Identification and functional expression in yeast of a prenylcysteine alpha-carboxyl methyltransferase gene from Arabidopsis thaliana.

Most isoprenylated proteins are alpha-carboxyl-methylated. However, despite numerous studies linking protein isoprenylation in plants to cell cycle control, meristem development, and phytohormone signaling, alpha-carboxyl methylation of isoprenylated plant proteins has not been characterized in detail. Here, we report the cloning of a prenylcysteine alpha-carboxyl methyltransferase gene (AtSTE14) from Arabidopsis thaliana. AtSTE14 restores fertility and enzymatic activity to a ste14 mutant of Saccharomyces cerevisiae, confirming its identity as a bona fide prenylcysteine alpha-carboxyl methyltransferase gene. Furthermore, the presence of AtSTE14 transcripts in various Arabidopsis organs suggests a ubiquitous role for the AtSTE14 protein in plant growth and development. These results demonstrate that Arabidopsis thaliana possesses a functional prenylcysteine alpha-carboxyl methyltransferase involved in post-isoprenylation protein processing.

Expression and processing of a hormonally regulated beta-expansin from soybean.

Expansin proteins are essential components of acid-induced cell wall loosening in plants. Beta-expansins, which constitute a subfamily of related expansin proteins, include the group I grass pollen allergens. To provide a better description of beta-expansin expression, we have characterized a cytokinin-inducible beta-expansin from soybean (Glycine max cv Mandarin) called Cim1. Our results demonstrate that the hormones cytokinin and auxin act synergistically to induce the accumulation and proteolytic processing of Cim1. Carboxyl terminal truncation of a 35-kD form of Cim1 is predicted to remove the putative cellulose binding domain from the amino terminal cysteine-rich domain, resulting in a 20-kD form of the protein. Furthermore, the identical amino termini of the 35- and 20-kD forms of Cim1 correspond to a position 11 amino acids downstream of the predicted signal sequence cleavage site, suggesting proteolysis of a short amino terminal propeptide after removal of the signal peptide. This propeptide fragment contains a consensus site for N-glycosylation and our data suggest that it is glycosylated by a tunicamycin-sensitive mechanism in cultured soybean cells. The onset of Cim1 expression correlates with increased growth of soybean cultures. Ultimately, Cim1 is rapidly and specifically proteolyzed as soybean cultures reach stationary phase. These findings are consistent with the hypothesis that beta-expansin proteins are extensively modified by post-translational N-glycosylation and proteolysis.

Functional implications of protein isoprenylation in plants.

Plant protein isoprenylation has received considerable attention in the past decade. Since the initial discovery of isoprenylated plant proteins and their respective protein isoprenyltransferases, several research groups have endeavored to understand the physiological significance of this process in plants. Various experimental approaches, including inhibitor studies, systematic methods of protein identification, and mutant analyses in Arabidopsis thaliana, have enabled these groups to elucidate important roles for isoprenylated proteins in cell cycle control, signal transduction, cytoskeletal organization, and intracellular vesicle transport. This article reviews recent progress in understanding the functional implications of protein isoprenylation in plants.

A new class of proteins capable of binding transition metals.

Ion uptake, transport, and sequestration are essential to meet the nutritional requirements for plant growth and development. Furthermore, regulation of these processes is critical for plants to tolerate toxic levels of ions. The examination of isoprenylated proteins encoded by Arabidopsis thaliana and Glycine max cDNAs revealed a unique family of proteins containing putative metal-binding motifs (the core sequence is M/LXCXXC). Here, we describe this new class of proteins, which are capable of being isoprenylated and binding transition metal ions. Members of this family contain consensus isoprenylation (CaaX) sites, which we demonstrate are efficiently isoprenylated in vitro. ATFP3, a representative of the Arabidopsis family, was expressed in Escherichia coli and examined for metal-binding activity in vitro. Analysis of the interaction of ATFP3 with metal-chelating columns (IMAC) suggested that it binds to Cu2+, Ni2+, or Zn2+. To test whether proteins with these characteristics are present in other plant species, tobacco BY2 cells were labeled in vivo with [14C]mevalonate and the resulting mevalonate-labeled proteins were tested for metal-binding activity. Several soluble, isoprenylated proteins which bound copper-IMAC columns were revealed. Consistent with a wide-spread distribution of these proteins in plants, their presence was observed in Arabidopsis, soybean, and tobacco.

Cytokinin regulates the expression of a soybean beta-expansin gene by a post-transcriptional mechanism.

The cytokinin-inducible soybean mRNA Cim1 accumulates 20-60-fold upon cytokinin addition to cytokinin-starved soybean suspension cultures. In this report, we demonstrate that cytokinin-induced stability of the Cim1 mRNA plays an important role in the accumulation of the message. We also present evidence that cytokinin-induced Cim1 stability is blocked by the addition of the protein phosphatase inhibitor okadaic acid. Thus, we suggest that protein phosphatase activity is required for the cytokinin-induced stability and subsequent accumulation of Cim1 in soybean cells. The deduced amino acid sequence of the Cim1 protein product is similar to the group I pollen allergens from various plants, which constitute a subfamily of expansin proteins. The relatedness between Cim1 and the expansins supports our hypothesis that the protein product of Cim1 is localized to the cell wall and suggests a role for Cim1 in cytokinin-regulated cell wall expansion. Thus, post-transcriptional regulation of Cim1 by cytokinin may represent a molecular link between cytokinin and changes in cell shape and size.

Prenylation of oncogenic human PTP(CAAX) protein tyrosine phosphatases.

Many isoprenylated proteins are known to participate in signal transduction, but not all have been identified. Using an in vitro prenylation screen, two human cDNAs (PTP(CAAXI) and PTP(CAAX2)) homologous to the rat PRL-1 and human OV-1 protein tyrosine phosphatase genes were identified. PTP(CAAXI) and PTP(CAAX2) were farnesylated in vitro by mammalian farnesyl:protein transferase, and epitope-tagged PTP(CAAX2) was prenylated in epithelial cells. Overexpression of PTP(CAAXI) and PTP(CAAX2) in epithelial cells caused a transformed phenotype in culture and tumor growth in nude mice. Thus, PTP(CAAXI) and PTP(CAAX2) represent a novel class of isoprenylated, oncogenic protein tyrosine phosphatases.

Positive and negative regulation of a sterol biosynthetic gene (ERG3) in the post-squalene portion of the yeast ergosterol pathway.

Regulation of sterol biosynthesis in the terminal portion of the pathway represents an efficient mechanism by which the cell can control the production of sterol without disturbing the production of other essential mevalonate pathway products. We demonstrate that mutations affecting early and late steps in sterol homeostasis modulate the expression of the ERG3 gene: a late step in sterol biosynthesis in yeast. Expression of ERG3 is increased in response to a mutation in the major isoform of HMG CoA reductase which catalyzes the rate-limiting step of sterol biosynthesis. Likewise, mutations in non-auxotrophic ergosterol biosynthetic genes downstream of squalene production (erg2, erg3, erg4, erg5, and erg6) result in an up-regulation of ERG3 expression. Deletion analysis of the ERG3 promoter identified two upstream activation sequences: UAS1 which when deleted reduces ERG3 gene expression 3-4-fold but maintains sterol regulation and UAS2, which when deleted further reduces ERG3 expression and abolishes sterol regulation. The recent isolation of two yeast genes responsible for the esterification of intracellular sterol (ARE1 and ARE2) has enabled us to directly analyze the relationship between sterol esterification and de novo biosynthesis. Our results demonstrate that the absence of sterol esterification leads to a decrease in total intracellular sterol and ERG3 is a target of this negative regulation.

Identification and isoprenylation of plant GTP-binding proteins.

To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies).

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.

Changes in Protein Isoprenylation during the Growth of Suspension-Cultured Tobacco Cells.

Isoprenylation facilitates the association of proteins with intracellular membranes and/or other proteins. In mammalian and yeast cells, isoprenylated proteins are involved in signal transduction, cell division, organization of the cytoskeleton, and vesicular transport. Recently, protein isoprenylation has been demonstrated in higher plants, but little is currently known about the functions of isoprenylated plant proteins. We report that inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (lovastatin) or prenyl:protein transferases (perilly alcohol) severely impair the growth of cultured tobacco (Nicotiana tabacum) cells but only when added within the first 2 d following transfer to fresh medium, before any increase in culture volume is detectable. This "window" of sensitivity to inhibitors of protein isoprenylation correlates temporally with an increase in [14C]mevalonate incorporation into tobacco cell proteins in vitro. We have also observed a marked increase in farnesyl:protein transferase activity at this early time in the growth of tobacco cultures. In contrast, type I geranylgeranyl:protein transferase activity does not change significantly during culture growth. Although these events coincide with the replication of DNA, I [mu]M lovastatin-treated cells are capable of DNA synthesis, suggesting that lovastatin-induced cell growth arrest is not due to inhibition of DNA replication. Together, these data support the hypothesis that protein isoprenylation is necessary for the early stages of growth of tobacco cultures.

Novel isoprenylated proteins identified by an expression library screen.

Isoprenylated proteins are involved in eukaryotic cell growth and signal transduction. The protein determinant for prenylation is a short carboxyl-terminal motif containing a cysteine, to which the isoprenoid is covalently attached via thioether linkage. To date, isoprenylated proteins have almost all been identified by demonstrating the attachment of an isoprenoid to previously known proteins. Thus, many isoprenylated proteins probably remain undiscovered. To identify novel isoprenylated proteins for subsequent biochemical study, colony blots of a Glycine max cDNA expression library were [3H]farnesyl-labeled in vitro. Proteins identified by this screen contained several different carboxyl termini that conform to consensus farnesylation motifs. These proteins included known farnesylated proteins (DnaJ homologs) and several novel proteins, two of which contained six or more tandem repeats of a hexapeptide having the consensus sequence (E/G)(G/P)EK(P/K)K. Thus, plants contain a diverse array of genes encoding farnesylated proteins, and our results indicate that fundamental differences in the identities of farnesylated proteins may exist between plants and other eukaryotes. Expression library screening by direct labeling can be adapted to identify isoprenylated proteins from other organisms, as well as proteins with other post-translational modifications.

Cytokinin regulation of a soybean pollen allergen gene.

Cytokinin treatment of suspension-cultured soybean cells stimulated the accumulation of an mRNA, called cim 1, by a factor of ca. 20 within 4 h. Induction of cim 1 mRNA accumulation occurred at benzyladenine concentrations as low as 10(-8) M. Furthermore, cim 1 mRNA accumulation was stimulated in the absence of cytokinin by staurosporine (an inhibitor of protein kinases) and inhibited in the presence of cytokinin by okadaic acid (an inhibitor of protein phosphatases 1 and 2a), suggesting that cim 1 accumulation in response to cytokinin is dependent on cytokinin-induced dephosphorylation of one or more cellular proteins. The deduced amino acid sequence of the cim 1 protein product, derived from the complete nucleotide sequence of a cim 1 cDNA, was 40% identical to that of a perennial rye grass pollen allergen cDNA (Lol Pl). This sequence also indicated that the cim 1 protein product contains a putative signal peptide followed by predominantly hydrophilic residues, consistent with the hypothesis that it is exported to the apoplast.

Protein isoprenylation in suspension-cultured tobacco cells.

Many mammalian and yeast proteins, including small ras-like GTP binding proteins, heterotrimeric G protein gamma subunits, and nuclear lamins, have been shown to be covalently linked to isoprenoid derivatives of mevalonic acid. Isoprenylation of these proteins is required for their assembly into membranes and, hence, for their biological activity. In this report, it is shown that cultured tobacco cells, when pretreated with an inhibitor of endogenous mevalonic acid synthesis (lovastatin), incorporate radioactivity from 14C-mevalonic acid into proteins. Most of these proteins are membrane associated, and many are similar in mass to mammalian ras-like GTP binding proteins and nuclear lamins. Furthermore, it is shown that tobacco cell extracts catalyze the transfer of radioactivity from 3H-farnesyl pyrophosphate and 3H-geranylgeranyl pyrophosphate to protein substrates in vitro. These studies indicate the presence of at least two distinct prenyl:protein transferases in tobacco extracts: one that utilizes farnesyl pyrophosphate and preferentially modifies a substrate protein with a CAIM carboxy terminus (farnesyl:protein transferase) and one that utilizes geranylgeranyl pyrophosphate and preferentially modifies a substrate protein with a CAIL carboxy terminus (geranylgeranyl:protein transferase type I). This work provides a basis for future work on the role of protein isoprenylation in plant cell growth, signal transduction, and membrane biogenesis.

Levels and location of expression of the Agrobacterium tumefaciens pTiA6 ipt gene promoter in transgenic tobacco.

The location of gene expression of the Agrobacterium tumefaciens ipt gene promoter in transgenic tobacco plants was examined using the beta-glucuronidase (GUS) reporter gene. Expression of GUS was detected in every organ and most cell types examined. The highest levels of GUS activity were found in roots. To further examine the transcriptional basis of this broad expression pattern, deletions in the 5' non-coding region of the gene were translationally fused to two promoterless reporter genes, encoding the enzymes chloramphenicol acetyl transferase (CAT) and beta-glucuronidase (GUS). Reporter enzyme assays revealed the existence of an upstream segment required for maximal promoter function, the 5' end of which is between -442 and -408 of the Pipt ATG codon. This upstream segment is required for maximal levels of GUS expression in roots, but not in other organs, and a tobacco suspension-cultured cell line. The implications of broad ipt expression on the process of crown gall tumorigenesis are discussed.

Inhibition of growth of cultured tobacco cells at low concentrations of lovastatin is reversed by cytokinin.

De novo synthesis of mevalonic acid, which is catalyzed by 3-hydroxy-3-methylglutaryl coenzyme A reductase, is the first committed step in the formation of isoprenoid compounds. Various studies have shown that mevalonic acid-derived compounds are required for growth of plant and animal cells, a conclusion supported by the observation that cells treated with lovastatin (a potent inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A reductase) cease growth. We show that Nicotiana tabacum BY-2 cells, which require exogenous auxin for growth in culture but do not require exogenous cytokinin, are growth inhibited by 1 mum lovastatin. However, these cells are capable of growing in the presence of 1 mum lovastatin if 8 mum zeatin is supplied in the medium. Furthermore, benzyladenine, kinetin, and thidiazuron effectively reverse the inhibition of growth of these cells at 1 mum lovastatin, whereas adenine and 6-methyladenine have no effect. These results demonstrate that restoration of growth to lovastatin-treated cells is cytokinin specific and is not caused by metabolism of cytokinin into other isoprenoid compounds. Cytokinin does not effectively reverse the effects of higher concentrations of lovastatin, but mevalonic acid does, consistent with the hypothesis that cytokinin biosynthesis is more sensitive to lovastatin than the biosynthesis of other essential isoprenoid compounds in tobacco cells. This observation suggests that lovastatin can be used to induce cytokinin dependence in cytokinin-autonomous tobacco cell cultures.

Characterization of a stress-induced, developmentally regulated gene family from soybean.

We describe a family of stress-induced, developmentally regulated soybean genes for which cDNAs have been obtained from two different cultivars (Glycine max cv. Mandarin and Glycine max cv. Williams). The mRNAs corresponding to these cDNAs, called SAM22 and H4, respectively, accumulate predominantly in the roots of soybean seedlings but are present at high levels in the roots and leaves of mature plants. SAM22 accumulation is especially dramatic in senescent leaves. In addition, SAM22 accumulation can be induced on young leaves by wounding or by transpiration-mediated uptake of salicylic acid, methyl viologen, fungal elicitor, hydrogen peroxide or sodium phosphate (pH 6.9). Taken together, these data indicate that the genes corresponding to SAM22 and H4 are induced by various stresses and developmental cues. Southern blot analysis indicates that multiple copies of sequences related to SAM22 exist in the soybean genome. We also show that the nucleotide sequences of the cDNAs corresponding to SAM22 and H4 are 86% identical at the nucleotide level to each other and 70% identical at the amino acid level to the 'disease resistance response proteins' of Pisum sativum.

Induction of Specific mRNAs in Cultured Soybean Cells during Cytokinin or Auxin Starvation.

We report the isolation of five cDNA clones whose corresponding mRNAs accumulate in cultured soybean cells (Glycine max cv Mandarin) during cytokinin or auxin starvation. The levels of three of these mRNAs decrease rapidly after addition of 5 micromolar zeatin to cytokinin-starved cells or after addition of 10 micromolar alpha-naphthaleneacetic acid to auxin-starved cells. These mRNAs also exhibit various patterns of accumulation in the tissues of intact soybean plants. Partial nucleotide sequence analysis demonstrates that one of the cDNAs in the collection, called SAM46, is 46% identical at the amino acid level to the iron superoxide dismutase gene of Escherichia coli. Expression of this cDNA in Escherichia coli cells results in detectable iron superoxide dismutase activity, confirming the identity of the cDNA.

Cytokinin-induced mRNAs in cultured soybean cells.

We have isolated 20 cDNA clones for which the corresponding mRNAs accumulate 2- to 20-fold within 4 hr of cytokinin addition to cytokinin-starved soybean suspension cultures. These changes in mRNA levels precede the greening and cell growth induced by cytokinin in this system. Treatment with cycloheximide before cytokinin addition enhances the accumulation of these messages. In addition, the abundance of these mRNAs is influenced by auxin. We demonstrate that the mRNAs corresponding to the cDNAs in this collection display a variety of patterns of accumulation in the tissues of an intact plant. We also show that different cytokinins induce similar increases in message levels. However, analogs of cytokinin that are much less active in bioassays induce lower levels of message accumulation. Sequence analyses indicate that two of the cDNAs correspond to ribosomal protein genes. The remaining cDNAs are likely to represent previously undiscovered genes that function early in the initiation of plant cell growth and/or chloroplast development.