A Molecular Evolution Approach to Study the Roles of Tropomyosin in Fission Yeast

Abstract

Tropomyosin, a coiled-coil protein that binds along the length of the actin filament, is a universal regulator of the actin cytoskeleton. We have taken a bioinformatics/proteomic approach to studying structure-function relationships in this protein. The presence of a single, essential tropomyosin gene, cdc8, in fission yeast, Schizosaccharomyces pombe, enables a systems-based approach to define the residues that are important for cellular functions. Using molecular evolution methodologies we identified the most conserved residues and related them to the coiled coil structure. Mutants in which one or more of 21 of the most conserved surface residues was mutated to Ala were tested for the ability to rescue growth of a temperature-sensitive cdc8 mutant when overexpressed at the restrictive temperature. Based on altered morphology of the septum and actin cytoskeleton, we selected three sets of mutations for construction of mutant cdc8 strains using marker reconstitution mutagenesis and analysis of recombinant protein in vitro: D16A.K30A, V114S.E117A.H118A and R121A.D131A.E138A. The mutations have sequence-specific effects on cellular morphology including cell length, organization of cytoskeletal structures (actin patches, actin cables and contractile rings), and in vitro actin affinity, lending credence to the proteomic approach introduced here. We propose that bioinformatics is a valid analysis tool for defining structure-function relationships in conserved proteins in this model organism.

Myosin motor isoforms direct specification of actomyosin function by tropomyosins

“…These three positions are not involved with the folding and stability of the coiled‐coil and are consequently available for association with other proteins. Similar conserved acidic and basic residues are also found at b , c , and f positions in Cdc8p and other fungal tropomyosins (Cranz‐Mileva et al, ). Interestingly, amino acid substitutions at some of these sites in Cdc8p were recently found to compromise actin association and in vivo function (Cranz‐Mileva et al, ).…”

“…This is surprising given the large evolutionary distance between yeast and mammals reflected by the limited homology between tropomyosins from these two distinct systems. This regulation most likely reflects some conserved features of the tropomyosin sequence in general, which appear to extend from human to yeast tropomyosins (Cranz‐Mileva et al, ). Striated muscle α‐tropomyosin/Tpm1.1st possesses a conserved periodic series of acidic and basic residues that establish association with actin (Hitchcock‐DeGregori et al, ; Singh and Hitchcock‐DeGregori, ; Barua et al, ), while other conserved acidic residues on the tropomyosin surface influence SKMM‐II regulation (Oguchi et al, ; Barua et al, ).…”

Tropomyosin 3.5 protects the F-actin networks required for tissue biomechanical properties

“…Tropomyosins (Tpms) are conserved F-actin-binding proteins that stabilize filaments and regulate their interactions with a variety of actin-binding proteins, including cofilin/ADF filament-severing proteins, α-actinin and fimbrin (plastin) filament-crosslinking proteins, tropomodulin (Tmod) filament minus ('pointed')-endcapping proteins and force-producing myosin motors (Christensen et al, 2017;Gunning et al, 2015; Hitchcock-DeGregori and Barua, 2017;Kostyukova, 2008;Nakano and Mabuchi, 2006;Ono and Ono, 2002;Winkelman et al, 2016;Yamashiro et al, 2012). Tpms are expressed in all animals and fungi (Barua et al, 2011;Cranz-Mileva et al, 2013), and in mammals, Tpms are expressed from four genes with alternative splicing that produces more than 40 variants in different tissues (Geeves et al, 2015;Pittenger et al, 1994;Vindin and Gunning, 2013). In vitro studies demonstrate that F-actin assembly, elongation and disassembly rates depend on Tpm (Gunning et al, 2015; Hitchcock-DeGregori and Barua, 2017) and that Tpm isoforms direct assembly of different types of F-actin populations (Gateva et al, 2017;Janco et al, 2016).…”

A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion

“…We then took opportunity of a collection of point mutations in predicted surface--exposed Cdc8 residues conserved in fungi [41,42] to screen for non--conditional mutants that would hinder cell fusion when homothallic. This identified two alleles each carrying a single point mutation, cdc8 R121A and cdc8 E104A , with reduced fusion efficiency ( Figure 5D).…”