A regulator of nutritional copper signaling in Chlamydomonas is an SBP domain protein that recognizes the GTAC core of copper response element

Abstract

The CRR1 (Copper Response Regulator) locus, required for both activating and repressing target genes of a copper- and hypoxia-sensing pathway in Chlamydomonas, encodes a 1,232-residue candidate transcription factor with a plant-specific DNA-binding domain named SBP, ankyrin repeats, and a C-terminal Cys-rich region, with similarity to a Drosophila metallothionein. The recombinant SBP domain of Crr1 shows zinc-dependent binding to functionally defined copper-response elements associated with the CYC6 and CPX1 promoters that contain a critical GTAC core sequence. Competition experiments indicate equivalent selectivity for copper-response elements from either promoter and 10-fold greater selectivity for the wild-type sequence vs. a sequence carrying a single mutation in the GTAC core. The SBP domain of Chlamydomonas Crr1 binds also to a related GTAC-containing sequence in the Arabidopsis AP1 promoter that is the binding site of a defining member of the SBP family of DNA-binding proteins. Chlamydomonas Crr1 is most similar to a subset of the Arabidopsis SBP domain proteins, which include SPL1, SPL7, and SPL12. The abundance of the CRR1 mRNA is only marginally copper-responsive, and although two mRNAs that differ with respect to splicing of the first intron are detected, there is no indication that the splicing event is regulated by metal nutrition or hypoxia. It is likely that the dramatic copper-responsive action of Crr1 occurs at the level of the polypeptide.

Fig. 1.

Rescue of crr1 growth by the cloned gene. (A) Growth of strains on TAP medium with (+) or without (-) 6 μM supplemental copper. The strains [wild-type (CC125), crr1-1, crr1-2, and a complemented crr1-1 strain (crr1-1:B)] were grown for 1 week at 20°C under 100 to 125 μmol photons·m^-2·s^-1. (B) Fluorescence rise and decay kinetics from strains grown without supplemental copper.

Fig. 2.

Rescue of all crr1 expression phenotypes by the cloned gene. Total RNA was prepared from the indicated allelic mutant strains crr1-1 and crr1-2, a wild-type strain (CC425), or strains rescued with either the 9,201-bp EcoRI fragment (:E) or the 6,096-bp BamHI fragment (:B), and analyzed for the expression of target genes CYC6, CPX1, CRD1/CHL27A, or CTH1/CHL27B and a loading control CBLP by hybridization (specific activity of probes were in the range 2.4 to 11 × 10⁸ cpm/μg DNA). CTH1 produces two transcripts, the 2-kb form (white arrow) in +Cu cells encodes the protein, whereas the 3-kb form (black arrow) occurs only in -Cu cells dependent on Crr1 function. (A) The strains were grown in copper-supplemented (+) or copper-deficient (-) TAP medium. (B) The strains were grown in +Cu TAP medium. NiCl₂ was added (+) or not (-) to a final concentration of 25 μM, and RNA was isolated 5 h later. (C) The strains were grown at room temperature on a shaker with aeration (100% air) (+O₂) or were transferred for 24 h to hypoxic conditions (96%N₂/2% air/2% CO₂)(-O₂) before RNA isolation.

Fig. 3.

Structure of the CRR1 locus. (A) Structure of the CRR1 gene. The EcoRI (E) and BamHI (B) sites are indicated. The double-headed arrow indicates the length scale (500 bp). The gray rectangles indicate exons, and the thin lines intervening indicate introns. The numbers mark the corresponding amino acids encoded by the exons. The dashed line indicates an “inefficient” splicing event (see text). The diagram of the mRNA is shown below with the ORF indicated from ATG to a TAA codon. (B) Diagram of the protein. The numbers mark the amino acyl residues in the protein. The red box marks the DNA-binding SBP domain, and the pale pink box indicates an extended region of conservation consisting of a conserved sequence WL(X)₃P(X)₃E(X)₂IRPGC found in a subset of SBP domain proteins in Arabidopsis and rice. A putative nuclear localization signal is indicated in green within the SBP domain. Ankyrin repeats are shown as purple arrows, and a C-terminal cysteine-rich sequence is in turquoise. A gray rectangle at the N terminus of the protein represents the core of a region that shows sequence characteristics of an “AHA motif” found in a subset of SBP domain proteins. In strains crr1-1 and crr1-2, there is a single nucleotide deletion at codon 895 or 780, respectively, resulting in a frame shift and, presumably, premature termination of translation.

Fig. 4.

Expression of CRR1. RNA blot hybridization. RNA was isolated from copper-supplemented (+) or copper-deficient (-) wild-type (CC425), crr1-1, crr1-2,or crr1-1 complemented with the 6-kb BamHI genomic DNA fragment (crr1-1:B) strains and analyzed for CRR1 expression by using fragment A (see Fig. 7) as a probe for hybridization. CβLP was used as a loading control. The size of the CRR1 mRNA was estimated by comparison to size markers (0.24–9.5 kb RNA ladder, Invitrogen). The difference in mRNA abundance between mutant and wild-type strains evident in this figure was not observed in six other experiments (e.g., Table 1).

Fig. 5.

The SBP domain of Crr1 binds to the CuRE. (A) Recombinant His-6-Crr1-SBP was incubated with radiolabeled DNA corresponding to a CuRE (position -134 to -110) from the CYC6 promoter (lanes 2–10) in the presence of increasing concentration (5×, 10×, 50×, and 100× molar ratio) of unlabeled wild-type binding site (lanes 3–6) or a binding site carrying a T to A change in the GTAC core (lanes 7–10). (B) Radiolabeled DNA representing the CuRE from the CPX1 promoter (position -49 to -26 relative to the 5′ end of the longest CPX1 mRNA) (lanes 2–10) was incubated with increasing concentrations (5×,10×,50×, and 100× molar ratio) of unlabeled wild-type binding site (lanes 3–6) or a mutated binding site where the nucleotides of the GTAC core were rearranged to ACTG (lanes 7–10). The specific band corresponding to the DNA–protein complex is marked with an arrow. The other band is probably a primer-trimer.

Fig. 6.

Alternative models for copper-responsive modification of Crr1 function. (A) In copper-deficient cells, Crr1 binds the CuRE through the SBP and activates transcription by interaction with the transcription apparatus (double-headed arrows). In copper-replete cells, the structure of Crr1 is modified, either by binding of copper to the SBP domain (B) or to the C-terminal Cys-rich region (C), such that the protein no longer binds the CuRE or is no longer able to activate transcription.