Interaction of yeast importin alpha with the NLS of prothymosin alpha is insufficient to trigger nuclear uptake of cargos

A proliferation-related human protein prothymosin alpha displays exclusivel y nuclear localization when produced in human and Saccharomyces cerevisiae cells, whereas its isolated bipartite NLS confers nuclear targeting of the GFP reporter in human but not in yeast cells. To test whether this observat ion is indicative of the existence of specific requirements for nuclear tar geting of proteins in yeast, a sea of prothymosin alpha deletion mutants wa s constructed. Subcellular localization of these mutants fused to GFP was d etermined in yeast and compared with their ability to bind yeast importin a lpha (Srp1p) in vitro. The NLS of prothymosin alpha turned out to be both n ecessary and sufficient to provide protein recognition by importin alpha. H owever, the NLS-importin alpha interaction did not ensure nuclear targeting of prothymosin alpha derivatives. This defect could be complemented by add ing distinct prothymosin alpha sequences to the NLS-containing import subst rate, possibly by providing binding site(s) for additional components of th e yeast nuclear import machinery. (C) 2000 Academic Press.