Neurospora Crassa possesses a novel ultra short tropomyosin

Abstract

Tropomyosins are alpha-helical coiled coil proteins. They interact with actin filaments; binding along the major grooves, forming a continuous filament along the actin strand. In higher eukaryotes their most well understood role is in the regulation of muscle contraction, where they regulate the myosin II - actin interaction that generates force. Their non-muscular functions are not well characterised, and in lower eukaryotes regulatory function is less clear. However, it has been clearly shown they are fundamental to maintaining the actin cyoskeleton in yeast. We have identified that Neurospora crassa possess two tropomyosins: a 161 residue, 4 actin spanning protein, and a 123 residue, 3 actin spanning protein. The latter isofrom is shorter than any previously identified naturally occurring tropomyosin. The only tropomyosin of similar size previously characterised was an artificial construct that was shown to possess novel acto-myosin regulatory properties. The two isoforms are produced by alternative splicing from a single gene. We have shown that the shorter isoform is expressed at much lower levels than the larger one. We have cloned the two tropomyosins and characterised their actin binding and biophysical properties. As has been found with other recombinantly expressed tropomyosins, both isoforms need the addition of an N-terminal Ala-Ser dipeptide to bind to actin. This replaces the function of the N-acetyl group present in native tropomyosins and it is hence presumed the native forms are acetylated. As flexibility has been intimately related to tropomyosin function, we determined the thermal stability of these novel tropomyosins using circular dichroism. Surprisingly this was found to be significantly higher than that of the 161 residue S.cerevisiae and S.pombe tropomyosins, and the artificial 123 residue S.cerevisiae construct. We are currently assessing whether this indicative measurement is reflected in the acto-myosin regulation of reconstituted thin-filaments.