lys and Arg were mutated to Gly and Ala (GAGA). Summary of yeast two-hybrid interactions between a fragment of Spa2-1-145-GAGA containing mutated residues or its wild-type version, and the corresponding fragments of Cyk3 and Hof1.
Fig 4.
Spa2 protein interacts with secretory vesicle system in the absence of IPC component Hof1.
(A) SPA2-GFP (YMF167) and SPA2-GFP iqg1-td (YMF183) strains were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 37°C to deplete Iqg1-td. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with single rings of Spa2 at the cleavage site (ii). Example of cell with Spa2-GFP single ring at the bud-neck at 75 minutes is shown (iii). Scale bar: 2 μm. (B) SPA2-GFP hof1-td (YMF713) and SPA2-GFP cyk3-td (YMF1104) strains were grown in YPRaff as in (A) and released from G1 arrest at 37°C after depletion of Hof1-td or Cyk3-td. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with Spa2 rings at the cleavage site (ii). (C) TAP-SPA2 (YMF1301) and TAP-SPA2 hof1-td (YMF1299) strains were grown as in (A) and cell extracts were prepared 75 minutes after the release, before the immunoprecipitation of Spa2 and detection of the indicated proteins by immunoblotting. (D) SPA2-GFP (YMF167) and SPA2-GFP myo2-td (YMF716) strains were arrested in G1 phase at 24°C in YPRaff. Cells were then shifted to YPRaff medium containing 0.2 M hydroxyurea to arrested them in early S phase. Cells were allowed to fully grow their buds and were then transferred to YPGal containing 0.2 M hydroxyurea at 37°C in order to deplete Myo2-td. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i), the percentage of cells with rings of Spa2 at the cleavage site (ii) and the percentage of cells localising Spa2 at the bud tips (iii).
Fig 5.
IPC component Hof1 and secretory vesicle transport protein Myo2 both contribute to the localisation of Spa2 at the site of division.
(A) SPA2-GFP (YMF167) and SPA2-GFP hof1-td myo2-td (YMF1418) strains were arrested in G1 phase at 24°C in YPRaff and then synchronously shifted to YPRaff medium containing 0.2 M hydroxyurea and arrested in early S phase. Before cells were transferred to YPGal containing 0.2 M hydroxyurea at 37°C in order to deplete Hof1-td and Myo2-td, they were allowed to grow their buds. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with Spa2 rings at the cleavage site (ii). An example of a cell with Spa2-GFP ring at the bud-neck at 60 minutes is shown (iii). Scale bar: 2μm. (B) Indicated diploid strains TAP-SPA2/SPA2-5FLAG (YMF1448) and SPA2/SPA-5FLAG (YMF1449) were grown asynchronously at 24°C in YPD medium and cell extracts were prepared before immunoprecipitation of TAP-Spa2 and detection of the indicated proteins by immunoblotting. (C) Summary of yeast two-hybrid interactions between the different fragments of Spa2, one containing SHD-I region (Spa2-1-145) and another one containing SHD-II (Spa2-421-552). (D) SPA2-GFP (YMF117), ΔC-SPA2-GFP (YMF967, Spa2-1-552-GFP) and ΔN-SPA2-GFP (YMF1023, Spa2-553-1466-GFP) strains were arrested in G1 phase at 24°C in YPD and then released from G1 arrest to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Spa2 or its truncations at the cleavage site (ii). (E) Summary of yeast two-hybrid interactions between the C-terminal fragments of Spa2 and Hof1.
Fig 6.
Spa2 interacts directly with the chitin synthase Chs2.
(A) Tetrad analysis of the meiotic progeny from the indicated diploid strain (YMF741) shows that CHS2-V377I allows spa2Δ cyk3Δ cells to grow. Spores of the indicated genotypes were grown for 20 hours on YPD plates at 24°C. Scale bar: 20 μm. (B) Tetrad analysis of the meiotic progeny from the indicated diploid strain (YMF866) shows that CHS2-V377I allows spa2Δ hof1Δ cells to grow. Spores of the indicated genotypes were grown for 20 hours on YPD plates at 24°C. Scale bar: 20 μm. (C) Scheme representing the genetical relationship between Cyk3, Hof1, Spa2 and Chs2-V377I proteins (i). Serial dilutions of strains YMF140 (1) and YMF1307 (2) were plated on YPD medium or YPGal medium and incubated as indicated (ii). Serial dilutions of strains YMF759 (3), YMF1401 (4) and YMF1399 (5) were plated on YPD medium or YPGal medium and incubated as indicated (iii). (D) Summary of yeast two-hybrid interactions between the different fragments of Spa2 and the Chs2. Truncated allele of Chs2 containing the catalytic domain (Chs2-215-629) was used to show that this region of Chs2 (Chs2-215-629) interacts in a yeast two-hybrid assay with Spa2 via different SHD-containing fragments. (E) Pairs of E. coli cell cultures expressing Strep-tag-Chs2-215-629 and 6His-Spa2-1-552 were mixed and used to purify putative protein complexes via Strep-Tactin Superflow resin. The final purified fractions were analysed by SDS-PAGE and the tagged proteins were detected with anti-Strep or anti-His antibodies. His-tag was only used for protein detection, not for purification purposes.
Fig 7.
Spa2 localisation at the site of division requires the presence of Hof1 and Cyk3.
(A) SPA2-GFP (YMF167) and SPA2-GFP hof1-td cyk3-td (YMF1088) strains were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 37°C to deplete Hof1-td and Cyk3-td simultaneously. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Spa2 at the cleavage site (ii). Example of cell with Spa2-GFP ring at the bud-neck is shown for the 75 minute time-point (iii). Scale bar: 2 μm. (B) CHS2-GFP (YMF330) and CHS2-GFP hof1-td cyk3-td (YMF1076) strains were grown in YPRaff as in (A). Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Chs2 at the cleavage site (ii). Example of cell with Chs2-GFP ring at the bud-neck at 75 minutes (iii). Scale bar: 2 μm. (C) CHS2-GFP hof1-td cyk3-td (YMF1076) and CHS2-V377I-GFP hof1-td cyk3-td (YMF1329) strains were grown in YPRaff as in (A). Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Chs2 or Chs2-V377I at the cleavage site (ii). Example of cell with Chs2-V377I-GFP ring at the bud-neck at 60 minutes (iii). Scale bar: 2μm.
Fig 8.
Higher-affinity binding of Chs2-V377I to Inn1 promotes recruitment of Spa2 at the site of division in the absence of Hof1 and Cyk3.
(A) Serial dilutions of strains YASD522 (1), YMF1375 (2), YMF1394 (3), YMF1370 (4), YMF140 (5) and YMF1307 (6) were plated on YPD medium or YPGal medium and incubated as indicated. (B) Chs2-V377I has a higher affinity for binding to Inn1. Pairs of E. coli cell cultures expressing either Strep-tag-Chs2-215-629 or Strep-tag-Chs2-215-629-V377I were mixed with cultures expressing 6His-Inn1 and used to purify putative protein complexes via Strep-Tactin Superflow resin. The final purified fractions were analysed by SDS-PAGE and the tagged proteins were detected with anti-Strep or anti-His antibodies. His-tag was only used for protein detection, but not for purification purposes. (C) SPA2-GFP CHS2 hof1-td cyk3-td (YMF1088) and SPA2-GFP CHS2-V377I hof1-td cyk3-td (YMF1403) strains were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 37°C to deplete Hof1-td and Cyk3-td simultaneously. Subsequently, cells were released to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with rings of Spa2 at the cleavage site (ii). Example of cell with Spa2-GFP ring at the bud-neck at 60 minutes (iii). Scale bar: 2 μm.
Fig 9.
Spa2 coordinates delivery of Chs2 to the site of division.
(A) CHS2-GFP (YASD819) and CHS2-GFP GAL-SPA2 (YMF1660) strains were arrested in G1 phase at 24°C in YPRaff and then shifted to YPGal at 24°C to induce expression of Spa2. Subsequently, cells were released from G1 block to allow progression through the cell cycle. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and the percentage of cells with Chs2 rings at the cleavage site (ii) and (iii). (B) CHS2-GFP SPC42-EQFP (YAD380) strain together with CHS2-GFP GAL-SPA2 (YMF1660) were grown as in Fig 8A. 75 minutes after the release from G1 block cells were shifted to SC medium before being placed on the time-lapse slide to examine the localisation of Chs2 every 2 minutes as cells completed cell division at 24°C (see Methods for details). A z-stack of images was gathered. A two-dimensional projection of the three-dimensional data is shown. Scale bar: 2 μm. The grey and black circles denote the timing of the actomyosin ring contraction. (C) chs3Δ control (YMF505) and chs3Δ GAL-SPA2 (YMF1534) cells were grown in YPRaff medium at 24°C and synchronised in G1 with alpha factor. Subsequently, cells were released in YPGal for 135 minutes from G1 block in the presence of calcofluor to visualise primary septum deposition. Samples were taken at the indicated times to determine the proportion of binucleate cells (i) and 100 cells with primary septum for each sample were examined. Examples of live cells grown with calcofluor are shown in (ii). Scale bar: 2 μm. The relative signal intensity of primary septum was measured for 100 cells and compared with control cells, whose signal intensity was set to 100% (iii).
Fig 10.
Spa2 protein coordinates specific delivery of secretory vesicles to the site of division during cytokinesis.
Secretory vesicles are driven to the cleavage site by type V myosin-based transport. Spa2 interacts with the secretory vesicle system and IPC components Hof1 and Cyk3. We suggest that the three proteins coordinate the incorporation of chitin synthase Chs2 into the IPCs before the start of actomyosin ring contraction.