Abstract
γ-Tubulin has a well-established role in nucleating the assembly of microtubules, yet how phosphorylation regulates its activity remains unclear. Here, we use a time-resolved, fitness-based SGA approach to compare two γ-tubulin alleles, and find that the genetic interaction profile of γtub-Y362E is enriched in spindle positioning and cell polarity genes relative to that of γtub-Y445D, which is enriched in genes involved in spindle assembly and stability. In γtub-Y362E cells, we find a defect in spindle alignment and an increase in the number of astral microtubules at both spindle poles. Our results suggest that the γtub-Y362E allele is a separation-of-function mutation that reveals a role for γ-tubulin phospho-regulation in spindle alignment. We propose that phosphorylation of the evolutionarily conserved Y362 residue of budding yeast γ-tubulin contributes to regulating the number of astral microtubules associated with spindle poles, and promoting efficient pre-anaphase spindle alignment.
Highlights
The synthetic genetic interactions (SGIs) profile for the γtub-Y362E mutation showed convergence with the γtub-Y445D mutant for a subset of genes involved in mitosis, and had a distinct set of SGIs with genes acting in spindle positioning and alignment relative to the future plane of cell division, as well as cell polarity
The viability of tub[4] alleles and their dependence on SAC activity was tested by analysis of progeny obtained from diploids heterozygous for γ-tubulin alleles γtub-S360D, γtub-Y445D or γtub-Y362E and the mad2∆ null mutation which blocks SAC activation. γ-Tubulin mutations S360D, Y445D and Y362E are viable under normal growth conditions (25–30 °C), with a weak growth defect observed for γtub-S360D and γtubY445D (16,17 and Fig. 1c)
SGIs specific to γtub-Y362E included components of the polarisome and actin cables that are critical for both Kar[9] and dynein dependent spindle positioning. These results suggested that the γtub-Y362E mutation likely perturbs a function of γ-tubulin that is directly linked to spindle positioning and actin organization rather than the previously characterized role in spindle assembly
Summary
An SGA-based approach for detection of separation-of-function mutations must be sensitive to differences in relative fitness of both single and double mutants, and be precise in measurements such that SGI profiles for multiple alleles of the same gene can be compared. The SGI profile for the γtub-Y362E mutation showed convergence with the γtub-Y445D mutant for a subset of genes involved in mitosis, and had a distinct set of SGIs with genes acting in spindle positioning and alignment relative to the future plane of cell division, as well as cell polarity. Our results provide new evidence that the phosphorylation of an evolutionarily conserved tyrosine residue (Y362) contributes to γ-tubulin function in controlling the number of astral MTs associated with both SPBs of the metaphase spindle, and influences the efficiency of pre-anaphase spindle alignment relative to the future plane of cytokinesis
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