Expression cloning in Fe2+ transport defective yeast of a novel maize MYC transcription factor.

Abstract

A complementation approach of the yeast fet3fet4 mutant strain, defective in both low- and high-affinity iron transport, was initiated as an attempt to characterize the Fe(III)-mugineic acid (MA) transporter from grasses. A maize cDNA encoding a novel MYC transcription factor, named 7E, was cloned by screening an iron-deficient maize root cDNA expression library on a minimum media containing Fe(III)-deoxyMA as a unique iron source. 7E expression restored growth specifically to the fet3 fet4 mutant strain. It did not affect growth rate of a trk1trk2 potassium transport defective yeast strain or parental W303 strain growth rate. No 55Fe uptake increase was observed in 7E transformed fet3 fet4 yeast during short-term kinetics. However, the iron accumulation in these cells was 1.3-fold higher than in untransformed cells after a 24-h period. The 7E protein contained 694 amino acids and had a predicted molecular mass of 74.2kDa. It had 44% identity with the RAP-1 protein, a 67.9-kDa MYC-like protein from Arabidopsis thaliana which binds the G-box sequence via a basic region helix-loop-helix (bHLH), without requiring heterodimerization with MYB proteins. Phylogenic comparisons revealed that the maize 7E protein was related to the Arabidopsis thaliana RAP-1 protein and to the Phaseolus vulgaris PG1. This similarity was particularly evident for the bHLH domain, which was 95% identical between maize 7E and Arabidopsis thaliana RAP-1. 7E, RAP-1 and PG-1 proteins revealed a plant MYC-like sub-family that was more related to the maize repressor-like IN1 than to maize R proteins. 7E mRNA was detected in both roots and leaves by the Northern analysis. The amount of 7E mRNA increased, in response to iron starvation, by 20 and 40% in roots and leaves, respectively. The relationship between iron metabolism and myc expression in animal cells is discussed.