FRET-detectable interactions between the ARE binding proteins, HuR and p37AUF1.

A number of highly regulated gene classes are regulated post-transcriptionally at the level of mRNA stability. A central feature in these mRNAs is the presence of A+U-rich elements (ARE) within their 3' UTRs. Two ARE binding proteins, HuR and AUF1, are associated with mRNA stabilization and destabilization, respectively. Previous studies have demonstrated homomultimerization of each protein and the capacity to bind simultaneous or competitively to a single ARE. To investigate this possibility further, cell biological and biophysical approaches were undertaken. Protein-protein interaction was monitored by fluorescence resonance energy transfer (FRET) and by immunocytochemistry in live and fixed cells using fluorescently labeled CFP/YFP fusion proteins of HuR and p37AUF1. Strong nuclear FRET between HuR/HuR and AUF1/AUF1 homodimers as well as HuR/AUF1 heterodimers was observed. Treatment with the MAP kinase activator, anisomycin, which commonly stabilizes ARE-containing mRNAs, caused rapid nuclear to cytoplasmic shuttling of HuR. AUF1 also underwent shuttling, but on a longer time scale. After shuttling, HuR/HuR, AUF1/AUF1, and HuR/AUF1, FRET was also observed in the cytoplasm. In further studies, arsenite rapidly induced the formation of stress granules containing HuR and TIA-1 but not AUF1. The current studies demonstrate that two mRNA binding proteins, HuR and AUF1, are colocalized and are capable of functional interaction in both the nucleus and cytoplasm. FRET-based detection of AUF1/HuR interaction may serve as a basis of opening up new dimensions in delineating the functional interaction of mRNA binding proteins with RNA turnover.

Mitochondrial cytochrome oxidase produces nitric oxide under hypoxic conditions: implications for oxygen sensing and hypoxic signaling in eukaryotes.

Eukaryotic cells respond to low-oxygen concentrations by upregulating hypoxic nuclear genes (hypoxic signaling). Although it has been shown previously that the mitochondrial respiratory chain is required for hypoxic signaling, its underlying role in this process has been unclear. Here, we find that yeast and rat liver mitochondria produce nitric oxide (NO) at dissolved oxygen concentrations below 20 microM. This NO production is nitrite (NO2-) dependent, requires an electron donor, and is carried out by cytochrome c oxidase in a pH-dependent fashion. Mitochondrial NO production in yeast is influenced by the YHb flavohemoglobin NO oxidoreductase, stimulates expression of the hypoxic nuclear gene CYC7, and is accompanied by an increase in protein tyrosine nitration. These findings demonstrate an alternative role for the mitochondrial respiratory chain under hypoxic or anoxic conditions and suggest that mitochondrially produced NO is involved in hypoxic signaling, possibly via a pathway that involves protein tyrosine nitration.

Effects of a transition from normoxia to anoxia on yeast cytochrome c oxidase and the mitochondrial respiratory chain: implications for hypoxic gene induction.

Previous studies have demonstrated that the mitochondrial respiratory chain and cytochrome c oxidase participate in oxygen sensing and the induction of some hypoxic nuclear genes in eukaryotes. In addition, it has been proposed that mitochondrially-generated reactive oxygen and nitrogen species function as signals in a signaling pathway for the induction of hypoxic genes. To gain insight concerning this pathway, we have looked at changes in the functionality of the yeast respiratory chain as cells experience a shift from normoxia to anoxia. These studies have revealed that yeast cells retain the ability to respire at normoxic levels for up to 4 h after a shift and that the mitochondrial cytochrome levels drop rapidly to 30--50% of their normoxic levels and the turnover rate of cytochrome c oxidase (COX) increases during this shift. The increase in COX turnover rate cannot be explained by replacing the aerobic isoform, Va, of cytochrome c oxidase subunit V with the more active hypoxic isoform, Vb. We have also found that mitochondria retain the ability to respire, albeit at reduced levels, in anoxic cells, indicating that yeast cells maintain a functional mitochondrial respiratory chain in the absence of oxygen. This raises the intriguing possibility that the mitochondrial respiratory chain has a previously unexplored role in anoxic cells and may function with an alternative electron acceptor when oxygen is unavailable.