Abstract
Tropomyosin is a coiled coil protein that binds and regulates actin filaments. Actin is highly conserved and we reasoned that tropomyosin residues critical for binding and regulation of actin would also be conserved. In the fission yeast, S. pombe, there is a single tropomyosin (SpTm) that is essential for cell division, cable formation, endocytosis, vesicle movement and mating. We mutated 21 surface-exposed SpTm residues that are evolutionarily-conserved in fungi. The resulting tropomyosin variants were tested to identify the contribution of the respective residues to the cellular function of tropomyosin. Overexpression of any of the mutants rescued growth of a cdc8-temperature sensitive strain (cdc8-27) at the restrictive temperature. Rescued cells were analyzed for abnormalities and we selected SpTm-16/30 (E16A, K30A), SpTm-114/117/118 (V114S, E117A, H118A) and SpTm-121/131/138 (R121A, D131A, E138A) for further study. Actin affinity was measured by cosedimentation assays with bacterially-expressed proteins. Actin affinity of SpTm-16/30 was similar to SpTm-wt, but affinity of SpTm-114/117/118 and SpTm-121/131/138 was too weak to measure using cosedimentation. We created three mutant fission yeast strains by replacing the endogenous tropomyosin gene with the variants. In this approach the protein is expressed at endogenous levels under cellular control. All strains were viable. The strains were analyzed for changes in cell size and shape, nuclear number and appearance of actin structures. All three strains showed altered appearance of actin structures. The strain SpTm-16/30 had very few actin cables. In SpTm-114/117/118 very few actin patches were visible; cables are abundant but abnormal. Expression of SpTm-121/131/138 caused abnormalities in contractile ring assembly, cables were nearly absent and the polar location of actin patches during interphase was disrupted. These results highlight that different regulatory functions can be traced to specific regions of tropomyosin. Supported by NIH.
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