Expression of Reticulomyxa filosa tubulins in Pichia pastoris: regulation of tubulin pools

Abstract

We expressed the α2- and β2-tubulin isoforms of the giant freshwater amoeba Reticulomyxa filosa in the methylotrophic yeast Pichia pastoris. Single expression lead to little or no detectable material. Coexpression of both tubulins, however, resulted in a significant increase of expressed proteins. At the same time, the detectable internal tubulins of the host yeast cell were downregulated. This finding indicates the functionality of the expressed amoeba tubulins. Further regulation phenomena were observed on the level of equilibrium between the two R. filosa tubulin isoforms and on the level of the total tubulin pool. The P. pastoris/ R. filosa system therefore seems to be an accessible system for the simultaneous study of the various mechanisms involved in tubulin regulation.