Polyproline, beta-turn helices. Novel secondary structures proposed for the tandem repeats within rhodopsin, synaptophysin, synexin, gliadin, RNA polymerase II, hordein, and gluten

Seven proteins each contain 8 to 52 tandem repeats of a unique class of oligopeptide. The consensus peptide for each is rhodopsin Tyr Pro Pro Gln Gly synaptophysin Tyr Gly Pro Gln Gly synexin Tyr Pro Pro Pro Pro Gly gliadin Tyr Pro Pro Pro Gln Pro RNA polymerase II Tyr Ser Pro Thr Ser Pro Ser hordein Phe Pro Gln Gln Pro Gln Gln Pro gluten Tyr Pro Thr Ser Pro Gln Gln Gly Tyr Although there is obvious variation of sequence and of length, the penta- to nonapeptides share an initial Tyr (or Phe) and have high Pro contents and abundant Gly, Gln, and Ser. We have evaluated helical models that both recognize the uniqueness of these sequence repeats and accommodate variations on the basic theme. We have developed a group of related helical models for these proteins with about three oligopeptide repeats per turn of 10-20 A. These models share several common features: Most of the phi dihedral angles are -54 degrees, to accommodate Pro at all positions except the first (Tyr). Except for the beta-turns, most psi dihedral angles are near +140 degrees as found in polyproline. Each oligopeptide has at least one beta-turn; several have two. Some contain a cis-Tyr, Pro peptide bond; a few have a cis-bond plus one beta-turn. Tyr side chains vary from totally exposed to buried within the helices and could move to accommodate either external hydrophobic interactions or phosphorylation. The several related structures seem to be readily interconverted without major change in the overall helical parameters, and therein may lie the key to their functions.     

P53 polymorphism and lung cancer susceptibility: a pooled analysis of 32 case–control studies

To explore the real association between p53 codon 72 polymorphism and lung cancer risk, a pooled analysis of 32 case–control studies involving 19,255 subjects was conducted. When all 32 studies were pooled into the analysis, significantly elevated lung cancer risks were associated with variant genotypes in all genetic models (for Pro/Arg vs. Arg/Arg: OR 1.21, 95% CI 1.01–1.23; for Pro/Pro vs. Arg/Arg: OR 1.20, 95% CI 1.03–1.39; for Pro/Pro + Pro/Arg vs. Arg/Arg: OR 1.14, 95% CI 1.03–1.25; for Pro/Pro vs. Arg/Arg + Pro/Arg: OR 1.06, 95% CI 1.01–1.12). In the subgroup analysis by ethnicity, histological type, or smoking status, significantly increased risks were found in subgroups such as Asians, Caucasians, lung adenocarcinoma patients, or smokers, respectively. In conclusion, our results suggest that the Pro allele at p53 codon 72 is emerging as a low-penetrance susceptibility allele for lung cancer development.     

Genetic Polymorphisms of DNA Repair Gene: XRCC1 Pro206Pro and Gln632Gln in a Chinese Han Population

DNA repair genes are increasingly studied because of their critical role in maintaining genome integrity. The base excision repair (BER) pathway is a DNA repair pathway that operates on small lesions, such as oxidized or reduced bases, fragmented or nonbulky adducts, or those produced by methylating agents. The XRCC1 polymorphic system is the key gene of the BER pathway. In this study, polymorphisms of XRCC1 Pro206Pro on exon 7 and Gln632Gln on exon 17 were analyzed in a northeastern Chinese Han population. Genomic DNA extracted from 303 unrelated individuals and the PCR-RFLP technique were used to identify variants. The allele frequencies were 0.90 (A) and 0.10 (G) for XRCC1 Pro206Pro and 0.88 (G) and 0.12 (A) for XRCC1 Gln632Gln. The genotype frequencies were 0.797 (AA), 0.203 (AG), and 0 (GG) for XRCC1 Pro206Pro and 0.007 (AA), 0.222 (AG), and 0.771 (GG) for XRCC1 Gln632Gln. The expected heterozygosity and PIC were 18 and 16.38% for Pro206Pro and 21.12 and 18.89% for Gln632Gln. The two polymorphisms were in strong linkage disequilibrium (D' = 0.921, r (2) = 0.735). The results are compared with those of other reported populations. They showed marked ethnic group differences. This study provides the first analysis of the distribution of allele frequency for XRCC1 Pro206Pro and Gln632Gln in a Chinese population.     

A p53 codon 72 polymorphism associated with prostate cancer development and progression in Japanese

An association between the Pro/Pro genotype of p53 codon 72 and a lower risk of prostate cancer in Caucasians was recently reported. However, the association of this polymorphism with prostate cancer risk in a Japanese population has not been clarified. We performed a case-control study consisting of 114 prostate cancer patients and 105 noncancer controls. Sixty-nine percent (79 of 114) of the patients had a positive family history. The genotypic frequencies in the controls were 39.0% for Arg/Arg, 54.3% for Arg/Pro and 6.7% for Pro/Pro; they were in Hardy-Weinberg equilibrium. When a comparison of the distribution of the p53 codon 72 polymorphism was made between patients with a first-degree family history and all control subjects, the adjusted odds ratios (ORs) for prostate cancer associated with the Arg/Arg, Arg/Pro and Pro/Pro genotypes were 1.00, 0.99 [95% confidence interval (CI) 0.53-1.88] and 2.80 (95% CI 1.04-7.53), respectively. When stratification of cases was performed based on clinical stage (localized or metastatic cancer) and pathological grade (a Gleason score of <7 or > or =7), there tended to be a greater number of patients with localized cancers among those patients with the Arg/Pro genotype than among those with the Arg/Arg genotype (overall cases: age-adjusted OR 0.36, 95% CI 0.13-1.00, p = 0.049; positive family history cases: age-adjusted OR 0.25, 95% CI 0.075-0.84, p = 0.025). In addition, there tended to be a greater number of patients with low-grade cancers among those with the Pro/Pro genotype than among those with other genotypes (overall cases: age-adjusted OR 0.41, 95% CI 0.13-1.30, p = 0.13; positive family history cases: age-adjusted OR 0.20, 95% CI 0.004-0.89, p = 0.035). The present findings suggest that the Pro/Pro genotype of p53 codon 72 played a role in prostate cancer susceptibility in a Japanese population. However, the Pro allele did not appear to worsen such clinical parameters as clinical stage or pathological grade.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

Enhanced preference for pi-bond containing substrates is correlated to Pro110 in the substrate-binding tunnel of Escherichia coli thioesterase I/protease I/lysophospholipase L(1)

Escherichia coli thioesterase I/protease I/lysophospholipase L(1) (TAP) possesses multifunctional enzyme with thioesterase, esterase, arylesterase, protease, and lysophospholipase activities. Leu109, located at the substrate-binding tunnel, when substituted with proline (Pro) in TAP, shifted the substrate-preference from medium-to-long acyl chains to shorter acyl chains of triglyceride and p-nitrophenyl ester, and increased the preference for aromatic-amino acid-derived esters. In the three-dimensional TAP structures, the only noticeable alteration of backbone and side chain conformation was located at the downstream Pro110-Ala123 region rather than at Pro109 itself. The residue Pro110, adjacent to Leu109 or Pro109, was found to contribute to the substrate preference of TAP enzymes for esters containing acyl groups with pi bond(s) or aromatic group(s). Some of the interactions between the enzyme protein and the substrate may be contributed by an attractive force between the Pro110 C-H donor and the substrate pi-acceptor.     

Proline Accumulation in Developing Grapevine Fruit Occurs Independently of Changes in the Levels of Δ1-Pyrroline-5-Carboxylate Synthetase mRNA or Protein

Mature fruit of grapevine (Vitis vinifera) contains unusually high levels of free proline (Pro; up to 24 μmol or 2.8 mg/g fresh weight). Pro accumulation does not occur uniformly throughout berry development but only during the last 4 to 6 weeks of ripening when both berry growth and net protein accumulation have ceased. In contrast, the steady-state levels of both the mRNA encoding V. vinifera Δ¹-pyrroline-5-carboxylate synthetase (VVP5CS), a key regulatory enzyme in Pro biosynthesis, and its protein product remain relatively uniform throughout fruit development. In addition, the steady-state protein levels of Pro dehydrogenase, the first enzyme in Pro degradation, increased throughout early fruit development but thereafter remained relatively constant. The developmental accumulation of free Pro late in grape berry ripening is thus clearly distinct from the osmotic stress-induced accumulation of Pro in plants. It is not associated with either sustained increases in steady-state levels of P5CS mRNA or protein or a decrease in steady-state levels of Pro dehydrogenase protein, suggesting that other physiological factors are important for its regulation.     

Functional consequences of proline mutations in the cytoplasmic and transmembrane sectors of the Ca2(+)-ATPase of sarcoplasmic reticulum

Site-specific mutagenesis was used to investigate whether Pro160, Pro195, Pro308, Pro312, Pro803, and Pro812 play essential roles in the function of the sarcoplasmic reticulum Ca2(+)-ATPase. All six prolines were substituted with alanine; and in addition, Pro308 was replaced by glycine and Pro312 by glycine as well as by leucine. Mutant cDNAs were expressed in COS-1 cells, and mutant Ca2(+)-ATPases located in the isolated microsomal fraction were examined with respect to Ca2+ uptake activity, Ca2+ dependence of phosphorylation from ATP, and the kinetic properties of the phosphoenzyme intermediates formed from both ATP and Pi. The enzymatic cycle was little affected by substitution of Pro160, Pro195, and Pro812, which are located in the cytoplasmic domain; but replacement of Pro308, Pro312, and Pro803, in the putative transmembrane helices, had a profound impact on the function of the enzyme. All mutations of Pro308 and Pro803 led to ATPases which were characterized by a reduced affinity for Ca2+. These prolines may therefore be involved in the structure of the high affinity Ca2(+)-binding sites in the enzyme. Substitution of Pro312 with alanine or glycine gave rise to mutants unable to transport Ca2+ even though their apparent affinities for Ca2+ in the phosphorylation reaction with ATP were increased. In these enzymes, the ADP-sensitive phosphoenzyme intermediate was stable for at least 5 min at 0 degrees C, whereas the ADP-insensitive phosphoenzyme intermediate decay at a rate similar to that of the wild type. Thus, the inability to transport Ca2+ could be accounted for by a block of ADP-sensitive to ADP-insensitive phosphoenzyme intermediate conformational transition. In contrast, substitution of Pro312 with leucine gave rise to a mutant enzyme that retained about 7% of the normal Ca2+ transport rate. Phosphoenzyme turnover in this mutant also occurred at a low but significant rate, suggesting that the leucine side chain can substitute to some extent for proline.     

Effects of free proline accumulation in petunias under drought stress

Petunias (Petunia hybrida cv. 'Mitchell') accumulate free proline (Pro) under drought-stress conditions. It is therefore believed that Pro acts as an osmoprotectant in plants subjected to drought conditions. Petunia plants were transformed by Delta(1)-pyrroline-5-carboxylate synthetase genes (AtP5CS from Arabidopsis thaliana L. or OsP5CS from Oryza sativa L.). The transgenic plants accumulated Pro and their drought tolerance was tested. The Pro content amounted to 0.57-1.01% of the total amino acids in the transgenic plants, or 1.5-2.6 times that in wild-type plants grown under normal conditions. The transgenic plant lines tolerated 14 d of drought stress, which confirms that both P5CS transgenes had full functionality. Exogenous L-Pro treatment caused the plants to accumulate Pro; plants treated with 5 mM L-Pro accumulated up to 18 times more free Pro than untreated plants. Exogenous L-Pro restricted the growth of wild-type petunias more than that of Arabidopsis plants. The capacity for free Pro accumulation might depend on the plant species. The growth of petunia plants was influenced not only by the Pro concentration in the plants, but by the ratio of the Pro content to the total amino acids, because the growth of the transgenic petunia plants appeared normal.     

p53基因多态性和脑外伤预后及临床意义

目的：研究颅脑外伤患者中P53等位基因与基因型的频率分布,探讨其与脑损伤预后的关联性,为脑损伤患者预后提供部分参数依据。方法：选择各型颅脑损伤患者141例,正常对照者144人,患者按GCS格拉斯哥评分法进行分类;采用PCR基因型检测法检测P53等位基因,基因计数法计算等位基因频率;统计学分析等位基因和基因型在脑外伤组与正常对照组、预后良好组与预后不良组的频率分布的差异,并综合分析患者年龄、性别、GCS以及P53各等位基因等因素对脑外伤患者预后的影响。结果：在颅脑外伤患者和正常对照者中P53等位基因和基因型的频率分布,差异均无统计学意义（P〉0.05）;等位基因Arg相对于Pro的OR为1.140,基因型Arg/Pro、Arg/Arg相对于Pro/Pro的OR分别为0.890和1.170。在重型脑外伤患者（3≤GCS≤8分）86例中,预后良好31例,预后不良55例,基因型对预后不良的影响：非Pro/Pro相对于Pro/Pro的OR为0.417,非Arg/Pro相对于Arg/Pro的OR为0.419,非Arg/Arg相对于Arg/Arg的OR为3.667。在所有脑外伤患者中,预后良好组和预后不良组的患者年龄、性别差异均无统计学意义（P〉0.05）;GCS、P53等位基因差异具有显著统计学意义（P〈0.01）。结论：在重型颅脑患者中,基因型Arg/Arg有增加预后不良的风险;脑外伤预后与患者的年龄、性别无关联,与GCS、P53基因密切相关。     

Widely distributed residues in thymosin beta4 are critical for actin binding

We have investigated the contributions of hydrophobic residues, the conserved and variable proline residues, and the conserved lysine residues to the affinity and kinetics of thymosin beta4 (Tbeta4) binding to MgATP-actin monomers. Pro4, Lys18, Lys19, Pro27, Leu28, Pro29, and Ile34 were substituted with alanine residues. Mutagenesis of Pro4 or Pro27 has little effect (or=10-fold, but the kinetic basis of the lower stability varies among the mutants. Substitution of the conserved lysine residues weakens the affinity by slowing association and accelerating dissociation. Substitution of hydrophobic residue Leu28 or Ile34 weakens the affinity by accelerating dissociation. These results favor a reaction mechanism in which Tbeta4 binds actin monomers following a two-step mechanism in which the formation of a bimolecular complex is followed by isomerization to a strong binding state that is coupled to the formation of widely distributed hydrophobic contacts. The isomerization equilibrium is slowed by mutagenesis of Pro29, as revealed by the double-exponential time course of association. Mutagenesis of Pro4 or Pro27 accelerates binding and dissociation but minimally affects the binding affinity (相似文献   

Processing of Escherichia coli alkaline phosphatase. Sequence requirements and possible conformations of the -6 to -4 region of the signal peptide

Analysis of the precursors of bacterial exported proteins revealed that those having bulky hydrophobic residues at position -5 have a high incidence of Pro residues at positions -6 and -4, Val at position -3, and Ser at positions -4 and -2. This led to a hypothesis that the previously observed inhibition of processing by bulky residues at position -5 can be suppressed by introduction of Pro, Ser, or Val in the corresponding nearby positions. Subsequent mutational analysis of Escherichia coli alkaline phosphatase showed that, as it was predicted, Pro on either side of bulky hydrophobic -5 Leu, Ile, or Tyr completely restores efficiency of the maturation. Introduction of Val at position -3 also partially suppresses the inhibition imposed by -5 Leu, while a Ser residue at position -4 or -2 does not restore processing. In addition, effective maturation of a mutant with Pro residues at positions from -6 throughout -4 proved that polyproline conformation of this region is permissive for processing. To understand the effects of the mutations, we modeled a peptide substrate into the active site of the signal peptidase using the known position of the beta-lactam inhibitor. The inhibitory effect of the -5 residue and its suppression by either Pro -6 or Pro -4 can be explained if we assume that Pro-containing -6 to -4 regions adopt a polyproline conformation whereas the region without Pro residues has a beta-conformation. These results permit us to specify sequence requirements at -6, -5, and -4 positions for efficient processing and to improve the prediction of yet unknown cleavage sites.     

The use of double mutants to detect structural changes in the active site of the tyrosyl-tRNA synthetase (Bacillus stearothermophilus)

In a previous study, a mutant of tyrosyl-tRNA synthetase in which a threonine residue (Thr51) was converted to proline dramatically improved the affinity of the enzyme for its ATP substrate. How does Pro51 improve the enzyme's affinity for ATP? A priori, Pro51 might interact directly with the ATP, or it might distort the polypeptide backbone and thereby force new or improved contacts elsewhere from the enzyme to ATP. By making mutants of the Pro51 enzyme at two residues that make hydrogen bonds to the ATP substrate, we show that Pro51 greatly improves the strength of one of these contacts. Thus the propagation of a structural change in an enzyme induced by mutation may be detected by the introduction of further mutations.     

The Pro33 isoform of integrin beta3 enhances outside-in signaling in human platelets by regulating the activation of serine/threonine phosphatases

Integrin beta(3) is polymorphic at residue 33 (Leu(33) or Pro(33)), and the Pro(33)-positive platelets display enhanced aggregation, P-selectin secretion, and shorter bleeding times. Because outside-in signaling is critical for platelet function, we hypothesized that the Pro(33) variant provides a more efficient signaling than the Leu(33) isoform. When compared with Pro(33)-negative platelets, Pro(33)-positive platelets demonstrated significantly greater serine/threonine phosphorylation of extracellular signal-regulated kinase (ERK2) and myosin light chain (MLC) but not cytoplasmic phospholipase A2 upon thrombin-induced aggregation. Tyrosine phosphorylation of integrin beta(3) and the adaptor protein Shc was no different in the fibrinogen-engaged platelets from both genotypes. The addition of Integrilin (alpha(IIb)beta(3)-fibrinogen blocker) or okadaic acid (serine/threonine phosphatase inhibitor) dramatically enhanced ERK2 and MLC phosphorylation in the Pro(33)-negative platelets when compared with Pro(33)-positive platelets, suggesting that integrin engagement during platelet aggregation activates serine/threonine phosphatases. The phosphatase activity of myosin phosphatase (MP) that dephosphorylates MLC is inactivated by phosphorylation of the myosin binding subunit of MP at Thr(696), and aggregating Pro(33)-positive platelets exhibited an increased Thr(696) phosphorylation of MP. These studies highlight a role for the dephosphorylation events via the serine/threonine phosphatases during the integrin outside-in signaling mechanism, and the Leu(33) --> Pro polymorphism regulates this process. Furthermore, these findings support a mechanism whereby the reported enhanced alpha granule secretion in the Pro(33)-positive platelets could be mediated by an increased phosphorylation of MLC, which in turn is caused by an increased phosphorylation and subsequent inactivation of myosin phosphatase.     

Isolation of Tryptic Peptides from the lie Chain of Ricin D and the Sequences of Some Tryptic Peptides Including N- and C-Terminal Peptides

Twenty tryptic peptides were isolated from the performic acid-oxidized He chain of ricin D by Dowex 1 × 2 column chromatography followed by paper chromatography. The amino acids contained in these peptides accounted for 218 out of 266 residues in the whole protein. The amino acid sequences of nine peptides were determined by manual liquid phase or automatic solid phase Edman degradation, and N- and C-terminal sequences of the He chain of ricin D were established to be NH₂–Ile–Phe–Pro–Lys–Gln–Tyr–Pro–Ile–Ile– and Cys–Ala–Pro–Pro–Pro–Ser–Ser–Gln–Phe, respectively.     

Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition

glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.     

Casein-derived tripeptide Ile-Pro-Pro improves angiotensin-(1-7)- and bradykinin-induced rat mesenteric artery relaxation

AimsMilk casein-derived bioactive tripeptides isoleucine–proline–proline (Ile–Pro–Pro) and valine–proline–proline (Val–Pro–Pro) lower blood pressure in animal models of hypertension and humans. In some studies, their angiotensin-converting enzyme (ACE)-inhibitory effect has been demonstrated. Besides classical ACE-angiotensin II-AT₁-receptor pathway (ACE-Ang II- AT₁), the significance of ACE2-angiotensin-(1–7)-Mas-receptor (ACE2-Ang-(1–7)-Mas) axis in the blood pressure regulation has now been acknowledged. The present study was aimed to further evaluate the renin–angiotensin system (RAS)-related vascular effects of Ile–Pro–Pro in vitro using rat mesenteric arteries.Main methodsSuperior mesenteric arteries of spontaneously hypertensive rat (SHR) were isolated, cut into rings and mounted in standard organ bath chambers. Endothelium-intact arterial rings were incubated in Krebs solution either with Ile–Pro–Pro, proline–proline (Pro–Pro), isoleucine (Ile), proline (Pro) or captopril for 6 h at + 37 °C and vascular reactivity was measured.Key findingsIn the presence of AT₁-antagonist valsartan, Ang II induced vasodilatation, which was more pronounced in the arteries incubated with Ile–Pro–Pro (P < 0.05) compared to the other compounds. Ang-(1–7)-induced vasodilatation was augmented by Ile–Pro–Pro or Pro (P < 0.001 vs. control). Mas-receptor antagonist A-779 did not alter the responses. Ile–Pro–Pro and Pro augmented also bradykinin-induced relaxations (P < 0.001 vs. control). Control arteries and arteries incubated with captopril showed only slight relaxations at higher bradykinin concentrations.SignificanceCasein-derived tripeptide Ile–Pro–Pro and amino acid Pro enhance the vasodilatory effect of Ang-(1–7) and bradykinin. The role of ACE2-Ang–(1–7)-Mas axis in the modulation of vascular tone by these compounds seems probable.     

Proline accumulation in maize (Zea mays L.) primary roots at low water potentials. II. Metabolic source of increased proline deposition in the elongation zone

The proline (Pro) concentration increases greatly in the growing region of maize (Zea mays L.) primary roots at low water potentials (ψ_w), largely as a result of an increased net rate of Pro deposition. Labeled glutamate (Glu), ornithine (Orn), or Pro was supplied specifically to the root tip of intact seedlings in solution culture at high and low ψ_w to assess the relative importance of Pro synthesis, catabolism, utilization, and transport in root-tip Pro deposition. Labeling with [³H]Glu indicated that Pro synthesis from Glu did not increase substantially at low ψ_w and accounted for only a small fraction of the Pro deposition. Labeling with [¹⁴C]Orn showed that Pro synthesis from Orn also could not be a substantial contributor to Pro deposition. Labeling with [³H]Pro indicated that neither Pro catabolism nor utilization in the root tip was decreased at low ψ_w. Pro catabolism occurred at least as rapidly as Pro synthesis from Glu. There was, however, an increase in Pro uptake at low ψ_w, which suggests increased Pro transport. Taken together, the data indicate that increased transport of Pro to the root tip serves as the source of low-ψ_w-induced Pro accumulation. The possible significance of Pro catabolism in sustaining root growth at low ψ_w is also discussed.