Regulation and function of proline oxidase under nutrient stress

Under conditions of nutrient stress, cells switch to a survival mode catabolizing cellular and tissue constituents for energy. Proline metabolism is especially important in nutrient stress because proline is readily available from the breakdown of extracellular matrix (ECM), and the degradation of proline through the proline cycle initiated by proline oxidase (POX), a mitochondrial inner membrane enzyme, can generate ATP. This degradative pathway generates glutamate and α‐ketoglutarate, products that can play an anaplerotic role for the TCA cycle. In addition the proline cycle is in a metabolic interlock with the pentose phosphate pathway providing another bioenergetic mechanism. Herein we have investigated the role of proline metabolism in conditions of nutrient stress in the RKO colorectal cancer cell line. The induction of stress either by glucose withdrawal or by treatment with rapamycin, stimulated degradation of proline and increased POX catalytic activity. Under these conditions POX was responsible, at least in part, for maintenance of ATP levels. Activation of AMP‐activated protein kinase (AMPK), the cellular energy sensor, by 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR), also markedly upregulated POX and increased POX‐dependent ATP levels, further supporting its role during stress. Glucose deprivation increased intracellular proline levels, and expression of POX activated the pentose phosphate pathway. Together, these results suggest that the induction of proline cycle under conditions of nutrient stress may be a mechanism by which cells switch to a catabolic mode for maintaining cellular energy levels. J. Cell. Biochem. 107: 759–768, 2009. © 2009 Wiley‐Liss, Inc.     

Inheritance of the snakeskin color pattern in the guppy, Poecilia reticulata

We made single-pair reciprocal crosses between the Green Snakeskin and Yellow Snakeskin domesticated strains of the guppy, Poecilia reticulata. The two snakeskin strains differ by a single autosomal gene, with the Green Snakeskin strain having the wild-type background coloration caused by the dominant gene (B), whereas the Yellow Snakeskin is homozygous for the recessive blond allele (bb). The snakeskin body and tail patterns characterizing males of these two strains are determined by two genes--Ssb and Sst--that are closely linked on the Y chromosome. The greenish-yellow tail color of the Green Snakeskin strain is mediated by an X-linked dominant gene, Grt. The recessive wild-type allele, Grt+, gives the hyaline tail color. In the Yellow Snakeskin strain, the Grt gene is expressed as a golden-yellow color as a result of the presence of the bb homozygous condition. The putative genotypes of the males and females of the Green Snakeskin strain are BB XGrt YSsb,Sst and BB XGrt XGrt, respectively. Males and females of the Yellow Snakeskin strain have the putative genotypes bb XGrt YSsb,Sst and bb XGrtXGrt, respectively. As a result of crossing over between the X and Y chromosomes, a few males and females of these two snakeskin strains may carry one or both snakeskin pattern genes (Ssb and Sst) on the X chromosome.     

Electrophysiological studies in Xenopus oocytes for the opening of Escherichia coli SecA-dependent protein-conducting channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5'-(beta,gamma-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.     

Electrophysiological Studies in Xenopus Oocytes for the Opening of Escherichia coli SecA-Dependent Protein-Conducting Channels

Protein translocation in Escherichia coli requires protein-conducting channels in cytoplasmic membranes to allow precursor peptides to pass through with adenosine triphosphate (ATP) hydrolysis. Here, we report a novel, sensitive method that detects the opening of the SecA-dependent protein-conducting channels at the nanogram level. E. coli inverted membrane vesicles were injected into Xenopus oocytes, and ionic currents were recorded using the two-electrode voltage clamp. Currents were observed only in the presence of E. coli SecA in conjunction with E. coli membranes. Observed currents showed outward rectification in the presence of KCl as permeable ions and were significantly enhanced by coinjection with the precursor protein proOmpA or active LamB signal peptide. Channel activity was blockable with sodium azide or adenylyl 5′-(β,γ-methylene)-diphosphonate, a nonhydrolyzable ATP analogue, both of which are known to inhibit SecA protein activity. Endogenous oocyte precursor proteins also stimulated ion current activity and can be inhibited by puromycin. In the presence of puromycin, exogenous proOmpA or LamB signal peptides continued to enhance ionic currents. Thus, the requirement of signal peptides and ATP hydrolysis for the SecA-dependent currents resembles biochemical protein translocation assay with E. coli membrane vesicles, indicating that the Xenopus oocyte system provides a sensitive assay to study the role of Sec and precursor proteins in the formation of protein-conducting channels using electrophysiological methods.     

Molecular basis for differential nucleotide binding of the nucleotide-binding domain of ABC-transporter CvaB

The cytoplasmic membrane protein CvaB, involved in colicin V secretion in Escherichia coli, belongs to the ABC-transporter family in which ATP hydrolysis is typically the driving force for substrate transport. However, our previous studies indicated that the nucleotide-binding domain of CvaB could also bind and hydrolyze GTP and, indeed, highly preferred GTP over ATP at low temperatures. In this study, we have examined the molecular basis of this preference. Sequence alignment and homology modeling of the CvaB nucleotide-binding domain predicted that the aromatic stacking region of CvaB (Y501DSQ loop) had a role in the differential binding of nucleotides, and Ser503 and Gln504 provided potential hydrogen bonds to GTP but not to ATP. Site-directed mutagenesis of the Y501DSQ loop, mutations S503A, Q504L, and double mutation S503A/Q504L, was made to test the predicted hydrogen bonds with GTP. The double mutation S503A/Q504L increased the affinity for ATP by 6-fold, whereas the affinity for GTP was reduced slightly: the ATP/GTP-binding ratio increased about 10-fold. The temperature effect assays on nucleotide binding and hydrolysis further indicated that the double mutant protein had largely eliminated the difference for substrates ATP and GTP, and behaved more similarly to the NBD of typical ABC-transporter HlyB. Therefore, we conclude that Ser503 and Gln504 in aromatic stacking region of CvaB block the ATP binding and are important for the GTP-binding preference.     

Tonoplast-located GmCLC1 and GmNHX1 from soybean enhance NaCl tolerance in transgenic bright yellow (BY)-2 cells

Genes encoding ion transporters that regulate ion homeostasis in soybean have not been carefully investigated. Using degenerate primers, we cloned a putative chloride channel gene (GmCLC1) and a putative Na+/H+ antiporter gene (GmNHX1) from soybean. Confocal microscopic studies using yellow fluorescent fusion proteins revealed that GmCLC1 and GmNHX1 were both localized on tonoplast. The expressions of GmCLC1 and GmNHX1 were both induced by NaCl or dehydration stress imposed by polyethylene glycol (PEG). Using mitochondrial integrity and cell death as the damage indicators, a clear alleviation under NaCl stress (but not PEG stress) was observed in both GmCLC1 and GmNHX1 transgenic cells. Using fluorescent dye staining and quenching, respectively, a higher concentration of chloride ion (Cl-) or sodium ion (Na+) was observed in isolated vacuoles in the cells of GmCLC1 and of GmNHX1 transgenic lines. Our result suggested that these vacuolar-located ion transporters function to sequester ions from cytoplasm into vacuole to reduce its toxic effects.     

The uptake of pyrroline 5-carboxylate. Group translocation mediating the transfer of reducing-oxidizing potential

The cellular uptake of pyrroline 5-carboxylate (P5C) is of interest because this nutritionally responsive constituent of human plasma can mediate the transfer of oxidizing potential into cells and stimulate the production of phosphoribosyl pyrophosphate. Using a cloned line of Chinese hamster ovary cells, we found that the uptake of P5C was saturable, temperature-dependent, and sensitive to metabolic inhibitors. Furthermore, this uptake of P5C exhibited unusual features. It was independent of sodium ion and had a pH optimum of 6.4. The kinetics characteristics of P5C uptake included an apparent Km of 0.46 +/- 0.04 mM and a Vmax of 19.6 +/- 1.8 nmol/min/mg. Although the Vmax for P5C was comparable to those for certain other amino acids, e.g. leucine, it was significantly higher than that for alpha-methylaminoisobutyric acid in these cells. Importantly, there was no interaction between these amino acids and the uptake mechanism for P5C. Twenty naturally occurring amino acids, each at a concentration of 5 mM, were without effect on the uptake of P5C. Interestingly, the uptake mechanism for P5C is unusual in that it is linked to the transfer of reducing-oxidizing potential. Over wide ranges of P5C concentration and duration of incubation, P5C entry is coupled to its conversion to proline and the concomitant oxidation of reduced pyridine nucleotide with stimulation of the pentose phosphate shunt. In fact, no free P5C derived from the medium could be detected in cells. Our interpretation of these findings is that P5C uptake occurs by its own unique mechanism, a group translocation that mediates the transfer of reducing-oxidizing potential.