Binding Proteins to Phytochrome A in Etiolated Pea Seedlings

In order to detect and characterize a putative receptor(s) fora signal from PhyA, proteins that bind to purified pea PhyAwere searched for in the crude extract of etiolated pea seedlingswith affinity chromatography. PhyA was coupled to the columnsubstrate either in P_R form (P_R column) or in red-irradiatedform (P_FR column). The coupled PhyA of both columns retainsits spectral reversibility between P_R and P_FR, although theirpeptide mapping by trypsin digestion suggests that the C-terminalhalf of PhyA in the P_FR column is partially fixed in P_FR structure.15 polypeptides were detected reproducibly in the elution fromthe P_FR column by silver-staining of SDS-PAGE. These 15 polypeptidesmay form two complexes judging from their elution profiles.Of the 15 polypeptides, the 6 major polypeptides have approximatemol wt of 80, 55, 53, 46, 40 and 35 kDa. On the other hand,only a trace amount of protein, which mainly consists of the46 kDa species, was eluted from P_R column, indicating the presenceof P_FR-specific BPs in the crude extract of etiolated pea seedlings.Of the 6 major polypeptides, the 40 kDa species binds to thePhyA in a photoreversible manner. (Received June 19, 1998; Accepted December 19, 1998)     

Identification and characterization of the UL56 gene product of herpes simplex virus type 2

The UL56 gene product of herpes simplex virus (HSV) has been shown to play an important role in viral pathogenicity. However, the properties and functions of the UL56 protein are little understood. We raised rabbit polyclonal antisera specific for the UL56 protein of HSV type 2 (HSV-2) and examined its expression and properties. The gene product was identified as three polypeptides with apparent molecular masses ranging from 32 to 35 kDa in HSV-2-infected cells, and at least one species was phosphorylated. Studies of their origins showed that the UL56 protein of HSV-2 is also translated from the upstream in-frame methionine codon that is not present in the HSV-1 genome. Synthesis was first detected at 6 h postinfection and was not abolished by the viral DNA synthesis inhibitor phosphonoacetic acid. Indirect immunofluorescence studies revealed that the UL56 protein localized to both the Golgi apparatus and cytoplasmic vesicles in HSV-2-infected and single UL56-expressing cells. Deletion mutant analysis showed that the C-terminal hydrophobic region of the protein was required for association with the cytoplasmic membrane and that the N-terminal proline-rich region was important for its translocation to the Golgi apparatus and cytoplasmic vesicles. Moreover, the results of protease digestion assays and sucrose gradient fractionation strongly suggested that UL56 is a tail-anchored type II membrane protein associated with lipid rafts. We thus hypothesized that the UL56 protein, as a tail-anchored type II membrane protein, may be involved in vesicular trafficking in HSV-2-infected cells.     

Diversity of Plant Methionine Sulfoxide Reductases B and Evolution of a Form Specific for Free Methionine Sulfoxide

Methionine can be reversibly oxidized to methionine sulfoxide (MetO) under physiological conditions. Organisms evolved two distinct methionine sulfoxide reductase families (MSRA & MSRB) to repair oxidized methionine residues. We found that 5 MSRB genes exist in the soybean genome, including GmMSRB1 and two segmentally duplicated gene pairs (GmMSRB2 and GmMSRB5, GmMSRB3 and GmMSRB4). GmMSRB2 and GmMSRB4 proteins showed MSRB activity toward protein-based MetO with either DTT or thioredoxin (TRX) as reductants, whereas GmMSRB1 was active only with DTT. GmMSRB2 had a typical MSRB mechanism with Cys121 and Cys 68 as catalytic and resolving residues, respectively. Surprisingly, this enzyme also possessed the MSRB activity toward free Met-R-O with kinetic parameters similar to those reported for fRMSR from Escherichia coli, an enzyme specific for free Met-R-O. Overexpression of GmMSRB2 or GmMSRB4 in the yeast cytosol supported the growth of the triple MSRA/MSRB/fRMSR (Δ3MSRs) mutant on MetO and protected cells against H₂O₂-induced stress. Taken together, our data reveal an unexpected diversity of MSRBs in plants and indicate that, in contrast to mammals that cannot reduce free Met-R-O and microorganisms that use fRMSR for this purpose, plants evolved MSRBs for the reduction of both free and protein-based MetO.     

Kinetics and product analysis of the reaction catalysed by recombinant homoaconitase from Thermus thermophilus

HACN (homoaconitase) is a member of a family of [4Fe-4S] cluster-dependent enzymes that catalyse hydration/dehydration reactions. The best characterized example of this family is the ubiquitous ACN (aconitase), which catalyses the dehydration of citrate to cis-aconitate, and the subsequent hydration of cis-aconitate to isocitrate. HACN is an enzyme from the alpha-aminoadipate pathway of lysine biosynthesis, and has been identified in higher fungi and several archaea and one thermophilic species of bacteria, Thermus thermophilus. HACN catalyses the hydration of cis-homoaconitate to (2R,3S)-homoisocitrate, but the HACN-catalysed dehydration of (R)-homocitrate to cis-homoaconitate has not been observed in vitro. We have synthesized the substrates and putative substrates for this enzyme, and in the present study report the first steady-state kinetic data for recombinant HACN from T. thermophilus using a (2R,3S)-homoisocitrate dehydrogenase-coupled assay. We have also examined the products of the reaction using HPLC. We do not observe HACN-catalysed 'homocitrate dehydratase' activity; however, we have observed that ACN can catalyse the dehydration of (R)-homocitrate to cis-homoaconitate, but HACN is required for subsequent conversion of cis-homoaconitate into homoisocitrate. This suggests that the in vivo process for conversion of homocitrate into homoisocitrate requires two enzymes, in simile with the propionate utilization pathway from Escherichia coli. Surprisingly, HACN does not show any activity when cis-aconitate is substituted for the substrate, even though other enzymes from the alpha-aminoadipate pathway can accept analogous tricarboxylic acid-cycle substrates. The enzyme shows no apparent feedback inhibition by L-lysine.     

The US11 gene product of herpes simplex virus has intercellular trafficking activity.

The US11 gene product of herpes simplex virus is an abundant virion structural protein with RNA-binding regulatory activity. Its carboxyl-terminal half consists of tandem tripeptide repeats of the sequence RXP. We demonstrate that the US11 protein has intercellular trafficking activity and accumulates in the nucleolus when singly expressed in cultured cells, and that the RXP repeats are responsible for this activity. These same properties were also observed in cells expressing a fusion protein linking US11 to the green fluorescent protein. Furthermore, exogenous US11 protein was internalized by cells at 4 degrees C, which suggests that US11 protein uptake occurs primarily through an energy-independent pathway.     

Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger

Ubiquitin-like with PHD and RING finger domain-containing protein 1 (UHRF1)-dependent DNA methylation is essential for maintaining cell fate during cell proliferation. Developmental pluripotency-associated 3 (DPPA3) is an intrinsically disordered protein that specifically interacts with UHRF1 and promotes passive DNA demethylation by inhibiting UHRF1 chromatin localization. However, the molecular basis of how DPPA3 interacts with and inhibits UHRF1 remains unclear. We aimed to determine the structure of the mouse UHRF1 plant homeodomain (PHD) complexed with DPPA3 using nuclear magnetic resonance. Induced α-helices in DPPA3 upon binding of UHRF1 PHD contribute to stable complex formation with multifaceted interactions, unlike canonical ligand proteins of the PHD domain. Mutations in the binding interface and unfolding of the DPPA3 helical structure inhibited binding to UHRF1 and its chromatin localization. Our results provide structural insights into the mechanism and specificity underlying the inhibition of UHRF1 by DPPA3.