Cloning, characterization, and expression of cDNAs encoding human delta 1-pyrroline-5-carboxylate dehydrogenase.
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Delta 1-pyrroline-5-carboxylate dehydrogenase (P5CDh; EC 1.5.1.12), a mitochondrial matrix NAD(+)-dependent dehydrogenase, catalyzes the second step of the proline degradation pathway. Deficiency of this enzyme is associated with type II hyperprolinemia (HPII), an autosomal recessive disorder characterized by accumulation of delta 1-pyrroline-5-carboxylate (P5C) and proline. As an initial step in understanding the biochemistry of human P5CDh and molecular basis of HPII, we utilized published peptide sequence data and degenerate primer polymerase chain reaction to clone two full-length human P5CDh cDNAs, differing in length by 1 kilobase pair (kb). Both cDNAs have the identical 1689-base pair open reading frame encoding a protein of 563 residues with a predicted molecular mass of 62 kDa. The long cDNA contains an additional 1-kb insert in the 3'-untranslated region that appears to be an alternatively spliced intron. The conceptual translation of human P5CDh has 89% sequence identity with the published human P5CDh peptide sequences and 42 and 26% identity with Saccharomyces cerevisiae and Escherichia coli P5CDhs, respectively, as well as homology to several other aldehyde dehydrogenases. Both P5CDh cDNA clones detect a single 3.2-kb transcript on Northern blots of multiple human tissues, indicating the long cDNA containing the 3'-untranslated intron represents the predominant transcript. The P5CDh structural gene appears to be single copy with a size of about 20 kb localized to chromosome 1. To confirm the identity of the putative P5CDh cDNAs, we expressed them in a P5CDh-deficient strain of S. cerevisiae. Both conferred measurable P5CDh activity and the ability to grow on proline as a sole nitrogen source. (+info)
The Agaricus bisporus pruA gene encodes a cytosolic delta 1-pyrroline-5-carboxylate dehydrogenase which is expressed in fruit bodies but not in gill tissue.
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A fortuitously cloned 3'-truncated cDNA encoding the Agaricus bisporus delta 1-pyrroline-5-carboxylate dehydrogenase was used to characterize the complete gene. The gene would encode a cytosolic polypeptide of 546 amino acids, and the basidiomycetous gene was evenly expressed in various parts of the mushroom except for the gills. No expression was detected in compost-grown mycelium. The steady-state mRNA level of the gene in the vegetative phase was determined on simple synthetic media and was two- to threefold higher with ammonium or proline as the sole nitrogen source compared to glutamate as the sole nitrogen source. Moreover, the steady-state mRNA level was not markedly influenced by addition of ammonium phosphate to proline- or glutamate-utilizing cultures. The results suggest that ammonium and the amino acids proline and glutamate are equally preferred nitrogen sources in this organism and are consistent with previous observations of H. M Kalisz, D.A. Wood, and D. Moore (Trans. Br. Mycol. Soc. 88:221-227, 1987) that A. bisporus continues to degrade protein and secrete ammonium even if ammonium and glucose are present in the culture medium. (+info)
Proline accumulation and salt-stress-induced gene expression in a salt-hypersensitive mutant of Arabidopsis.
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The sos1 mutant of Arabidopsis thaliana is more than 20 times more sensitive to NaCl stress than wild type Arabidopsis. Because proline (Pro) is generally thought to have an important role in plant salt tolerance, the sos1 mutant and the wild type were compared with respect to their capacity to accumulate Pro under NaCl stress, and sos1 mutant plants accumulated more Pro than wild-type. The P5CS gene, which catalyzes the rate-limiting step in Pro biosynthesis, is induced by salt stress to a higher level in sos1 than in the wild type. Although a defective high-affinity K uptake system in sos1 causes K deficiency and inhibits growth in NaCl-treated plants, this decrease is not a sufficient signal for Pro accumulation and P5CS gene expression. Not all salt-stress-induced genes have a higher level of expression in sos1. The expression levels of AtPLC and RD29A, which encode a phospholipase C homolog and a putative protective protein, respectively, are the same in sos1 as in the wild type. However, the expression of AtMYB, which encodes a putative transcriptional factor, is induced to a much higher level by salt stress in sos1. Thus, the SOS1 gene product serves as a negative regulator for the expression of P5CS and AtMYB, but has no effect on AtPLC and RD29A expression. (+info)
GTP hydrolysis is essential for protein import into the mitochondrial matrix.
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Protein import into the innermost compartment of mitochondria (the matrix) requires a membrane potential (delta psi) across the inner membrane, as well as ATP-dependent interactions with chaperones in the matrix and cytosol. The role of nucleoside triphosphates other than ATP during import into the matrix, however, remains to be determined. Import of urea-denatured precursors does not require cytosolic chaperones. We have therefore used a purified and urea-denatured preprotein in our import assays to bypass the requirement of external ATP. Using this modified system, we demonstrate that GTP stimulates protein import into the matrix; the stimulatory effect is directly mediated by GTP hydrolysis and does not result from conversion of GTP to ATP. Both external GTP and matrix ATP are necessary; neither one can substitute for the other if efficient import is to be achieved. These results suggest a "push-pull" mechanism of import, which may be common to other post-translational translocation pathways. (+info)
Mutations in the Delta1-pyrroline 5-carboxylate dehydrogenase gene cause type II hyperprolinemia.
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We surveyed Delta1-pyrroline 5-carboxylate dehydrogenase genes from four patients with hyperprolinemia type II using RT-PCR amplification, genomic PCR amplification and direct sequencing. We found four mutant alleles, two with frameshift mutations [A7fs(-1) and G521fs(+1)] and two with missense mutations (S352L and P16L). To test the functional consequences of three of these, we expressed them in a P5CDh-deficient strain of Saccharomyces cerevisiae . In contrast to wild-type human P5CDh, yeast expressing S352L and G521fs(+1) failed to grow on proline and had no detectable P5CDh activity. The P16L allele, however, produced fully functional P5CDh and subsequent analysis suggests that it is polymorphic in the relevant (Spanish) population. Interestingly, the G521fs(+1) allele segregates in the large Irish Traveller pedigree used to define the HPII phenotype. To our knowledge, this is the first description of the molecular basis for this inborn error. (+info)
Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for Delta1-pyrroline-5-carboxylate synthetase from tomato.
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We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Delta1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis. tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G. Garcia-Rios, T. Fujita, P.C. LaRosa, R.D. Locy, J.M. Clithero, R.A. Bressan, L.N. Csonka [1997] Proc Natl Acad Sci USA 94: 8249-8254), whereas tomPRO2 encodes a full-length P5CS. We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals. Treatment with 200 mM NaCl resulted in a >60-fold increase in Pro levels in roots and leaves. However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress. Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen. We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato. Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome. Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes. (+info)