Wobble modification differences and subcellular localization of tRNAs in Leishmania tarentolae: implication for tRNA sorting mechanism

In Leishmania tarentolae, all mitochondrial tRNAs are encoded in the nuclear genome and imported from the cytosol. It is known that tRNA(Glu)(UUC) and tRNA(Gln)(UUG) are localized in both cytosol and mitochondria. We investigated structural differences between affinity-isolated cytosolic (cy) and mitochondrial (mt) tRNAs for glutamate and glutamine by mass spectrometry. A unique modification difference in both tRNAs was identified at the anticodon wobble position: cy tRNAs have 5-methoxycarbonylmethyl-2- thiouridine (mcm(5)s(2)U), whereas mt tRNAs have 5- methoxycarbonylmethyl-2'-O-methyluridine (mcm(5)Um). In addition, a trace portion (4%) of cy tRNAs was found to have 5-methoxycarbonylmethyluridine (mcm(5)U) at its wobble position, which could represent a common modification intermediate for both modified uridines in cy and mt tRNAs. We also isolated a trace amount of mitochondria-specific tRNA(Lys)(UUU) from the cytosol and found mcm(5)U at its wobble position, while its mitochondrial counterpart has mcm(5)Um. Mt tRNA(Lys) and in vitro transcribed tRNA(Glu) were imported much more efficiently into isolated mitochondria than the native cy tRNA(Glu) in an in vitro importation experiment, indicating that cytosol-specific 2-thiolation could play an inhibitory role in tRNA import into mitochondria.     

Identification of the DNA-binding domains of the switch-activating-protein Sap1 from S.pombe by random point mutations screening in E.coli.

Mating type switching in fission yeast, Schizosaccharomyces pombe, is initiated by a site-specific double-strand break (DSB) at the mat1 locus. The DSB is controlled from a distance by cis- and trans-acting elements. The switch-activating protein, Sap1 binds to the SAS1 cis-acting element which controls the frequency of the DSB at the mat1 locus and, consequently the efficiency of mating type switching. We developed a general method for screening randomly mutagenized expression libraries of DNA-binding protein in E.coli. Sap1 gene was mutagenized by PCR under conditions of reduced Taq polymerase fidelity. The mutated DNA was expressed in E.coli and screened for SAS1-recognition. This method was used to isolated 16 point mutations that abolished SAS1 interaction together with 18 mutations that did not affect binding. The position of these point mutations allowed the identification of three protein domains located in the N-terminal part of Sap1 that are essential for DNA-binding. Deletions and biochemical analysis showed that Sap1 is a dimer both in solution and when bound to SAS1 sequence. The dimerization domain was localized C-terminally to the three domains described above and when used in exess it inhibited DNA binding.     

Enhancement of [C]Sucrose Export from Source Leaves of Vicia faba by Gibberellic Acid

The effect of gibberellic acid (GA₃) on sucrose export from source leaves was studied in broad bean (Vicia faba L.) plants trimmed of all but one source and one sink leaf. GA₃ (10 micromolar) applied to the source leaf, enhanced export of [¹⁴C]sucrose (generated by ¹⁴CO₂ fixation) to the root and to the sink leaf. Enhanced export was observed with GA treatments as short as 35 minutes. When GA₃ was applied 24 hours prior to the ¹⁴CO₂ pulse, the enhancement of sucrose transport toward the root was abolished but transport toward the upper sink leaf was unchanged. The enhanced sucrose export was not due to increased photosynthetic rate or to changes in the starch/sucrose ratio within the source leaf; rather, GA₃ increased the proportion of sucrose exported. After a 10-min exposure to [¹⁴C]GA₃, radioactivity was found only in the source leaf. Following a 2 hour exposure to [¹⁴C]GA₃, radioactivity was distributed along the entire stem and was present in both the roots and sink leaf. Extraction and partitioning of GA metabolites by thin layer chromatography indicated that there was a decline in [¹⁴C]GA₃ in the lower stem and root, but not in the upper stem. This pattern of metabolism is consistent with the disappearance of the GA₃ effect in the lower stem with time after treatment. We conclude that in the short term, GA₃ enhances assimilate export from source leaves by increasing phloem loading. In the long term (24 hours), the effect of GA₃ is outside the source leaf. GA₃ accumulates in the apical region resulting in enhanced growth and thus greater sink strength. Conversely, GA₃ is rapidly metabolized in the lower stem thus attenuating any GA effect.     

Interaction of cell turgor and hormones on sucrose uptake in isolated Phloem of celery

Phloem tissue isolated from celery (Apium graveolens L.) was used to investigate the regulation of sucrose uptake by turgor (manipulated by 50-400 milliosomolal solutions of polyethylene glycol) and hormones indoleacetic acid (IAA) and gibberillic acid (GA₃). Sucrose uptake was enhanced under low cellular turgor (increase in the V_max). Furthermore, enhancement of sucrose uptake was due to a net increase in influx rates since sucrose efflux was not affected by cell turgor. Manipulations of cell turgor had no effect on 3-O-methyl glucose uptake. When 20 millimolar buffer was present in uptake solutions, low turgor-induced effects were observed only at low pH range (4.5-5.5). However, the effect was extended to higher external pH (up to 7.5) when buffer was omitted from uptake solutions. A novel interaction between cellular turgor and hormone treatments was observed, in that GA₃ (10 micromolar) and IAA (0.1-100 micromolar) enhanced sucrose uptake only at moderate turgor levels. The hormones elicited little or no response on sucrose uptake under conditions of low or high cell turgor. Low cell turgor, IAA, GA₃, and fusicoccin caused acidification by isolated phloem segments in a buffer-free solution. It is suggested that enhanced sucrose uptake in response to low turgor and/or hormones was mediated through the plasmalemma H⁺-ATPase and most likely occurred at the site of loading.     

Turgor-Regulated Sugar Release from the Source Leaves of Sugarbeet (Beta vulgaris L.)

Under mild water stress conditions, a potential site of regulationfor distribution of sucrose between osmotic adjustment and exportmay be at the mesophyll plasmalemma and/or tonoplast. This possibilitywas examined in attached leaves of sugarbeet (Beta vulgarisL.), labeled with ¹⁴CO₂. Leaf discs were exposed to solutionscontaining 400 or 50 mM mannitol to generate "low" or "high"cellular turgor, respectively and release of labeled soluteswas monitored. Response to changes in cell turgor was rapidand reversible. High turgor increased solute efflux rates todouble those at low turgor conditions. Approximately 30% and55% of the released label was in the sugar (sucrose and hexose)fractions at low and high turgor, respectively. Paramercuribenzenesulfonic acid (PCMBS) had no effect on efflux, but N-ethylmaleimide(NEM) and carbonylcyanide-m-chlorophenyl hydrazone (CCCP) enhancedefflux, especially at high turgor. Presence of unlabeled sucrosegreatly enhanced efflux in a turgor-dependent manner; suggestinga sucrose exchange system. While influx across the plasmalemmais both turgor sensitive and carrier-mediated, turgor-regulatedplasmalemma efflux did not appear to involve a carrier. Boththe tonoplast and plasmalemma appeared to be involved in turgor-inducedsugar efflux. Turgor-regulated efflux of solutes from vacuole-containingcells (mesophyll), may contribute to the establishment of ahomeostatic turgor pressure in these cells. (Received June 9, 1989; Accepted September 5, 1989)     

Phloem loading of sucrose: Update and opportunities in molecular biology

Sucrose accumulates in the phloem against a concentration gradient via a presumed sucrose-specific carrier protein located at the plasmalemma of the sieve elements/companion cells. Recent evidence suggests that sucrose carrier in soybean is a 62-kDa protein. Immunocytochemical localization has shown the protein to be exclusively at the plasmalemma, which is also the site of sucrose transport. To enhance our understanding of the phenomenon, the structural gene of the sucrose carrier must be cloned and sequenced. Furthermore, development of appropriate probes should help answer long-standing questions relative to the molecular nature of sugar transport and phloem loading, the mechanism of induction/activation of sugar carriers, and developmental regulation of expression of genes encoding such carriers.