Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication - PubMed (original) (raw)
Spc29p is a component of the Spc110p subcomplex and is essential for spindle pole body duplication
S Elliott et al. Proc Natl Acad Sci U S A. 1999.
Abstract
In yeast, microtubules are organized by the spindle pole body (SPB). The SPB is a disk-like multilayered structure that is embedded in the nuclear envelope via its central plaque, whereas the outer and inner plaques are exposed to the cytoplasm and nucleoplasm, respectively. How the SPB assembles is poorly understood. We show that the inner/central plaque is composed of a stable SPB subcomplex, containing the gamma-tubulin complex-binding protein Spc110p, calmodulin, Spc42p, and Spc29p. Spc29p acts as a linker between the central plaque component Spc42p and the inner plaque protein Spc110p. Evidence is provided that the calmodulin-binding site of Spc110p influences the binding of Spc29p to Spc110p. Spc42p also was identified as a component of a cytoplasmic SPB subcomplex containing Spc94p/Nud1p, Cnm67p, and Spc42p. Spc29p and Spc42p may be part of a critical interface of nucleoplasmic and cytoplasmic assembled SPB subcomplexes that form during SPB duplication. In agreement with this, overexpressed Spc29p was found to be a nuclear protein, whereas Spc42p is cytoplasmic. In addition, an essential function of SPC29 during SPB assembly is indicated by the SPB duplication defect of conditional lethal spc29(ts) cells and by the genetic interaction of SPC29 with CDC31 and KAR1, two genes that are involved in SPB duplication.
Figures
Figure 2
Genetic interactions between SPC29 and CMD1, SPC110, and SPC42. (A) The chromosomal SPC29 of _cmd1_-1, _spc110_-2, and _spc42_-9 (sectors “ts”) mutant or wild-type cells (sector “wt”) was deleted in the presence of a plasmid containing SPC29 (_URA3_-based). Growth of mutant and wild-type cells with the control plasmid pRS315 (sector “pRS315”), or _spc29_-2 (sectors “_spc29_-_2_”), _spc29_-3 (sectors “_spc29_-_3_”), or SPC29 (sectors “_SPC29_”) on pRS315 were tested at 23°C on plates containing 5-fluoroorotic acid (5-FOA), which selects against the URA3_-based pRS316-SPC29 plasmid. Lack of growth on 5-FOA in the presence of pRS315 (sector “ts/pRS315”) shows that SPC29 is an essential gene. All cells grew with SPC29 on pRS315 (sectors “_ts/SPC29 ” or “_wt/SPC29_”). The failure of _cmd1_-1, _spc110_-2, or _spc42_-9 cells to grow with pRS315–_spc29_-2 (ts/_spc29_-2) or pRS315–_spc29_-3 (ts/_spc29_-3) on 5-FOA indicates synthetic lethality. In contrast, wild-type cells with spc29-2 (“_wt/spc29-2_”) or spc29-3 (“_wt/spc29-3_”) on pRS315 grew on 5-FOA. As a control, the strains were complemented with CMD1, SPC110, or SPC42 (sector named “_ts/TS_”) on pRS315, which allowed growth on synthetic complete medium at 37°C (data not shown). However, these cells did not grow on 5-FOA, because SPC29 is essential for growth. (B) Two-hybrid interactions of Spc29p with C–Spc110p and N–Spc42p. The indicated pACT2 and pEG202 derivatives were transformed into the indicator strain SGY37. β-galactosidase activity was determined by using a plate assay (18). Blue colony color indicates interaction. (C) _SPC110_811–944 in plasmid pEG202 of wild type or of SPC110 mutants carrying the indicated mutations in the Cmd1p-binding site were tested in the two-hybrid system for interaction with SPC29 in pACT2. Cmd1p binding to the Spc110p mutants has been determined before by using an in vitro binding assay (18).
Figure 1
An Spc110p-containing SPB subcomplex. (A) An Spc110p-containing SPB subcomplex was purified from cells with a protein A-tagged SPC110 (ProA–SPC110) (lane 2) by using IgG Sepharose. The protein bands were subjected to matrix-assisted laser desorption ionization (MALDI) MS analysis. Wild-type cells, containing Spc110p without a ProA tag (lane 1), were treated identically to identify proteins that bound nonspecifically to the IgG resin. The major proteins are encoded by SPC110 (band 1, ProA–Spc110p homodimer; band 2, ProA-Spc110p; bands 3 and 4, ProA–Spc110p degradation products), SPC42 (band 5), SPC29 (band 6), and CMD1 (band 7). (B) Coimmunoprecipitation of Spc29p and Spc110p. High-salt extracts from cells expressing _SPC110 SPC29_–3HA (lane 1) or _ProA–SPC110 SPC29_–3HA (lane 2) were incubated with IgG Sepharose. The immunoblots were probed with the indicated antibodies.
Figure 3
Cellular localization of Spc29p and Spc42p. (A) Localization of Spc29p-GFP after its overexpression from the Gal1 promoter for 3 hours at 30°C was determined by using fluorescence microscopy. DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI). (B and C) Electron microscopic picture of an SPB of _mps2_-1 cells with (C) or without (B) overexpression of SPC42. Cells of _mps2_-1 with or without Gal1–SPC42 were shifted to 37°C for 30 min in 2% raffinose medium followed by the induction of the Gal1 promoter by the addition of 2% galactose. ∗ in C indicates the Spc42p polymer. (D and E) Electron microscopic picture of an SPB from cells with (E) and without (D) cooverexpression of SPC29, SPC42, and SPC110. Cmd1p is probably present in excess and was therefore not overexpressed. ∗ in E indicates the Spc42p polymer. C, central plaque; Cy, cytoplasm; HB, half bridge; I, inner plaque; NE, nuclear envelope; Nu, nucleus; O, outer plaque; S, satellite. (Bar = 0.25 μm.). B_–_E are all of same magnification.
Figure 4
Spc94p/Nud1p, Cnm67p, and Spc42p are present in common complexes. (A) A lysate of cells expressing SPC94–ProA (lane 1) was separated by centrifuging (40,000 × g, 30 min, 4°C) into a cytoplasmic extract (lane 2) and a SPB-containing pellet (lane 3). This pellet was extracted with 1 M NaCl/1% Triton X-100/50 mM Tris⋅HCl (pH 7.5) followed by centrifuging, resulting in a supernatant (lane 4) and a pellet (lane 5). Both extracts (lanes 2 and 4) were incubated with IgG Sepharose. The bound proteins were eluted (lanes 7 and 9). Lanes 6 and 8 are controls of wild-type cells that were treated identically to lanes 7 and 9, respectively. Fractions were tested by immunoblotting using antibodies directed against the indicated proteins. (B) Two-hybrid interactions between Spc42p, Cnm67p, and Spc94p/Nud1p. Experiment was performed as described in Fig. 2_B_ legend.
Figure 5
spc29( ts ) cells are defective in SPB duplication. Wild-type cells and cells of _spc29_-2 and _spc29_-3 were incubated for 2.5 hours at 23°C with 0.01 mg/ml α-factor. α-Factor was removed by washing with prewarmed (37°C) medium to release the cell-cycle block. Cells were incubated for 3 hours (B_–_H) or for up to 4 hours (A) at 37°C. (A) The DNA content of cells of SPC29, _spc29_-2, and _spc29_-3 was determined by fluorescence-activated cell sorting analysis (FACS) before release or 1, 2, 3 and 4 hours after release of the cell-cycle block. (B) Microtubules and the outer plaque protein Spc72p of _spc29_-2 and SPC29 cells were visualized by using indirect immunofluorescence. DNA was stained with DAPI. Wild-type cells (C) and cells of _spc29_-2 (D and E) and of _spc29_-3 (F_–_H) were prepared for electron microscopy followed by serial sectioning. Abbreviations are as in Fig. 3. (Bar = 0.25 μm.). C_–_H are all of same magnification.
Figure 6
Model for the functions of Spc29p. Kar1p, and Cdc31p are components of half bridge (5, 7) that function in SPB duplication (4, 6). Spc72p anchors the yeast γ-tubulin complex containing Tub4p, Spc98p, and Spc97p to the outer plaque (11). The yeast γ-tubulin complex is bound to the inner plaque via Spc110p (20). Spc42p forms a layer within the SPB (2). Cnm67p and Spc94p/Nud1p are components of the cytoplasmic outer plaque (26). Based on our results, we propose that Cnm67p binds to Spc42p and Spc94p/Nud1p interacts with Cnm67p. Spc29p is bridging C–Spc110p and N–Spc42p. During SPB duplication, Spc29p, N–Spc42p, and C–Spc110p form a critical border of cytoplasmic and nuclear assembled SPB subcomplexes. Spc42p may also be part of the satellite that forms in G1 phase of the cell cycle (3).
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