Prolyl-4-hydroxylase domain (PHD) proteins are 2-oxoglutarate and dioxygen-dependent enzymes that mediate the rapid destruction of hypoxia-inducible factor ␣ subunits. Whereas PHD1 and PHD3 proteolysis has been shown to be regulated by Siah2 ubiquitin E3 ligase-mediated polyubiquitylation and proteasomal destruction, protein regulation of the main oxygen sensor responsible for hypoxia-inducible factor ␣ regulation, PHD2, remained unknown. We recently reported that the FK506-binding protein (FKBP) 38 specifically interacts with PHD2 and determines PHD2 protein stability in a peptidylprolyl cis-trans isomerase-independent manner. Using peptide array binding assays, fluorescence spectroscopy, and fluorescence resonance energy transfer analysis, we defined a minimal linear glutamate-rich PHD2 binding domain in the N-terminal part of FKBP38 and showed that this domain forms a high affinity complex with PHD2. Vice versa, PHD2 interacted with a nonlinear N-terminal motif containing the MYND (myeloid, Nervy, and DEAF-1)-type Zn 2؉ finger domain with FKBP38. Biochemical fractionation and immunofluorescence analysis demonstrated that PHD2 subcellular localization overlapped with FKBP38 in the endoplasmic reticulum and mitochondria. An additional fraction of PHD2 was found in the cytoplasm. In cellulo PHD2/FKBP38 association, as well as regulation of PHD2 protein abundance by FKBP38, is dependent on membraneanchored FKBP38 localization mediated by the C-terminal transmembrane domain. Mechanistically our data indicate that PHD2 protein stability is regulated by a ubiquitin-independent proteasomal pathway involving FKBP38 as adaptor protein that mediates proteasomal interaction. We hypothesize that FKBP38-bound PHD2 is constantly degraded whereas cytosolic PHD2 is stable and able to function as an active prolyl-4-hydroxylase.The heterodimeric ␣/␤ transcription factor complexes of hypoxia-inducible factors (HIFs) 4 are central regulators of the cellular, local, and systemic response to reduced oxygen partial pressure (pO 2 ) (1, 2). Under normoxic conditions, two highly conserved prolyl residues within the oxygen-dependent degradation domain of HIF␣ subunits are hydroxylated by members of the prolyl-4-hydroxylase domain (PHD) family (also called egg laying-defective nine homolog (EGLN) or HIF prolyl hydroxylase) (3-5). Hydroxylated prolines are then bound by an E3 ubiquitin ligase complex containing the von Hippel-Lindau tumor suppressor protein (pVHL) as recognition subunit, mediating polyubiquitylation and proteasomal degradation of HIF␣ subunits (6 -8). In addition, factor inhibiting HIF hydroxylates under normoxic conditions an asparaginyl residue in the C-terminal transcriptional transactivation domain of HIF␣ subunits, preventing the association with the CH1 domain of the p300 and cAMP-responsive element-binding protein-binding protein (CBP) co-activators (9, 10). PHDs and factor inhibiting HIF belong to the 2-oxoglutarate-and irondependent dioxygenase superfamily and act as cellular oxygen sensors by correlating the ...

It is known that various poplar species and cultivated poplar hybrids have the potential to interbreed and produce fertile offspring. Conservation strategies for the genetic resources of the endangered Eurasian black poplar (Populus nigra L.) thus rely on a monitoring which enables the identification and verification of the pure species status. At the same time, the risk of hybrid dispersal and introgressive gene flow has to be estimated. In the present study a combination of two molecular markers, one from chloroplast DNA and the other from nuclear DNA, was applied to evaluate a large P. nigra population on the Elbe River. Hybrid clones of P. · canadensis are scattered within this population and also occur as plantations in the surrounding landscape. By means of the DNA markers the taxonomic status of 208 adult trees in the population and 140 young poplars along the riverbank was monitored. From the analysed young poplars, almost 20 percent were found to exhibit at least one of the two P. deltoides or P. · canadensis diagnostic alleles or genotypes, respectively. Possible vegetative spreads of F1 hybrids and precedent mating scenarios are discussed. Most interestingly we found clear evidence for a small number of backcross hybrids where P. · canadensis acted as pollen donor. This case had long been debated and thought to be less probable, so far.

BackgroundCellular oxygen sensing is fundamental to all mammalian cells to adequately respond to a shortage of oxygen by increasing the expression of genes that will ensure energy homeostasis. The transcription factor Hypoxia-Inducible-Factor-1 (HIF-1) is the key regulator of the response because it coordinates the expression of hypoxia inducible genes. The abundance and activity of HIF-1 are controlled through posttranslational modification by hydroxylases, the cellular oxygen sensors, of which the activity is oxygen dependent.MethodsFluorescence resonance energy transfer (FRET) was established to determine the assembly of the HIF-1 complex and to study the interaction of the α-subunit of HIF-1 with the O2-sensing hydroxylase. New software was developed to improve the quality and reliability of FRET measurements.ResultsFRET revealed close proximity between the HIF-1 subunits in multiple cells. Data obtained by sensitized FRET in this study were fully compatible with previous work using acceptor bleaching FRET. Interaction between the O2-sensing hydroxylase PHD1 and HIF-1α was demonstrated and revealed exclusive localization of O2-sensing in the nucleus. The new software FRET significantly improved the quality and speed of FRET measurements.ConclusionFRET measurements do not only allow following the assembly of the HIF-1 complex under hypoxic conditions but can also provide important information about the process of O2-sensing and its localisation within a cell.MCS codes: 92C30, 92C05, 92C40