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The DCC is essential for Spc42 stability. A Left, schematic of Spc42 variant constructs. The ability to rescue the SPC42 deletion was tested as for Figure 3G ; the same phenotype was seen at all temperatures unpublished data. B Schematic showing smaller deletions within the first coiled-coil as well as replacement constructs that have heptad repeats from the coiled-coil of myosin. D Incorporation of constructs into a superplaque were quantitated as for Figure 3C.

E Levels of overexpressed protein from were analyzed as for Figure 3A. F Heptad repeats from the coiled-coil of myosin were added within the DCC to examine how increased length would affect Spc42 function, using a dilution assay as in A. Collectively, these data suggest that the first coiled-coil is critical for Spc42 function in vivo.

The ability of myosin to restore Spc42 assembly, but not SPC42 function, points to the possibility that the DCC is more than a structural linker between the central plaque and IL2. Consistent with idea, we found that extension of the DCC cannot be tolerated Figure 6F , with the exception of a residue myosin coiled-coil inserted between residues 80 and 81 at the end of the structural region of the DCC.

This implies that the functional interaction of the DCC with components of the central plaque and IL2 can be mostly associated with two distinct segments: 60—80 and 81— We further demonstrate that the DCC is not needed for the formation of a hexagonal array in vitro, but is functionally essential in vivo, which implies a structural redundancy. Moreover, our data reveal that Spc42 residues — are sufficient for lattice formation in vitro and, with the addition of UR4, for superplaque assembly in vivo, suggesting that the TCC, UR3, and ACC are major contributors to the hexagonal array observed at the SPB.

This dichotomy indicates that multiple interactions are important for SPB integrity. Two models were proposed by Bullitt et al. Spc42 was predicted to have three coiled-coil domains, but only the N-terminal DCC was found to self-associate in a fluorescent resonance energy transfer assay of coiled-coil peptides Zizlsperger et al. This observation led to preference for the trimer-of-dimers model, wherein three DCCs trimerize at the N-termini but split at the C-termini into six polypeptide chains that interact with adjacent units to form a hexagonal lattice Muller et al.

Evolution of interpretation of Spc42 architecture.

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A The trimer-of-dimers model, postulated before the structural data presented in this article, placed 12 Spc42 monomers in the hexagonal unit cell. In this model, the DCC, shown as blue spheres, form trimers of dimers, while the C-terminal regions of the protein, shown as green half-ellipsoids, dimerize with adjacent C-terminal regions Muller et al.

B Schematic representation of the P6 2D space group. C The TCC model incorporates the symmetry elements discerned from the crystallographic data presented here into a hexagonal lattice. In this model, the DCCs, shown as blue spheres, form dimers, while the TCCs, shown as green spheres, trimerize, and the ACCs, shown as yellow bars, dimerize in an antiparallel conformation.

This orientation requires only 6 Spc42 monomers in the hexagonal unit cell. Undetermined regions URs of Spc42 are not included in this model. Although the trimer-of-dimers model has been the prevailing model in the field for more than 20 yr Bullitt et al. Indeed, as shown here, the DCC is not necessary for Spc42 array formation in vitro or for superplaque assembly in vivo.

From Coil-Supported Seating to Coiling Storage Spaces

From first principles, a 2D lattice that exhibits sixfold symmetry and belongs to space group P6, as observed by Bullitt et al. The minimum number of protein subunits chains needed to fill the unit cell of this lattice is six. These six subunits must interact with one another around these axes of symmetry.

This arrangement of subunits generates a lattice with sixfold symmetry. In the earlier model Figure 7A , it was proposed that three DCCs six protein chains would be arranged around each threefold axis Muller et al. Because there are two threefold axes in each unit cell, this arrangement places 12 protein subunits into the cell.

The six alpha helices that make up the three DCCs arranged around a threefold axis would not exhibit sixfold symmetry. This model also suggested that two pairs of protein subunits a total of four chains would be related to each other across the twofold axes, as seen in Figure 7A. In any event, the observed twofold symmetry of the DCC is not used in the generation of the lattice. Our data from crystallographic structures, EM images of reconstructed Spc42 arrays, and analysis of superplaque formation in vivo are consistent with the TCC model Figures 7C and 8, A and B.


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In our hands, the real unit cell length derived from Bullitt et al. TCC model of Spc42 lattice organization.

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UR2—4 are depicted in gray. Both interpretations incorporate the symmetry elements in similar ways, but with differing connectivity; current data do not distinguish between these models. B Overhead view of the model incorporating an intramolecular role for the ACC, overlaid on the small globular fusion protein Gp7. Recall that these fusion proteins are necessary for improved solubility and stability of Spc42 constructs and do not affect the arrangement of Spc Unit cell dimensions were determined from single-particle analysis of EM images see Materials and Methods ; Table 2. C Spc42 array reconstruction by Bullitt et al.

Because these blocking domains lie beneath the segments of Spc42, it is not expected that they will interfere with the assembly of Spc Consistent with this conjecture, the lattice dimensions are independent of the size of the fusion domain. As noted earlier, distinguishing feature between the TCC and trimer-of-dimers models centers on Spc42 stoichiometry. The volume and dimensions of our fusion proteins, as well as GFP fusions in vivo, reveal that, while packing 6 protein subunits in the unit cell as a protein monolayer obeys sixfold symmetry Figure 8, D and E , it is difficult to conceive how 12 fusion protein subunits could be accommodated and use the observed Spc42 DCC, TCC, and ACC domains.

Based on the earlier model, this would allow for six copies of Spc29, Spc, and calmodulin in the central plaque, which is also thought to have hexagonal symmetry Bullitt et al. Given that the central plaque must contain six copies of each protein per unit cell to exhibit sixfold symmetry, our model suggests there must be a ratio between Spc42 and the other components.

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A recent study of nuclear envelope—associated nuclear pore complexes illustrates the caveats of calibrated GFP imaging in large structures in yeast Kim et al. This, combined with issues surrounding folding and maturation kinetics of fluorescent proteins within the SPB Lengefeld et al. It is possible that both Spc42 and Spc29 exist in the core and on the edge, tethering the soluble core to membrane components of the SPB.

Curiously, orthologues of Spc, calmodulin, and Cnm67 can be readily identified by sequence homology in virtually all fungal lineages and in many metazoans Ito and Bettencourt-Dias, In contrast, Spc42 and Spc29 are only found in closely related Saccharomycetaceae. In Schizosaccharomyces , the roles of both Spc29 and Spc42 appear to be fulfilled by a single protein, Ppc89 Rosenberg et al.

Homologues of Ppc89 can be found across Ascomycota, Pezizomycotina, and Taphrinomycotina yeasts, but are not found in Saccharomycotina, which contain both Spc29 and Spc This suggests that the function of Ppc89 split into two proteins during evolution and likely indicates that Spc29 and Spc42 function in a complex, similar to the ancestral Ppc While the requirements for superplaque formation in vivo and assembly of ordered array in vitro correlated in many cases, there were differences that are likely biologically significant. This is not in conflict with our structural models, because those components identified to be necessary for assembly are a subset of those required for function and, most likely, interaction with other components of the SPB.

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Our data suggest that the DCC is not the architect of hexagonal symmetry; however, it is more than just a linker between the central plaque and IL2. In doing so, the array formed by Spc42 could transmit hexagonal symmetry to the central plaque, as originally proposed Bullitt et al. In summary, structural analysis of the coiled-coil domains of Spc42 provides novel insight into the organization of this key scaffold at the SPB. The model for Spc42 organization is the simplest arrangement that incorporates the symmetry of the structural domains, their assembly properties in vitro, and their biological necessity in vivo.

The fact that multiple structural domains within Spc42 contribute to its organization illustrates the redundancy used by cells to ensure SPB integrity. Plasmids were assembled with QuikChange cloning such that sections of a gene of interest were directly inserted into a vector by linear amplification van den Ent and Lowe, ; Rocco et al.

All constructs contained an N-terminal H 6 -tag followed by a recombinant tobacco etch virus rTEV cleavage site Blommel and Fox, that lead into a coiled-coil solubilizing fusion protein Frye et al. The propensity and registration of coiled-coils in Spc42 and the predicted coiled-coil registration across the construct was assessed using the COILS algorithm Lupas, Sequences of all constructs were verified throughout the open reading frame Supplemental Table S1. Spc42 fusion proteins were expressed in E.


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The protein was eluted in 6 column volumes of buffer C buffer A, but generally with mM imidazole; Supplemental Table S1. Spc42 fusion constructs were eluted in 4 column volumes of buffer A, after which rTEV protease was eluted in buffer E buffer A, but with mM imidazole. For EM studies, the H 6 -tag was not removed. The exact storage buffers differed according to the stability of each construct and are listed in Supplemental Table S1.

Hexagonal crystals grew overnight. Layered rectangular sheet crystals spontaneously formed over roughly 1 wk. Rod-shaped hexagonal crystals formed over 1—3 d. Crystals were rapidly flash-frozen in liquid nitrogen. The x-ray data collection statistics are given in Table 1. Final refinement of both structures was done in Phenix Adams et al.

Subsequent cycles of refinement were executed using Refmac 5. Final refinement was done using Phenix. Refinement statistics are given in Table 1. Trilution LC 3. The 2D arrays of various Spc42 constructs were assembled with the aid of lipid monolayers following the procedures described previously Levy et al. Each was placed in a 6- or cm glass Petri dish with a wet circle of Whatman 2 filter paper cut to size.

The plate was incubated at room temperature for 1 h to allow for monolayer formation.