Isolation of a cDNA encoding a novel GTP-binding protein of Arabidopsis thaliana

A cDNA encoding for a 68 kDa GTP-binding protein was isolated from Arabidopsis thaliana (aG68). This clone is a member of a gene family that codes for a class of large GTP-binding proteins. This includes the mammalian dynamin, yeast Vps1p and the vertebrate Mx proteins. The predicted amino acid sequence was found to have high sequence conservation in the N-terminal GTP-binding domain sharing 54% identity to yeast Vps1p, 56% amino acid identity to rat dynamin and 38% identity to the murine Mx1 protein. The northern analysis shows expression in root, leaf, stem and flower tissues, but in mature leaves at lower levels. Southern analysis indicates that it may be a member of a small gene family or the gene may contain an intron.     

Cloning and characterization of a cDNA encoding a Rab1-like small GTP-binding protein from Petunia hybrida

The cloning of small GTP-binding proteins from Petunia hybrida was performed using a PCR-based strategy. Degenerate primers were designed from the DTAGQE and FMETSA consensus sequences. Three different cDNAs were amplified. The deduced polypeptide sequences PhPCRGP1 and PhPCRGP2 were homologous to RB11_HUMAN and PhPCRGP3 to RAB1A_HUMAN. Using PhPCRGP3 as a probe, 8 identical clones were selected from a Petunia leaf cDNA library. They all encode the same 22.5 kDa polypeptide, PhRAB1, able to bind GTP in vitro and 72% identical to RAB1A_HUMAN. Hybridizable mRNAs encoding PhRAB1 accumulated preferentially in opened flowers.     

Isolation and analysis of cDNAs encoding small GTP-binding proteins of Arabidopsis thaliana.

We previously isolated a DNA fragment from Arabidopsis thaliana homologous to the mammalian ras gene and named it ara [Matsui et al., Gene 76 (1989) 313-319]. Screening of cDNA clones homologous to ara in A. thaliana resulted in the isolation of four homologous genes. The products of these genes, ARA-2, ARA-3, ARA-4 and ARA-5, showed conservation of amino acids (aa) in four regions, all of which are present in small GTP-binding proteins, and are important for GTPase/GTP-binding activities. These products were highly homologous to those of the YPT genes of Saccharomyces cerevisiae and the ypt gene of Schizosaccharomyces pombe in the regions around aa 45, which is thought to be the site interacting with effector molecules. The products of these four genes showed characteristic aa sequence at their C termini, Cys-Cys-Xaa-Xaa. Another characteristic of this family is presence of Ser in place of Gly in the first conserved region (Gly12 of mammalian GTP-binding Ras protein).     

Analysis of a Nicotiana plumbaginifolia cDNA encoding a novel small GTP-binding protein.

Small GTP-binding proteins belonging to the Ras superfamily have been found in evolutionarily divergent organisms. Here, we report the isolation and analysis of a cDNA encoding a putative small GTP-binding protein, designated Rhn1, from the plant, Nicotiana plumbaginifolia. The 21.8-kDa protein has 60% amino acid similarity with the mammalian Rab5 proteins. The Rhn1 protein is encoded by a small multigene family. Northern analysis shows the highest steady-state mRNA levels to be in roots and flowers. Furthermore, the Rhn1 protein has 80% amino acid similarity with an Arabidopsis small GTP-binding protein, designated Rha1.     

Molecular characterization of an Arabidopsis thaliana cDNA coding for a monofunctional aspartate kinase

A cDNA clone encoding a monofunctional aspartate kinase (AK, ATP:L-aspartate 4-phosphotransferase, EC 2.7.2.4) has been isolated from an Arabidopsis thaliana cell suspension cDNA library using a homologous PCR fragment as hybridizing probe. Amplification of the PCR fragment was done using a degenerate primer designed from a conserved region between bacterial monofunctional AK sequences and a primer identical to a region of the A. thaliana bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH). By comparing the deduced amino acid sequence of the fragment with the bacterial and yeast corresponding gene products, the highest identity score was found with the Escherichia coli AKIII enzyme that is feedback-inhibited by lysine (encoded by lysC). The absence of HSDH-encoding sequence at the COOH end of the peptide further implies that this new cDNA is a plant lysC homologue. The presence of two homologous genes in A. thaliana is supported by PCR product sequences, Southern blot analysis and by the independent cloning of the corresponding second cDNA (see Tang et al., Plant Molecular Biology 34, pp. 287–294 [this issue]). This work is the first report of cloning a plant putative lysine-sensitive monofunctional AK cDNA. The presence of at least two genes is discussed in relation to possible different physiological roles of their respective product.     

Molecular cloning of cDNA encoding N-acetylglucosaminyltransferase II from Arabidopsis thaliana

N-acetylglucosaminyltransferase II (GnTII, EC 2.4.1.143) is a Golgi enzyme involved in the biosynthesis of glycoprotein-bound N-linked oligosaccharides, catalysing an essential step in the conversion of oligomannose-type to complex N-glycans. GnTII activity has been detected in both animals and plants. However, while cDNAs encoding the enzyme have already been cloned from several mammalian sources no GnTII homologue has been cloned from plants so far. Here we report the molecular cloning of an Arabidopsis thalianaGnTII cDNA with striking homology to its animal counterparts. The predicted domain structure of A. thalianaGnTII indicates a type II transmembrane protein topology as it has been established for the mammalian variants of the enzyme. Upon expression of A. thalianaGnTII cDNA in the baculovirus/insect cell system, a recombinant protein was produced that exhibited GnTII activity.     

Cloning and characterization of a cDNA encoding serine palmitoyltransferase in Arabidopsis thaliana

The first and committed step in de novo sphingolipid synthesis is catalysed by serine palmitoyltransferase (EC 2.3.1.50), which condenses serine and palmitoyl-CoA to form 3-ketosphinganine in a pyridoxal-5'-phosphate-dependent reaction. We have isolated and characterized a cDNA clone from Arabidopsis thaliana that is homologous to yeast and mammalian LCB2. For a functional identification, the A. thaliana homologous cDNA was expressed in Escherichia coli, which resulted in significant production of new sphinganine in E. coli cells.     

Molecular characterization of rgp2, a gene encoding a small GTP-binding protein from rice

Summary We previously reported the isolation of rgp1, a gene from rice, which encodes a ras-related GTP-binding protein, and subsequently showed that the gene induces specific morphological changes in transgenic tobacco plants. Here, we report the isolation and characterization of an rgp1 homologue, rgp2, from rice. The deduced rgp2 protein sequence shows 53% identity with the rice rgp1 protein, but 63% identity with both the marine ray ora3 protein, which is closely associated with synaptic vesicles of neuronal tissue, and the mammalian rab11 protein. Conservation of particular amino acid sequence motifs places rgp2 in the rab/ypt subfamily, which has been implicated in vesicular transport. Northern blot analysis of rgp1 and rgp2 suggests that both genes show relatively high, but differential, levels of expression in leaves, stems and panicles, but low levels in roots. In addition, whereas rgp1 shows maximal expression at a particular stage of plantlet growth, rgp2 is constitutively expressed during the same period. Southern blot analysis suggests that, in addition to rgp1 and rgp2, several other homologues exist in rice and these may constitute a small multigene family.     

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.     

Functional cloning of an Arabidopsis thaliana cDNA encoding cycloeucalenol cycloisomerase

Plants and certain protists use cycloeucalenol cycloisomerase (EC ) to convert pentacyclic cyclopropyl sterols to conventional tetracyclic sterols. We used a novel complementation strategy to clone a cycloeucalenol cycloisomerase cDNA. Expressing an Arabidopsis thaliana cycloartenol synthase cDNA in a yeast lanosterol synthase mutant provided a sterol auxotroph that could be genetically complemented with the isomerase. We transformed this yeast strain with an Arabidopsis yeast expression library and selected sterol prototrophs to obtain a strain that accumulated biosynthetic ergosterol. The novel phenotype was conferred by an Arabidopsis cDNA that potentially encodes a 36-kDa protein. We expressed this cDNA (CPI1) in Escherichia coli and showed by gas chromatography-mass spectrometry that extracts from this strain isomerized cycloeucalenol to obtusifoliol in vitro. The cDNA will be useful for obtaining heterologously expressed protein for catalytic studies and elucidating the in vivo roles of cyclopropyl sterols.