Identification of cDNAs encoding isoprenylated proteins

Isoprenylated proteins are involved in signal transduction, control of cell growth and differentiation, organization of the nuclear lamina and cytoskeleton, and vesicle sorting. The isoprenoid moiety facilitates the interaction of these proteins with membranes and/or other proteins. However, many isoprenylated proteins remain unidentified. A method is described for identifying novel and known cDNAs encoding isoprenylated proteins. Sufficient details of the screening procedure are given so that this method may be easily used to identify cDNAs encoding other covalently modified proteins or proteins possessing high affinity ligand binding sites.     

Identification of proteins that interact with NF-YA

We used the yeast two-hybrid system to show that the serum response factor (SRF) and zinc-fingers and homeobox 1 (ZHXI) proteins interact with the A subunit of nuclear factor-Y (NF-YA). GST pulldown assays revealed that both proteins interact specifically with NF-YA in vitro. Amino acids located between 272 and 564, a region that contains two homeodomains, are required for the interaction of ZHX1 with NF-YA. Two different domains of NF-YA, a glutamine-rich region and a serine/threonine-rich region, are necessary for the interactions with ZHX1 and SRF, respectively.     

Identification of two novel proteins that interact with germ-cell-specific RNA-binding proteins DAZ and DAZL1

The human DAZ (deleted in azoospermia) gene family on the Y chromosome and an autosomal DAZ-like gene, DAZL1, encode RNA-binding proteins that are expressed exclusively in germ cells. Their role in spermatogenesis is supported by their homology with a Drosophila male infertility gene boule and sterility of Daz11 knock-out mice. While all mammals contain a DAZL1 homologue on their autosomes, DAZ homologues are present only on the Y chromosomes of great apes and Old World monkeys. The DAZ and DAZL1 proteins differ in the copy numbers of a DAZ repeat and the C-terminal sequences. We studied the interaction of DAZ and DAZL1 with other proteins as an approach to investigate functional similarity between these two proteins. Using DAZ as bait in a yeast two-hybrid system, we isolated two DAZAP (DAZ-associated protein) genes. DAZAP1 encodes a novel RNA-binding protein that is expressed most abundantly in the testis, and DAZAP2 encodes a ubiquitously expressed protein with no recognizable functional motif. DAZAP1 and DAZAP2 bind similarly to both DAZ and DAZL1 through the DAZ repeats. The DAZAP genes were mapped to chromosomal regions 19p13.3 and 2q33-q34, respectively, where no genetic diseases affecting spermatogenesis are known to map.     

Identification of four nuclear transport signal-binding proteins that interact with diverse transport signals.

The transport of proteins into the nucleus requires not only the presence of a nuclear transport signal on the targeted protein but also the signal recognition proteins and the nuclear pore translocation apparatus. Complicating the search for the signal recognition proteins is the fact that the nuclear transport signals identified share little obvious homology. In this study, synthetic peptides homologous to the nuclear transport signals from the simian virus 40 large T antigen, Xenopus oocyte nucleoplasmin, adenovirus E1A, and Saccharomyces cerevisiae MAT alpha 2 proteins were coupled to a UV-photoactivable cross-linker and iodinated for use in an in vitro cross-linking reaction with cellular lysates. Four proteins, p140, p100, p70, and p55, which specifically interacted with the nuclear transport signal peptides were identified. Unique patterns of reactivity were observed with closely related pairs of nuclear transport signal peptides. Competition experiments with labeled and unlabeled peptides demonstrated that heterologous signals were able to bind the same protein and suggested that diverse signals use a common transport pathway. The subcellular distribution of the four nuclear transport signal-binding proteins suggested that nuclear transport involves both cytoplasmic and nuclear receptors. The four proteins were not bound by wheat germ agglutinin and were not associated tightly with the nuclear pore complex.     

Identification of leukocyte cationic proteins that interact with ceruloplasmin

Proteins from leukocytes were investigated for their ability to interact with ceruloplasmin (Cp), a copper-containing glycoprotein of human plasma. Extract from leukocytes was subjected to affinity chromatography on Cp-Sepharose, after which proteins were eluted from the resin with 0.5 M NaCl in Tris-HCl, pH 7.4. SDS-PAGE of the eluate revealed protein bands with molecular weights 78, 57, 40, 30, 16, and 12 kD. Among these, Western blotting detected myeloperoxidase (57, 40, and 12 kD) and lactoferrin (78 kD). Also, the 30-kD component had a sequence (1)I-(2)I/V-(3)G-(4)G-(5)R/H at the N-terminus that is likely to indicate the presence of neutrophilic elastase, cathepsin G, proteinase 3, and azurocidin (CAP 37) - all from the family of serprocidins. Mass spectrometry of tryptic fragments indicated the presence of the 16-kD eosinophilic cationic protein (seven peptides), 27-kD cathepsin G (eleven peptides), 27-kD azurocidin (eight peptides), 29-kD neutrophilic elastase (seven peptides), and 27-kD proteinase 3 (six peptides). Myeloperoxidase was represented by 57-, 40-, and 12-kD fragments (thirteen, ten, and four peptides, respectively). Thus, interaction with Cp of five cationic proteins, i.e. of eosinophilic cationic protein, cathepsin G, neutrophilic elastase, proteinase 3, and azurocidin is reported for the first time.     

Molecular characterization of tobacco cDNAs encoding two small GTP-binding proteins

We have isolated two cDNAs encoding small GTP-binding proteins from leaf cDNA libraries. These cDNAs encode distinct proteins which show considerable homology to members of the ras superfamily. Np-ypt3, a 1044 bp long Nicotiana plumbaginifolia cDNA, encodes a 24.4 kDa protein which shows 65% amino acid sequence similarity to the Schizosaccharomyces pombe ypt3 protein. The N-ypt3 gene is differentially expressed in mature flowering plants. Expression of this gene is weak in leaves, higher in stems and roots, but highest in petals, stigmas and stamens. Nt-rab5, a 712 bp long Nicotiana tabacum SR1 cDNA, encodes a 21.9 kDa protein which displays 65% amino acid sequence similarity to mammalian rab5 proteins. The expression pattern of the Nt-rab5 gene is very similar to that of the Np-ypt3 gene. The Nt-rab5 gene is virtually not expressed in leaves, higher in stems and roots, and highest in flowers. Both the Nt-rab5 and Np-ypt3 proteins were expressed in Escherichia coli and shown to bind GTP.     

Identification of cDNAs encoding two novel rat pancreatic serine proteases

Serine proteases (SPs) are a family of physiologically important and versatile enzymes. We designed degenerated oligodeoxyribonucleotide primers derived from the consensus amino acid aa sequences of the active site of mammalian SPs, to selectively amplify in a polymerase chain reaction (PCR) cDNA fragments coding for SPs. We used poly(A) ⁺ RNA from rat pancreas to obtain the cDNA. Two of the amplified cDNA fragments encode novel SPs. The full-lenght nucleotide sequence of both cDNAs was also obtained by PCR. The high degree of homology to trypsins and elastases suggests that the cDNAs encode a trypsin-like and an elastase-like SP, respectively. Both mRNAs were also found to occur, to a lesser extent, in spleen, as was the case for the mRNAs of other rat pancreatic SPs.     

Crystal structures of Drosophila mutant translin and characterization of translin variants reveal the structural plasticity of translin proteins

Translin protein is highly conserved in eukaryotes. Human translin binds both ssDNA and RNA. Its nucleic acid binding site results from a combination of basic regions in the octameric structure. We report here the first biochemical characterization of wild-type Drosophila melanogaster (drosophila) translin and a chimeric translin, and present 3.5 A resolution crystal structures of drosophila P168S mutant translin from two crystal forms. The wild-type drosophila translin most likely exists as an octamer/decamer, and binds to the ssDNA Bcl-CL1 sequence. In contrast, ssDNA binding-incompetent drosophila P168S mutant translin exists as a tetramer. The structures of the mutant translin are identical in both crystal forms, and their C-terminal residues are disordered. The chimeric protein, possessing two nucleic acid binding motifs of drosophila translin, the C-terminal residues of human translin, and serine at position 168, attains the octameric state and binds to ssDNA. The present studies suggest that the oligomeric status of translin critically influences the DNA binding properties of translin proteins.     

Molecular characterization of cDNAs encoding low-molecular-weight heat shock proteins of soybean

Three cDNA clones (GmHSP23.9, GmHSP22.3, and GmHSP22.5) representing three different members of the low-molecular-weight (LMW) heat shock protein (HSP) gene superfamily were isolated and characterized. A fourth cDNA clone, pFS2033, was partially characterized previously as a full-length genomic clone GmHSP22.0. The deduced amino acid sequences of all four cDNA clones have the conserved carboxyl-terminal LMW HSP domain. Sequence and hydropathy analyses of GmHSP22, GmHSP22.3, and GmHSP22.5, representing HSPs in the 20 to 24 kDa range, indicate they contain amino-terminal signal peptides. The mRNAs from GmHSP22, GmHSP22.3, and GmHSP22.5 were preferentially associated in vivo with endoplasmic reticulum (ER)-bound polysomes. GmHSP22 and GmHSP22.5 encode strikingly similar proteins; they are 78% identical and 90% conserved at the amino acid sequence level, and both possess the C-terminal tetrapeptide KQEL which is similar to the consensus ER retention motif KDEL; the encoded polypeptides can be clearly resolved from each other by two-dimensional gel analysis of their hybrid-arrest translation products. GmHSP22.3 is less closely related to GmHSP22 (48% identical and 70% conserved) and GmHSP22.5 (47% identical and 65% conserved). The fourth cDNA clone, GmHSP23.9, encodes a HSP of ca. 24kDa with an amino terminus that has characteristics of some mitochondrial transit sequences, and in contrast to GmHSP22, GmHSP22.3, and GmHSP22.5, the corresponding mRNA is preferentially associated in vivo with free polysomes. It is proposed that the LMW HSP gene superfamily be expanded to at least six classes to include a mitochondrial class and an additional endomembrane class of LMW HSPs.     

Identification of DNA replication and cell cycle proteins that interact with PCNA.

The identity of DNA replication proteins and cell cycle regulatory proteins which can be found in complexes involving PCNA were investigated by the use of PCNA immobilized on Sepharose 4B. A column containing bovine serum albumin (BSA) bound to Sepharose was used as a control. Fetal calf thymus extracts were chromatographed on PCNA-Sepharose and BSA-Sepharose. The columns were washed and then eluted with 0.5 M KCl. The salt eluates were examined for the presence of both DNA replication proteins (Pol alpha, delta, straightepsilon, PCNA, RFC, RFA, DNA ligase I, NDH II, Topo I and Topo II) and cell cycle proteins (Cyclins A, B1, D1, D2, D3, E, CDK2, CDK4, CDK5 and p21) by western blotting with specific antibodies. The DNA replication proteins which bound to PCNA-Sepharose included DNA polymerase delta and straightepsilon, PCNA, the 37 and 40 kDa subunits of RFC, the 70 kDa subunit of RPA, NDH II and topoisomerase I. No evidence for the binding of DNA polymerase alpha, DNA ligase I or topoisomerase II was obtained. Of the cell cycle proteins investigated, CDK2, CDK4 and CDK5 were bound. This study presents strong evidence that PCNA is a component of protein complexes containing DNA replication, repair and cell cycle regulatory proteins.     

Identification and characterization of cDNAs encoding pentatricopeptide repeat proteins in the basal land plant, the moss Physcomitrella patens

A large gene family encoding proteins with a pentatricopeptide repeat (PPR) motif exists in flowering plants but not in algae, fungi, or animals. This suggests that PPR protein genes expanded vastly during the evolution of the land plants. To investigate this possibility, we analysed PPR protein genes in the basal land plant, the moss Physcomitrella patens. An extensive survey of the Physcomitrella expressed sequence tag (EST) databases revealed 36 ESTs encoding PPR proteins. This indicates that a large gene family of PPR proteins originated before the divergence of the vascular plant and moss lineages. We also characterized five full-length cDNAs encoding PPR proteins, designated PPR513-10, PPR566-6, PPR868-14, PPR986-12, and PPR423-6. Intracellular localization analysis demonstrated two PPR proteins in chloroplasts (cp), whereas the cellular localization of the other three PPR proteins is unclear. The genes of the cp-localized PPR513-10 and PPR566-6 were expressed differentially in protonemata grown under different light-dark conditions, suggesting they have distinctive functions in cp. This is the first report and analysis of genes encoding PPR proteins in bryophytes.