Molecular cloning and structural analysis of genes from Zea mays (L.) coding for members of the ras-related ypt gene family.

We have isolated, cloned, and characterized two cDNAs from Zea mays (L.), denoted yptm1 and yptm2, encoding proteins related to the ypt protein family. Amino acid similarity scores with YPT1 from yeast and ypt from mouse are in the range of 70% for yptm1 and 74% for yptm2, respectively, whereas similarities with p21 ras and other ras-related proteins are less than 40%. Most amino acid residues showing identity are clustered in the GTP/GDP binding domain. In addition, two cysteine residues close to the C-terminal ends, known to be palmitoylated and necessary for membrane binding in all eukaryotic ras-related proteins that have been characterized so far, are conserved in the maize genes as well. Northern blot hybridization analysis of poly(A)+ mRNA from etiolated maize coleoptiles revealed single mRNA species of approximately the same size as the isolated cDNAs. The gene for yptm1 is expressed at very low levels in maize coleoptiles and tissue culture cells. The gene for yptm2 is expressed at higher levels and is differentially represented in RNAs isolated from various organs of maize plants, with its highest level in leaves and flowers. The structural similarity of the genes identified suggests that they could be involved in the control of secretory processes.     

Nuclear import of Agrobacterium VirD2 and VirE2 proteins in maize and tobacco.

Previously, we have shown that Agrobacterium-plant cell transferred DNA (T-DNA) transport into the host cell nucleus is likely mediated by two specific bacterial proteins, VirD2 and VirE2. Here, we used these proteins to study molecular pathways of nuclear import. First, the role of VirE2 nuclear localization signals (NLSs) in the T-DNA transport pathway was examined by using tobacco plants transgenic for deletion mutants of VirE2. In these plants, the virulence of wild-type Agrobacterium was reduced possibly by competition for the cellular nuclear import machinery. Second, we analyzed the nuclear localization of VirE2 and VirD2 in the nonhost monocot maize. Part of the known recalcitrance of monocots to transformation by Agrobacterium could be due to a potential selectivity in nuclear import pathways in monocotyledonous and dicotyledonous plants. Nuclear transport of VirD2 and VirE2 in maize leaves and roots was compared to that in tobacco protoplasts and roots. Both proteins accumulated in maize leaf and tobacco protoplast nuclei as well as in nuclei of immature root cells. In contrast, VirD2 and VirE2 expressed in mature roots of maize and tobacco remained cytoplasmic. Point mutations of VirE2 nuclear localization signals, NSE 1 and NSE 2, also revealed that, in maize, the NSE 1 signal was mainly responsible for nuclear import; in contrast, both signals functioned independently in tobacco protoplasts.     

EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana

Shortly after the release of singlet oxygen ((1)O2), drastic changes in nuclear gene expression occur in the conditional flu mutant of Arabidopsis that reveal a rapid transfer of signals from the plastid to the nucleus. In contrast to retrograde control of nuclear gene expression by plastid signals described earlier, the primary effect of (1)O2 generation in the flu mutant is not the control of chloroplast biogenesis but the activation of a broad range of signaling pathways known to be involved in biotic and abiotic stress responses. This activity of a plastid-derived signal suggests a new function of the chloroplast, namely that of a sensor of environmental changes that activates a broad range of stress responses. Inactivation of the plastid protein EXECUTER1 attenuates the extent of (1)O2-induced up-regulation of nuclear gene expression, but it does not fully eliminate these changes. A second related nuclear-encoded protein, dubbed EXECUTER2, has been identified that is also implicated with the signaling of (1)O2-dependent nuclear gene expression changes. Like EXECUTER1, EXECUTER2 is confined to the plastid. Inactivation of both EXECUTER proteins in the ex1/ex2/flu triple mutant is sufficient to suppress the up-regulation of almost all (1)O2-responsive genes. Retrograde control of (1)O2-responsive genes requires the concerted action of both EXECUTER proteins within the plastid compartment.     

Chlorophyll regulates accumulation of the plastid-encoded chlorophyll apoproteins CP43 and D1 by increasing apoprotein stability.

Chlorophyll apoprotein accumulation in higher plant chloroplasts is controlled by light-dependent chlorophyll formation. Dark-grown plants lack chlorophyll and chlorophyll apoproteins. However, the plastid genes encoding the chlorophyll apoproteins are transcribed; chlorophyll apoprotein mRNA accumulates and associates with polysomes in plastids of dark-grown plants. Pulse-labeling assays revealed a population of short-lived proteins in plastids of dark-grown plants. One of these transiently labeled proteins was CP43, a chlorophyll apoprotein associated with photosystem II. Pulse-chase assays showed that newly synthesized CP43 was rapidly degraded in plastids of dark-grown plants, which lack chlorophyll. In contrast, CP43 synthesized in plastids from illuminated plants was stable. The synthesis of D1, a chlorophyll apoprotein of the photosystem II reaction center, was also analyzed in plastids of dark-grown and illuminated plants. Radiolabel accumulation into full-length D1 was only detected in plastids of illuminated plants. However, D1 translation intermediates of 15-25 kDa were detected in both plastid populations. Pulse-chase assays showed that the 15- to 25-kDa D1 translation products were precursors of mature D1 in plastids of illuminated plants. In contrast, in plastids of dark-grown plants, the 15- to 25-kDa translation intermediates were converted into a 23-kDa polypeptide previously suggested to be a proteolytic product of D1. These results indicate that chlorophyll produced in illuminated plants stabilizes D1 nascent polypeptides, which allows accumulation of mature D1.     

Juvenile hormone receptors in insect larval epidermis: identification by photoaffinity labeling

Tritiated photoaffinity analogs of the natural lepidopteran juvenile hormones, JH I and II [epoxy[3H]bishomofarnesyl diazoacetate ([3H]EBDA) and epoxy[3H]homofarnesyl diazoacetate ([3H]EHDA)], and of the JH analog methoprene [[3H]methoprene diazoketone ([3H]MDK)] were synthesized and used to identify specific JH binding proteins in the larval epidermis of the tobacco hornworm (Manduca sexta). EBDA and EHDA specifically photolabeled a 29-kDa nuclear protein (pI 5.8). This protein and a second 29-kDa protein (pI 6.0) were labeled by MDK, but excess unlabeled methoprene or MDK only prevented binding to the latter. These 29-kDa proteins are also present in larval fat body but not in epidermis from either wandering stage or allatectomized larvae, which lack high-affinity JH binding sites. A 29-kDa nuclear protein with the same developmental specificity as this JH binder bound the DNA of two larval endocuticle genes. A 38-kDa cytosolic protein was also specifically photolabeled by these photoaffinity analogs. The 29-kDa nuclear protein is likely the high-affinity receptor for JH that mediates its genomic action, whereas the 38-kDa cytosolic protein may serve as an intracellular carrier for these highly lipophilic hormones and hormone analogs.     

Lateral gene transfer and the evolution of plastid-targeted proteins in the secondary plastid-containing alga Bigelowiella natans

Chlorarachniophytes are amoeboflagellate algae that acquired photosynthesis secondarily by engulfing a green alga and retaining its plastid (chloroplast). An important consequence of secondary endosymbiosis in chlorarachniophytes is that most of the nuclear genes encoding plastid-targeted proteins have moved from the nucleus of the endosymbiont to the host nucleus. We have sequenced and analyzed 83 cDNAs encoding 78 plastid-targeted proteins from the model chlorarachniophyte Bigelowiella natans (formerly Chlorarachnion sp. CCMP621). Phylogenies inferred from the majority of these genes are consistent with a chlorophyte green algal origin. However, a significant number of genes ( approximately 21%) show signs of having been acquired by lateral gene transfer from numerous other sources: streptophyte algae, red algae (or algae with red algal endosymbionts), as well as bacteria. The chlorarachniophyte plastid proteome may therefore be regarded as a mosaic derived from various organisms in addition to the ancestral chlorophyte plastid. In contrast, the homologous genes from the chlorophyte Chlamydomonas reinhardtii do not show any indications of lateral gene transfer. This difference is likely a reflection of the mixotrophic nature of Bigelowiella (i.e., it is photosynthetic and phagotrophic), whereas Chlamydomonas is strictly autotrophic. These results underscore the importance of lateral gene transfer in contributing foreign proteins to eukaryotic cells and their organelles, and also suggest that its impact can vary from lineage to lineage.     

Cloning of the cDNA and functional expression of the 47-kilodalton cytosolic component of human neutrophil respiratory burst oxidase.

Neutrophil NADPH oxidase is a multicomponent enzyme that is activated to generate superoxide anion and is defective in the cells of patients with chronic granulomatous disease. It requires both membrane and cytosolic components, the latter including 47- and 67-kDa proteins recognized by the polyclonal antiserum B-1. Immunoscreening of an induced HL-60 lambda ZAP cDNA library yielded seven cross-hybridizing cDNAs encoding the 47-kDa component. Fusion proteins of 22-50 kDa were recognized by B-1. Antiserum against a fusion protein recognized a 47-kDa protein in normal neutrophils but not in those from patients with autosomal chronic granulomatous disease who lack the 47-kDa cytosolic oxidase component. In a cell-free NADPH oxidase system full-length and C-terminal fusion proteins augmented superoxide generation and reconstituted the cytosolic defect of a patient missing the 47-kDa protein. The cDNA hybridized with a 1.4-kilobase mRNA from induced HL-60 cells. The longest cDNA contained an open reading frame encoding a protein of 41,440 Da with a calculated pI of 10.4, an N-terminal glycine, sites favorable for phosphorylation, a nucleotide binding domain, and a region of homology to the src protein kinases, phospholipase C, and alpha-fodrin. These structural features are pertinent to proposed functional roles of the protein in the respiratory burst oxidase.     

Replication factor encoded by a putative oncogene, set, associated with myeloid leukemogenesis.

DNA replication of the adenovirus genome complexed with viral core proteins is dependent on the host factor designated template activating factor I (TAF-I) in addition to factors required for replication of the naked genome. Recently, we have purified TAF-I as 39- and 41-kDa polypeptides from HeLa cells. Here we describe the cloning of two human cDNAs encoding TAF-I. Nucleotide sequence analysis revealed that the 39-kDa polypeptide corresponds to the protein encoded by the set gene, which is the part of the putative oncogene associated with acute undifferentiated leukemia when translocated to the can gene. The 41-kDa protein contains the same amino acid sequence as the 39-kDa protein except that short N-terminal regions differ in both proteins. Recombinant proteins, which were purified from extracts of Escherichia coli, expressing the proteins from cloned cDNAs, possessed TAF-I activities in the in vitro replication assay. A particular feature of TAF-I proteins is the presence of a long acidic tail in the C-terminal region, which is thought to be an essential part of the SET-CAN fusion protein. Studies with mutant TAF-I proteins devoid of this acidic region indicated that the acidic region is essential for TAF-I activity.     

S2 episome of maize mitochondria encodes a 130-kilodalton protein found in male sterile and fertile plants

The mitochondrial genome of the S-type male-sterile cytoplasm of maize contains two linear episomes, S1 (6397 base pairs) and S2 (5453 base pairs). The S2 episome contains two large unidentified open reading frames, URF1 (3512 base pairs) and URF2 (1017 base pairs). We have demonstrated that a polypeptide with an apparent molecular mass of 130 kDa is the gene product of URF1. This polypeptide was first detected in Coomassie blue-stained protein gels of cms-S (where cms = cytoplasmic male sterile) but not in those of cms-T, cms-C, or normal mitochondrial proteins. The protein product of a translational fusion containing the 5′ end of Escherichia coli lacZ and an internal segment from URF1 of S2 was recognized by antisera raised against the 130-kDa variant polypeptide. The mitochondria of fertile F₁ hybrids of cms-S × Ky21 (the male parent carrying nuclear fertility restoration genes) contain as much of the 130-kDa protein as is found in cms-S mitochondria of sterile plants. Spontaneous fertile cytoplasmic revertants from cms-S in a WF9 nuclear background also synthesized the 130-kDa polypeptide. Therefore, the mere presence or absence of the URF1 gene product of S2 does not determine the fertility status of maize plants, because male sterile and male fertile (nuclear restored and revertant) plants can contain equivalent amounts of the 130-kDa polypeptide.     

Cytoplasmic and nuclear Fos protein forms regulate resumption of spermatogenesis in the frog, Rana esculenta.

The role of Fos proteins in the regulation of germ cell progression during spermatogenesis has been studied in the frog, Rana esculenta. A peculiarity of this animal model is the finding of Fos in cytoplasmic compartment of primary spermatogonia during the resting period of the annual reproductive cycle. Interestingly, Fos is localized in the nuclear compartment when spermatogenesis resumes. Using Western blot analysis, we show that a 52-kDa Fos protein occurs in testicular cytosolic preparations, whereas two different Fos signals of 43 and 68 kDa are typical of the nuclear compartment. The 68-kDa Fos immunoreactive protein increases in nuclear extracts in concomitance with spermatogonia (SPG) proliferation either during the annual sexual cycle or in experimental animal groups where SPG proliferation was induced by thermal stimulus (24 C). Indeed, an increase in proliferating cell nuclear antigen was detectable after thermal induction of mitotic activity. A decrease in the 52-kDa signal and a concomitant increase in the 68-kDa signal is observed in testes of 24 C treated groups. The use of alkaline phosphatase and alkaline phosphatase inhibitors indicates that the 68-kDa protein is a phosphorylated form. Estrogens, which are able to induce SPG proliferation, are responsible for the appearance of the 43-kDa Fos form in nuclear testicular extracts. In conclusion, our results show, for the first time in a vertebrate species, that storage in the cytoplasm, on the one hand, and appearance as well as phosphorylation of Fos proteins in the nucleus of germ cells, on the other hand, regulate spermatogenesis progression during the seasonal breeding. Moreover, the phosphorylated 68-kDa Fos form may be involved in mechanisms underlying SPG proliferation.     

(125I)iodoazidococaine, a photoaffinity label for the haloperidol-sensitive sigma receptor.

A carrier-free radioiodinated cocaine photo-affinity label, (-)-3-(125I)iodo-4-azidococaine [(125I)IACoc], has been synthesized and used as a probe for cocaine-binding proteins. Photoaffinity labeling with 0.5 nM (125I)IACoc resulted in selective derivatization of a 26-kDa polypeptide with the pharmacology of a sigma receptor in membranes derived from whole rat brain, rat liver, and human placenta. Covalent labeling of the 26-kDa polypeptide was inhibited by 1 microM haloperidol, di(2-tolyl)guanidine (DTG), 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP), dextromethorphan, and carbetapentane. Stereoselective protection of (125I)IACoc photolabeling by 3-PPP [(+)-3-PPP more potent than (-)-3-PPP] was observed. (125I)IACoc labeling of the 26-kDa polypeptide was also inhibited by 10 microM imipramine, amitriptyline, fluoxetine, benztropine, and tetrabenazine. The size of the (125I)I-ACoc-labeled proteins is consistent with the size of proteins photolabeled in guinea pig brain and liver membranes by using the sigma photolabel azido-[3H]DTG. Kinetic analysis of (125I)IACoc binding to rat liver microsomes revealed two sites with Kd values of 19 and 126 pM, respectively. The presence or absence of proteolytic inhibitors during membrane preparation did not alter the size of the photolabeled sigma receptor, indicating that the 26-kDa polypeptide was not derived from a larger protein. In summary, (125I)IACoc is a potent and highly specific photoaffinity label for the haloperidol-sensitive sigma receptor and will be useful for its biochemical and molecular characterization.