Animals regulate iron metabolism largely through the action of the iron regulatory proteins (IRPs). IRPs modulate mRNA utilization by binding to ironresponsive elements (IRE) in the 5 or 3 untranslated region of mRNAs encoding proteins involved in iron homeostasis or energy production. IRP1 is also the cytosolic isoform of aconitase. The activities of IRP1 are mutually exclusive and are modulated through the assembly͞disassembly of its [4Fe-4S] cluster, reversibly converting it between an IRE-binding protein and cytosolic aconitase. IRP1 is also phosphoregulated by protein kinase C, but the mechanism by which phosphorylation posttranslationally increases IRE binding activity has not been fully defined. To investigate this, Ser-138 (S138), a PKC phosphorylation site, was mutated to phosphomimetic glutamate (S138E), aspartate (S138D), or nonphosphorylatable alanine (S138A). The S138E IRP1 mutant and, to a lesser extent, the S138D IRP1 mutant were impaired in aconitase function in yeast when grown aerobically but not when grown anaerobically. Purified wild-type and mutant IRP1s could be reconstituted to active aconitases anaerobically. However, when exposed to oxygen, the [4Fe-4S] cluster of the S138D and S138E mutants decayed 5-fold and 20-fold faster, respectively, than was observed for wild-type IRP1. Our findings suggest that stability of the Fe-S cluster of IRP1 can be regulated by phosphorylation and reveal a mechanism whereby the balance between the IRE binding and [4Fe-4S] forms of IRP1 can be modulated independently of cellular iron status. Furthermore, our results show that IRP1 can function as an oxygen-modulated posttranscriptional regulator of gene expression.

Conjugation of multiple ubiquitins serves as a committed step in the degradation of a variety of intracellular eukaryotic proteins by the 26S proteasome. Conjugates are formed via a three-enzyme cascade; the initial step requires ubiquitin-activating enzyme (E1), which couples ubiquitin activation to ATP hydrolysis. Previously, we showed that many higher plants contain multiple E1 proteins and described several E1 genes from wheat. To facilitate understanding of the roles of the different plant E1s, we characterized the E1 gene and protein family from Arabidopsis thaliana. Arabidopsis E1s are encoded by two genes (AtUBA1 and AtUBA2) that synthesize approximately 123-kDa proteins with 81% amino acid sequence identity to each other and 44-75% sequence identity with confirmed E1s from other organisms. Like other E1 proteins, AtUBA1 and 2 contain a cysteine residue in the putative active site for forming the ubiquitin thiol-ester intermediate. Enzymatic analysis of the corresponding proteins expressed in Escherichia coli demonstrated that both proteins activate ubiquitin in an ATP-dependent reaction and transfer the activated ubiquitin to a variety of Arabidopsis E2s with near equal specificity. Expression studies by quantitative RT-PCR and histochemistry with transgenic plants containing AtUBA promoter-beta-glucuronidase-coding region fusions showed that the AtUBA1 and 2 genes are co-expressed in most, if not all, Arabidopsis tissues and cells. Collectively, the data indicate that E1 proteins, and presumably the rest of the ubiquitin pathway, are present throughout Arabidopsis. They also show that the AtUBA1 and 2 genes are not differentially expressed nor do they encode E1s with dramatically distinct enzymatic properties.

Covalent attachment of ubiquitin to other cellular proteins has been implicated in a multitude of diverse physiological processes in eukaryotes including selective protein degradation. This attachment is carried out by a multi-enzyme pathway consisting of three classes of enzymes: ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin-protein ligases (E3s). E2s accept activated ubiquitin from E1 and conjugate it to target proteins with or without the participation of specific E3s. Previously, we have isolated wheat cDNAs encoding 16 and 23 kDa E2s, TaUBC1 and TaUBC4, respectively. TaUBC1 shows structural homology to the yeast RAD6 E2 that is essential for DNA repair whereas TaUBC4 is related to the yeast ScUBC8 E2, both of which effectively conjugate ubiquitin to histones in vitro but as yet are without a known in vivo function. Here, we report the isolation of genomic and cDNA homologues of these genes from Arabidopsis thaliana. In Arabidopsis, both of these E2s are encoded by three member gene families. Members of the AtUBC1 gene family, comprising AtUBC1, 2 and 3, encode 150-152 amino acid proteins that are 83-99% identical to each other and TaUBC1 and contain four introns that are conserved with respect to position. Members of the AtUBC4 gene family, comprising AtUBC4, 5 and 6, encode 187-191 amino acid proteins that are 73-88% identical to each other and TaUBC4 and contain five introns that are conserved with respect to position.(ABSTRACT TRUNCATED AT 250 WORDS)

The Arabidopsis thaliana ecotype Columbia ubiquitin gene family consists of 14 members that can be divided into three types of ubiquitin genes; polyubiquitin genes, ubiquitin-like genes and ubiquitin extension genes. The isolation and characterization of eight ubiquitin sequences, consisting of four polyubiquitin genes and four ubiquitin-like genes, are described here, and their relationships to each other and to previously identified Arabidopsis ubiquitin genes were analyzed. The polyubiquitin genes, UBQ3, UBQ10, UBQ11 and UBQ14, contain tandem repeats of the 228-bp ubiquitin coding region. Together with a previously described polyubiquitin gene, UBQ4, they differ in synonymous substitutions, number of ubiquitin coding regions, number and nature of nonubiquitin C-terminal amino acid(s) and chromosomal location, dividing into two subtypes; the UBQ3/UBQ4 and UBQ10/UBQ11/UBQ14 subtypes. Ubiquitin-like genes, UBQ7, UBQ8, UBQ9 and UBQ12, also contain tandem repeats of the ubiquitin coding region, but at least one repeat per gene encodes a protein with amino acid substitutions. Nucleotide comparisons, Ks value determinations and neighbor-joining analyses were employed to determine intra- and intergenic relationships. In general, the rate of synonymous substitution is too high to discern related repeats. Specific exceptions provide insight into gene relationships. The observed nucleotide relationships are consistent with previously described models involving gene duplications followed by both unequal crossing-over and gene conversion events.