Tropomyosins, N-terminal acetylation and their impact on yeast cytoskeletal function: A characterisation of novel tropomyosins from N. crassa and the N-terminal acetyltransferase, Nat3p

Abstract

PhDWhile the fundamental role of tropomyosins (Tms) in the maintenance of the actin cytoskeleton in yeast is established, details of their exact regulatory functions in lower eukaryotes remains to be deciphered. Here, two novel Tms have been identified from the filamentous yeast Neurospora crassa: a 161 residue protein spanning 4 actin monomers (crTm161p), and a 123 residue protein which spans 3 actin monomers (crTm123p). The latter isoform is the shortest naturally occurring Tm known. The isoforms are produced as a result of alternative splicing from a single gene- a phenomenon that has not previously been observed in yeast Tms. Both Tms were cloned, purified and crystallised. They were also characterised biochemically and biophysically, giving some insight into their role in fungal cytoskeleton regulation. The crystals produced provide the potential for future structural studies, as a high resolution structure of a complete Tm is still not available. The N-terminal acetylation of Tms is essential for their function, and is catalysed in Saccaromyces cerevisiae by the N-terminal N-acetyltransferase (NAT) Nat3p. Nat3p was expressed and purified. Its functionality was investigated via acetyl coenzyme A binding assays. The molecular structure of Nat3p was modelled using existing data from structural homologues. The closely related N-terminal NAT, Nat5p, was also expressed, purified and its structure modelled. Nat3p was largely insoluble while Nat5p was soluble and was successfully crystallised. Structural insights from molecular modelling were able to provide some justification for these differences. Finally the in vivo effects of genetic knockouts of the TPM1 and NAT3 genes in yeast were analysed quantitatively. Complementation of the defective knockout phenotypes by over-expression of various Tm and Nat3p constructs was also investigated. Quantifying the overlapping phenotypes of the NAT3Δ and TPM1Δ mutaunts has clarified their distinct impacts upon the cytoskeleton. The ability of the crTms to rescue TPM1Δ phenotypes implies they have roles similar to those of Tpm1p