Non-AUG translation initiation of a fungal RING finger repressor involved in photocarotenogenesis

The RING finger protein CrgA acts as a negative regulator of light-induced carotene biosynthesis in the fungus Mucor circinelloides. Sequence analysis of the crgA coding region upstream of the first AUG codon revealed the existence of an additional non-canonical RING finger domain at the most N-terminal end of the protein. The newly identified RING finger domain is required for CrgA to regulate photocarotenogenesis, as deduced from site-directed mutagenesis experiments. The role of both RING finger domains in the stability of CrgA has been investigated in a yeast system. Wild type CrgA, but not the RING finger deleted forms, is highly unstable and is stabilized by inhibition of the proteasome function, which suggests that native CrgA is degraded by the proteasome and that active RING finger domains are required for proteasome-mediated CrgA degradation. To identify the translation start of CrgA, a mutational analysis of putative initiation codons in the 5' region of the crgA gene was accomplished. We demonstrated that a GUG codon located upstream of the first AUG is the sole initiator of CrgA translation. To our knowledge, this is the first report of a naturally occurring non-AUG start codon for a RING finger regulatory protein. A combination of suboptimal translation initiation and proteasome degradation may help to maintain the low cellular levels of CrgA observed in wild type cells, which is probably required for accurate regulation of photocarotenogenesis.