Functionally testing the localization and oligomerization of ASR1, a stress-inducible protein

MARTINIANO M. RICARDI; RODRIGO M. GONZALEZ; JOSE M. ESTEVEZ; NORBERTO D. IUSEM

San Luis

Congreso; XLVII Reunión Anual de la Sociedad Argentina de Investigación en Bioquímica y Biología Molecular; 2011

Sociedad Argentina de Investigacion dne Bioquimica y Biologia Molecular

The ASR protein family is present in numerous economically relevant crops but absent in Arabidopsis. We explored two independent in vivo features of ASR1, one of its members, a small 14-kDa polypeptide, by means of transient expression in Nicotiana benthamiana. 1) ASR1 from tomato has a putative nuclear localization signal (NLS). However, it was detected not only in nucleus-enriched fractions but also in cytosolic fractions of tomato leaves and roots. This prompted us to test the functionality of its NLS by fusing ASR1 constructs to either the rather small GFP (green fluorescent protein) or the larger GUS-GFP chimera. Whereas the ASR-GFP fusion proteins readily entered the nucleus, GUS-GFP did not, even when attaching the NLS from ASR1. Therefore, nuclear localization of native ASR1 cannot be attributed to that amino acid tract but rather to simple diffusion. As a control, a proven viral NLS was able to target most of the GUS-GFP fluorescence to the nucleus. We thus conclude that the previously so-called “NLS” of ASR1 is not such. 2) Recombinant ASR1 can form homo-dimers in vitro. Its ability for in vivo oligomerization was tested by means of Bimolecular Fluorescence Complementation (BiFC) using split non-fluorescent variants of EYFP, each fused to ASR1. Oligomerization was inferred since fluorescence was evident only when both ASR1-fused halves were present at the same time.