Temporal regulation of Cat-1 (cationic amino acid transporter-1) gene transcription during endoplasmic reticulum stress.

Expression of the Cat-1 gene (cationic amino acid transporter-1) is induced in proliferating cells and in response to a variety of stress conditions. The expression of the gene is mediated via a TATA-less promoter. In the present study we show that an Sp1 (specificity protein 1)-binding site within a GC-rich region of the Cat-1 gene controls its basal expression and is important for induction of the gene during the UPR (unfolded protein response). We have shown previously that induction of Cat-1 gene expression during the UPR requires phosphorylation of the translation initiation factor eIF2alpha (eukaryotic initiation factor 2alpha) by PERK (protein-kinase-receptor-like endoplasmic reticulum kinase), one of the signalling pathways activated during the UPR. This leads to increased translation of the transcription factor ATF4 (activating transcription factor 4). We also show that a second signalling pathway is required for sustained transcriptional induction of the Cat-1 gene during the UPR, namely activation of IRE1 (inositol-requiring enzyme 1) leading to alternative splicing of the mRNA for the transcription factor XBP1 (X-box-binding protein 1). The resulting XBP1s (spliced XBP1) can bind to an ERSE (endoplasmic-reticulum-stress-response-element), ERSE-II-like, that was identified within the Cat-1 promoter. Surprisingly, eIF2alpha phosphorylation is required for accumulation of XBP1s. We propose that the signalling via phosphorylated eIF2alpha is required for maximum induction of Cat-1 transcription during the UPR by inducing the accumulation of both ATF4 and XBP1s.

Effect of the cauliflower Or transgene on carotenoid accumulation and chromoplast formation in transgenic potato tubers.

Transgenic plants have facilitated our understanding of the functional roles of genes and the metabolic processes affected in plants. Recently, the Or gene was isolated from an orange cauliflower mutant and it was shown that the Or gene could serve as a novel genetic tool to enrich carotenoid content in transgenic potato tubers. An in-depth characterization of these Or transgenic lines is presented here. It was found that the Or transgene may facilitate the identification of potential rate-limiting step(s) of the carotenoid biosynthetic pathway. The Or transgenic tubers accumulated not only increased levels of carotenoids that normally are present in the controls, but also three additional metabolite intermediates of phytoene, phytofluene, and zeta-carotene, indicating that the desaturation steps became limiting following the expression of the Or transgene. Moreover, we observed that long-term cold storage greatly enhanced carotenoid content in the Or transgenic tubers to a level of 10-fold over controls. Expression of the Or transgene in the transgenic plants caused no dramatic changes in the transcript levels of the endogenous carotenoid biosynthetic genes, which is in agreement with the Or gene not directly controlling carotenoid biosynthesis. Microscope analysis revealed that the Or transgene conferred the formation of chromoplasts containing carotenoid sequestering structures in a heterologous system. Such structures were not observed in tubers of potato cultivars that accumulate high levels of carotenoids. Collectively, these results provide direct evidence demonstrating that the Or gene indeed controls chromoplast differentiation and that regulation of chromoplast formation can have a profound effect on carotenoid accumulation in plants.

Phytoene desaturase is present in a large protein complex in the plastid membrane.

Phytoene desaturase (PDS; EC 1.14.99.-) represents one of the key enzymes in the carotenoid biosynthetic pathway and is present in nearly all types of plastids in plants. To further characterize PDS, we isolated the PDS cDNA from cauliflower (BoPDS) and confirmed its function by heterologous expression in a strain of Escherichia coli containing a carotenoid-producing plasmid. The BoPDS cDNA encodes a predicted mature protein of approximately 55 kDa. In comparison with PDS from a few other plant species, BoPDS exhibited a high enzyme activity in E. coli, and its expression in plastids was independent of carotenoid levels. Plastids were purified from tissues of different plant species including cauliflower curds, tomato fruits, carrot roots and Arabidopsis leaves. By employing both Blue Native PAGE and SDS-PAGE approaches in conjunction with Western blot analysis, it was found that PDS in these plants existed in two forms. The plastid membrane form was present in a large protein complex of approximately 350 kDa, whereas the stroma version was in an approximately 660 kDa complex.

A feedback transcriptional mechanism controls the level of the arginine/lysine transporter cat-1 during amino acid starvation.

The adaptive response to amino acid limitation in mammalian cells inhibits global protein synthesis and promotes the expression of proteins that protect cells from stress. The arginine/lysine transporter, cat-1, is induced during amino acid starvation by transcriptional and post-transcriptional mechanisms. It is shown in the present study that the transient induction of cat-1 transcription is regulated by the stress response pathway that involves phosphorylation of the translation initiation factor, eIF2 (eukaryotic initiation factor-2). This phosphorylation induces expression of the bZIP (basic leucine zipper protein) transcription factors C/EBP (CCAAT/enhancer-binding protein)-beta and ATF (activating transcription factor) 4, which in turn induces ATF3. Transfection experiments in control and mutant cells, and chromatin immunoprecipitations showed that ATF4 activates, whereas ATF3 represses cat-1 transcription, via an AARE (amino acid response element), TGATGAAAC, in the first exon of the cat-1 gene, which functions both in the endogenous and in a heterologous promoter. ATF4 and C/EBPbeta activated transcription when expressed in transfected cells and they bound as heterodimers to the AARE in vitro. The induction of transcription by ATF4 was inhibited by ATF3, which also bound to the AARE as a heterodimer with C/EBPbeta. These results suggest that the transient increase in cat-1 transcription is due to transcriptional activation caused by ATF4 followed by transcriptional repression by ATF3 via a feedback mechanism.

Polymorphism and selection at the SerH immobilization antigen locus in natural populations of Tetrahymena thermophila.

The SerH locus of Tetrahymena thermophila is one of several paralogous loci with genes encoding variants of the major cell surface protein known as the immobilization antigen (i-ag). The locus is highly polymorphic, raising questions concerning functional equivalency and selective forces acting on its multiple alleles. Here, we compare the sequences and expression of SerH1, SerH3, SerH4, SerH5, and SerH6. The precursor i-ags are highly similar. They are rich in alanine, serine, threonine, and cysteine and they share nearly identical ER translocation and GPI addition signals. The locations of the 39 cysteines are highly conserved, particularly in the 3.5 central, imperfect tandem repeats in which 8 periodic cysteines punctuate alternating short and long stretches of amino acids. Hydrophobicity patterns are also conserved. Nevertheless, amino acid sequence identity is low, ranging from 60.7 to 82.9%. At the nucleotide level, from 9.7 to 26.7% of nucleotide sites are polymorphic in pairwise comparisons. Expression of each allele is regulated by temperature-sensitive mRNA stability. H mRNAs are stable at <36 degrees but are unstable at >36 degrees. The H5 mRNA, which is less affected by temperature, has a different arrangement of the putative mRNA destabilization motif AUUUA. Statistical analysis of SerH genes indicates that the multiple alleles are neutral. Significantly low ratios of the rates of nonsynonymous to synonymous amino acid substitutions suggest that the multiple alleles are subject to purifying (negative) selection enforcing constraints on structure.

Transcription regulation is demonstrated for five key enzymes in Giardia intestinalis cyst wall polysaccharide biosynthesis.

The cyst wall of Giardia intestinalis contains proteins and a novel N-acetylgalactosamine (GalNAc) polysaccharide, which is its major constituent. GalNAc is not present in growing trophozoites, but is synthesized during encystment via an inducible pathway of enzymes that produce UDP-GalNAc from fructose 6-phosphate. This report focuses on the regulation of these enzymes and thus the genes for glucosamine 6-phosphate N-acetyltransferase (GNA), phosphoacetylglucosamine mutase (AGM), UDP-N-acetylglucosamine pyrophosphorylase (UAP), and UDP-N-acetylglucosamine 4-epimerase (UAE) were cloned and expressed in Escherichia coli. Each of these expressed enzymes had the predicted activity and was used to generate antibodies. Northern and Western blot analyses demonstrated that both the mRNA and protein levels for all of these enzymes increase during encystment. Nuclear run-on assays of these and the previously analyzed glucosamine 6-phosphate deaminase (GNP; glucosamine 6-P isomerase) showed that all of the genes responsible for UDP-GalNAc synthesis during encystment are induced at the transcription level.

UDP-N-acetylglucosamine 4'-epimerase from the intestinal protozoan Giardia intestinalis lacks UDP-glucose 4'-epimerase activity.

The protozoan parasite Giardia intestinalis has a simple life cycle consisting of an intestinal trophozoite stage and an environmentally resistant cyst stage. The cyst is formed when a trophozoite encases itself within an external filamentous covering, the cyst wall, which is crucial to the cyst's survival outside of the host. The filaments in the cyst wall consist mainly of a beta (1-3) polymer of N-acetylgalactosamine. Its precursor, UDP-N-acetylgalactosamine, is synthesized from fructose 6-phosphate by a pathway of five inducible enzymes. The fifth, UDP-N-acetylglucosamine 4'-epimerase, epimerizes UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine reversibly. The epimerase of G. intestinalis lacks UDP-glucose/UDP-galactose 4'-epimerase activity and shows characteristic amino acyl residues to allow binding of only the larger UDP-N-acetylhexosamines. While the Giardia epimerase catalyzes the reversible epimerization of UDP-N-acetylglucosamine to UDP-N-acetylgalactosamine, the reverse reaction apparently is favored. The enzyme has a higher Vmax and a smaller Km in this direction. Therefore, an excess of UDP-N-acetylglucosamine is required to drive the reaction towards the synthesis of UDP-N-acetylgalactosamine, when it is needed for cyst wall formation. This forms the ultimate regulatory step in cyst wall biosynthesis.

The cauliflower Or gene encodes a DnaJ cysteine-rich domain-containing protein that mediates high levels of beta-carotene accumulation.

Despite recent progress in our understanding of carotenogenesis in plants, the mechanisms that govern overall carotenoid accumulation remain largely unknown. The Orange (Or) gene mutation in cauliflower (Brassica oleracea var botrytis) confers the accumulation of high levels of beta-carotene in various tissues normally devoid of carotenoids. Using positional cloning, we isolated the gene representing Or and verified it by functional complementation in wild-type cauliflower. Or encodes a plastid-associated protein containing a DnaJ Cys-rich domain. The Or gene mutation is due to the insertion of a long terminal repeat retrotransposon in the Or allele. Or appears to be plant specific and is highly conserved among divergent plant species. Analyses of the gene, the gene product, and the cytological effects of the Or transgene suggest that the functional role of Or is associated with a cellular process that triggers the differentiation of proplastids or other noncolored plastids into chromoplasts for carotenoid accumulation. Moreover, we demonstrate that Or can be used as a novel genetic tool to induce carotenoid accumulation in a major staple food crop. We show here that controlling the formation of chromoplasts is an important mechanism by which carotenoid accumulation is regulated in plants.

Ribosome stalling regulates IRES-mediated translation in eukaryotes, a parallel to prokaryotic attenuation.

It was previously shown that the mRNA for the cat-1 Arg/Lys transporter is translated from an internal ribosome entry site (IRES) that is regulated by cellular stress. Amino acid starvation stimulated cat-1 translation via a mechanism that requires translation of an ORF in the mRNA leader and remodeling of the leader to form an active IRES (the "zipper model" of translational control). It is shown here that slowing of the leader peptide elongation rate, either by cycloheximide or the introduction of rare codons, stimulated translation of the downstream ORF. These results suggest that ribosome stalling in the upstream ORF causes mRNA remodeling and formation of an active IRES. This control is reminiscent of translation attenuation in prokaryotic operons, where inhibition of translation elongation can regulate both mRNA translation and gene transcription by altering mRNA structure.

Giardia intestinalis glucosamine 6-phosphate isomerase: the key enzyme to encystment appears to be controlled by ubiquitin attachment.

The cyst wall of the parasitic protozoan, Giardia intestinalis, is composed of a polymer of N-acetylgalactosamine, the precursor of which is synthesized by an inducible enzyme pathway. The first enzyme in this pathway, glucosamine 6-phosphate isomerase, is transcriptionally regulated. During encystment and in mature cysts this isomerase appears to be modified by ubiquitin attachment. Thus, it might be targeted for destruction by an ubiquitin-mediated pathway, suggesting that glucosamine 6-phosphate isomerase expression is tightly regulated.

Transcriptional control of the arginine/lysine transporter, cat-1, by physiological stress.

Cells respond to physiological stress by phosphorylating the alpha subunit of the translation initiation factor eIF2. This adaptive response inhibits protein synthesis and up-regulates genes essential for cell survival. Cat-1, the transporter for the essential amino acids, arginine and lysine, is one of the up-regulated genes. We previously showed that stress increases cat-1 expression by coordinated stabilization of the mRNA and increased mRNA translation. This induction is triggered by amino acid depletion and the unfolded protein response (UPR), which is caused by unfolded proteins in the endoplasmic reticulum. We show here that cat-1 gene transcription is also increased by cellular stress. Our studies demonstrate that the cat-1 gene promoter/regulatory region is TATA-less and is located in a region that includes 94 bases of the first exon. Transcription from this promoter is stimulated 8-fold by cellular stress. An amino acid response element within the first exon is shown to be required for the response to amino acid depletion but not to the UPR. The stimulation of transcription by amino acid depletion requires activation of GCN2 kinase, which phosphorylates eIF2alpha. This phosphorylation also induces translation of the cat-1 mRNA, demonstrating that stress-induced transcriptional and translational control of cat-1 are downstream targets of a signaling pathway initiating with eIF2alpha phosphorylation. Our studies show that the increase in cat-1 gene expression by cellular stress involves at least three types of coordinate regulation: regulation of transcription, regulation of mRNA stability, and regulation of mRNA translation.