Cell-cycle dependent phosphorylation of yeast pericentrin regulates γ-TuSC-mediated microtubule nucleation
- Universität Heidelberg, Germany
- University of Heidelberg, Germany
Figures
Figure 1 with 2 supplements
Phosphorylation of N-Spc110 is required for the γ-TuSC oligomerization.
(A) Diagram of Spc110's functional domain organization and the position of phospho-sites investigated in this study. These sites are located at the N-terminal domain of Spc110, which directly …
Figure 1—figure supplement 1
Phosphorylation of Spc1101–220 from insect cells.
(A) Table of mass-spectrometry identified phosphopeptides of Spc1101–220 purified from baculovirus-insect cell expression system. (B) Mass spectra of identified Mps1 sites (S60 and T68). (C) Mass …
Figure 1—figure supplement 2
γ-TuSC does not oligomerize in TB150 buffer.
(A–C) Purified γ-TuSC (A) spontaneously oligomerizes in BRB80 buffer (B) but not in TB150 buffer (C). Proteins were analyzed by Superose 6 10/300 and subsequently gel filtration fractions were …
Figure 2 with 6 supplements
Mps1 and Cdk1 phosphorylation of N-Spc110 stimulates γ-TuSC oligomerization.
(A) Summary of combination of non-phosphorylatable and phospho-mimicking mutations in Spc110 used in this study. The indicated Spc1101–220 variants were expressed, purified from E. coli, and then …
Figure 2—figure supplement 1
Purification of Spc1101–200 variants.
(A) Purified GST-Spc1101–220 variants. ∼1 μg of protein was loaded to each lane. The SDS-PAGE was stained with Coomassie Blue. (B) Calibration of Superose 6 10/300 gel filtration column. Following …
Figure 2—figure supplement 2
Phosphomimetic but not SPM defective N-Spc110 proteins induce oligomerization of γ-TuSC.
(A) Overlapped gel filtration chromatograms with molecular weight markers. The peak of the void-volume (V0, boxed area) corresponds to molecular weight fractions higher than 5000 kDa. Note that the …
Figure 2—figure supplement 3
Non-phosphorylatable mutations in Mps1 or Cdk1 sites of Spc110 are neutral to γ-TuSC oligomerization induced by phosphomimetic mutations.
Gel filtration chromatograms of γ-TuSC incubated with indicated Spc1101–220 variants in TB150 buffer. Non-phosphorylatable mutations in addition to phosphomimetic mutations were introduced on Spc1101…
Figure 2—figure supplement 4
EM single particle analysis of oligomerized γ-TuSC.
(A) Additional examples of ring-like and filament-like γ-TuSC-Spc1101–220 complexes. Scale bar: 50 nm. (B) Summary of particle numbers of γ-TuSC oligomerized by Spc1101–220 phospho-mimicking …
Figure 2—figure supplement 5
Mutations of T18 abolish the γ-TuSC oligomerization promoting activity by inactivating the SPM motif.
(A) Gel filtration chromatograms of γ-TuSC incubated with phosphomimetic Spc1101–220-5D with additional T18 mutations. Complexes were analyzed by Superose 6 10/300 chromatography in TB150 buffer. (B)…
Figure 2—figure supplement 6
Multiple sequence alignment of CM1 motif-containing proteins.
Multiple sequence alignment of selected γ-TuSC receptor family members containing CM1 motifs. Two of the most conserved residues within CM1 motif are marked with asterisks and mutated to disrupt CM1 …
Figure 3
Phosphorylation of N-Spc110 regulates the affinity to γ-TuSC.
(A) GST pull-down assays were performed between γ-TuSC (containing His-tagged Spc97-6His and Spc98-6His) and the GST-tagged Spc1101–220 proteins. The bound proteins were eluted with sample buffer …
Figure 4
Spc1101–220 phosphorylation enhances MT nucleation activity in vitro.
(A) Enhancement of MT nucleation activity of Spc110 by Mps1 and Cdk1 phosphorylation. Representative image fields of Alexa546-labeled microtubules from the nucleation assay polymerized in the …
Figure 5 with 1 supplement
Cell cycle dependent phosphorylation of Spc110.
(A and B) Two phospho-specific antibodies were generated from guinea pigs to recognize phosphorylation of Cdk1 sites (pS36-pS91) (A) and Mps1 sites (p60-p64-pT68) (B). In vitro kinase assays were …
Figure 5—figure supplement 1
Phosphorylation of T18 most likely affects γ-TuSC oligomerization promoting activity of Spc110 in a negative manner.
(A) Phospho-specific antibody against phospho-T18 (pT18) was generated from guinea pigs. In vitro kinase assays were performed in the presence of Cdk1as1 and Spc1101–220, either wild type (WT) or …
Figure 6 with 1 supplement
Cells with spc110 phospho-, SPM-, or CM1-mutant alleles have defects in spindle formation.
(A) Growth of 10-fold serial dilutions of SPC110 shuffle strains with integration vector encoding SPC110 (WT) or SPC110 mutants with and without the SAC gene MAD2. Growth was tested either on …
Figure 6—figure supplement 1
Phenotype of SPC110 mutants.
(A) There was no significant difference in the protein levels of Spc110 variants. Asynchronous cells grown to OD600 0.5 were harvested and subjected to TCA extraction for whole cell protein lysates. …
Figure 7 with 2 supplements
The N-terminus of Spc98 mediates binding to N-Spc110.
(A) Alignment of the amino acid sequence of GCP3 homologues from yeast to human. Shown are the putative α-helical regions H1–H5. Residues are marked according to the ClustalX colour scheme. (B) …
Figure 7—figure supplement 1
The importance of the N-terminal region of Spc98.
(A) Structure and functional domain organization of budding yeast Spc98 and human GCP3. Both Spc98 and GCP3 share the conserved N-terminal domain (NTD), the GCP core body and the divergent, …
Figure 7—figure supplement 2
γ-TuSCSpc98Δ1–156 maintains self-oligomerization capability in BRB80 buffer.
(A) γ-TuSCSpc98Δ1–177 was purified from insect cells, incubated in TB150 buffer, and then analyzed by Superose 6 10/300. The left panel presents the Coomassie Blue stained SDS-gel of purified γ-TuSCS…
Figure 8 with 1 supplement
Two types of γ-TuCRs defined by the N-terminal γ-TuSC binding motifs: SPM-CM1 and CM1-only.
(A) Graphical representations of the patterns of CM1 motif within the multiple sequence alignment of γ-TuCR protein sequences. The CM1 motif sequence logos were shown for γ-TuCR protein sequences …
Figure 8—figure supplement 1
Raw sequence input for the sequence logo of CM2, Spc110 PACT, and Spc72 CM1 motifs.
(A) Multiple sequence alignment of selected γ-TuSC receptor family members containing CM2 motif. Residues are marked according to the ClustalX colour scheme. The occurrence of each amino acid in …
Figure 9
γ-TuCRs are classified into three subgroups based on N-terminal γ-TuSC binding motifs and C-terminal MTOC targeting motifs.
(A) Graphical representations of the patterns of C-terminal MTOC targeting motifs within the multiple sequence alignment of γ-TuCR protein sequences. The MASC and PACT motif sequence logos were …
Figure 10
Role of Spc110 phosphorylation during SPB duplication.
(A) MT nucleation by the SPB during the cell cycle. See ‘Discussion’ for details. (B) Cell cycle dependent, stimulatory phosphorylations of Spc110 by Mps1 and Cdk1-Clb5 (early S phase to early M). (C…
Author response image 1
Additional files
-
Supplementary file 1
Plasmids used in this study.
- https://doi.org/10.7554/eLife.02208.026
-
Supplementary file 2
Strains used in this study.
- https://doi.org/10.7554/eLife.02208.027
