[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: Mucor circinelloides responds to blue light by activating the biosynthesis of carotenoids. Gene crgA acts as a repressor of this light-regulated process, as its inactivation leads to overaccumulation of carotenoids in both the dark and the light. The predicted CrgA protein contains different recognizable structural domains, including a RING-finger zinc-binding motif, several glutamine-rich regions, a putative nuclear localization signal and an isoprenylation domain. To gain insight into the specific mode of action of the CrgA protein, we sought to define the CrgA domains critical for the light regulation of carotenogenesis. For this, mutant crgA alleles harbouring missense or deletion mutations in conserved residues of those domains were generated, and their functionality was assessed by testing their ability to complement a null crgA mutation. Point mutations of the amino-terminal RING-finger domain abrogated the ability of CrgA to repress carotenogenesis in the dark, as did the deletion of a poly glutamine-rich region at the carboxyl domain of CrgA. In contrast, mutations of the isoprenylation domain only slightly affected the CrgA function in carotenogenesis. The results identify two functional domains presumably involved in protein-protein interaction in the CrgA protein and suggest a role for the ubiquitin-proteasome pathway in the light regulation of carotenogenesis in fungi.
[Show abstract][Hide abstract] ABSTRACT: Mucor circinelloides responds to blue light by activating carotene biosynthesis. Wild-type strains grown in darkness contain minimal amounts of beta-carotene because of the low levels of transcription of the structural genes for carotenogenesis. When exposed to a light pulse, the level of transcription of these genes increases strongly, leading to the formation of high concentrations of beta-carotene. The crgA gene is involved in the regulation of light-induced carotenoid biosynthesis. This gene, originally identified as a 3'-truncated ORF which causes carotene over-accumulation in the dark, encodes a protein with a cysteine-rich, zinc-binding, RING-finger motif, as found in diverse groups of regulatory proteins. The expression of the crgA gene is activated by a light pulse, with a time course similar to that of the structural genes for carotenogenesis. To understand the regulatory role of the crgA gene in carotenogenesis, we have used a genetic approach based on the construction of crgA null mutants by gene replacement. Lack of the crgA function provokes the over-accumulation of carotenoids both in the dark and the light. Introduction of the wild-type crgA allele into these mutants restores the wild-type phenotype for carotenogenesis. The high levels of carotenoid accumulation shown by the null crgA mutants are correlated with an increase in the expression of carotenogenic structural genes. These results strongly indicate that crgA acts as a negative regulator of light-inducible carotenogenesis in M. circinelloides.