ABSTRACT31P., 13C-, and "IN-nuclear magnetic resonance spectroscopy were used to determine the roles of malate, succinate, Ala, Asp, Glu, Gin, and y-aminobutyrate (GABA) in the energy metabolism and regulation of cytoplasmic pH in hypoxic maize (Zea mays L.) root tips. Nitogen status was manipulated by perfusing root tips with ammonium sulfate prior to hypoxia; this pretreatment led to enhanced synthesis of Ala early in hypoxia, and of GABA at later times. We show that: (a) the ability to regulate cytoplasmic pH during hypoxia is not significantly affected by enhanced Ala synthesis. (b) Independent of nitrogen status, decarboxylation of Glu to GABA is greatest after several hours of hypoxia, as metabolism collapses. (c) Early in hypoxia, cytoplasmic malate is in part decarboxylated to pyruvate (leading to Ala, lactate, and ethanol), and in part converted to succinate. It appears that activation of malic enzyme serves to limit cytoplasmic acidosis early in hypoxia. (d) Ala synthesis in hypoxic root tips under these conditions is due to transfer of nitrogen ultimately derived from Asp and Gin, present in oxygenated tissue. We describe the relative contributions of glycolysis and malate decarboxylation in providing Ala carbons. (e) Succinate accumulation during hypoxia can be attributed to metabolism of Asp and malate; this flux to succinate is energetically negligible. There is no detectable net flux from GIc to succinate during hypoxia. The significance of the above metabolic reactions relative to ethanol and lactate production, and to flooding tolerance, is discussed. The regulation of the pattems of metabolism during hypoxia is considered with respect to cytoplasmic pH and redox state.

The large subunit of Saccharomyces cerevisiae DNA polymerase , Pol2, comprises two essential functions. The N terminus has essential DNA polymerase activity. The C terminus is also essential, but its function is unknown. We report here that the C-terminal domain of Pol2 interacts with polymerase (Pol ), a recently identified, essential nuclear nucleotidyl transferase encoded by two redundant genes, TRF4 and TRF5. This interaction is functional, since Pol stimulates the polymerase activity of the Pol holoenzyme significantly. Since Trf4 is required for sister chromatid cohesion as well as for completion of S phase and repair, the interaction suggested that Pol , like Pol , might form a link between the replication apparatus and sister chromatid cohesion and/or repair machinery. We present evidence that pol2 mutants are defective in sister chromatid cohesion. In addition, Pol2 interacts with SMC1, a subunit of the cohesin complex, and with ECO1/CTF7, required for establishing sister chromatid cohesion; and pol2 mutations act synergistically with smc1 and scc1. We also show that trf5⌬ mutants, like trf4⌬ mutants, are defective in DNA repair and sister chromatid cohesion.Sister chromatid cohesion is the process by which newly replicated DNA duplexes are held together in the period between the end of S phase and the beginning of mitosis to ultimately ensure faithful transfer of parent genes to daughters. During DNA replication, cohesion is established via the formation of bridges thought to be composed of a multiprotein complex known as cohesin (12,24,45,56,58). Saccharomyces cerevisiae cohesin consists of at least four distinct proteins, Scc1/Mcd1, Scc3, Smc1, and Smc3; however, Scc2 and Scc4 are required for association with chromatin, and Pds5 may also be associated with the complex (23,57,60,64). Smc1 and Smc3

Saccharomyces cerevisiae DNA polymerase epsilon (pol ⑀) is essential for chromosomal replication. A major form of pol ⑀ purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2p⅐Dpb2p and Dpb3p⅐Dpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ⑀ may be dimeric in vivo. Gel filtration of the Pol2p⅐Dpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2p⅐Dpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication.DNA polymerases (pol) 1 play essential roles in the duplication of genetic material and DNA repair in both prokaryotes and eukaryotes (1). In Saccharomyces cerevisiae there exist three essential nuclear DNA polymerases, pol ␣, ␦, and ⑀ (2-4). Despite years of study, several perhaps equally plausible models exist for the function of each polymerase during DNA replication. Pol ␣ plays the role of a primase in the initiation of DNA replication on both leading and lagging strands in the simian virus 40 in vitro system (5). Pol ␦ and pol ⑀ are required for the bulk of the replication on the leading and lagging strands (6 -8). The precise location of pol ␦ and pol ⑀ on leading and lagging strands, however, is not known. Pol ␣, pol ␦, and pol ⑀ have also been shown to participate in DNA repair (1).Polymerase ⑀ was the first proofreading polymerase purified from yeast (2). Since then it has been purified from a number of sources including Schizosaccharomyces pombe, the silkworm Bombyx mori, and HeLa cells (3-7). The S. cerevisiae pol ⑀ consists of four subunits, Pol2p, Dpb2p, Dpb3p, and Dpb4p, with an estimated stoichiometry of 1:1:4:4 (4). These four proteins are encoded by the POL2, DPB2, DPB3, and DPB4 genes (4, 8). 2 The POL2 gene encodes the 256-kDa catalytic subunit of pol ⑀ (8). Mammalian and S. pombe Pol2p show strong sequence similarity to yeast Pol2p (7, 9). The DPB2, DPB3, and DPB4 genes encode the remaining 80-, 34-, and 29-kDa subunits of the yeast pol ⑀ holoenzyme, respectively (4, 10, 11). 2 A functional and structural...

In vivo pyruvate synthesis by malic enzyme (ME) and pyruvate kinase and in vivo malate synthesis by phosphoenolpyruvate carboxylase and the Krebs cycle were measured by 13 C incorporation from [1-13 C]glucose into glucose-6-phosphate, alanine, glutamate, aspartate, and malate. These metabolites were isolated from maize (Zea mays L.) root tips under aerobic and hypoxic conditions. 13 CNuclear magnetic resonance spectroscopy and gas chromatographymass spectrometry were used to discern the positional isotopic distribution within each metabolite. This information was applied to a simple precursor-product model that enabled calculation of specific metabolic fluxes. In respiring root tips, ME was found to contribute only approximately 3% of the pyruvate synthesized, whereas pyruvate kinase contributed the balance. The activity of ME increased greater than 6-fold early in hypoxia, and then declined coincident with depletion of cytosolic malate and aspartate. We found that in respiring root tips, anaplerotic phosphoenolpyruvate carboxylase activity was high relative to ME, and therefore did not limit synthesis of pyruvate by ME. The significance of in vivo pyruvate synthesis by ME is discussed with respect to malate and pyruvate utilization by isolated mitochondria and intracellular pH regulation under hypoxia.The role of malate in many important metabolic processes contributing to energy production, biosynthesis, and mineral nutrition in plants has long been recognized (Lance and Rustin, 1984), yet our quantitative understanding of how the many reactions of malate metabolism contribute to plant function is rudimentary. One notable example concerns the fueling of mitochondria in plant cells oxidizing carbohydrate. It has been widely considered that malate is an important substrate for mitochondria, such that a significant fraction of glycolytic products enters the Krebs cycle via the combined action of PEPC, malate dehydrogenase, and ME rather than via PK (see Fig. 1) (Fowler, 1974; Day and Hanson, 1977;Wiskich, 1980; Bryce and ap Rees, 1985). However, the validity of this model remains to be unequivocally demonstrated in intact plant cells (for review, see ap Rees, 1990; Douce and Neuburger, 1990; Lambers, 1990).One general strategy to solve this problem relies on using NMR and/or GC-MS to observe the metabolism of 13 C-labeled substrates, and then analyzing labeling patterns by various models to deduce metabolic fluxes (for review, see Kü nnecke, 1995). We previously presented qualitative evidence that in [1-13 C]Glc-labeled maize (Zea mays L.) root tips ME is active under hypoxia (Roberts et al., 1992), and may play a role in cytoplasmic pH regulation via proton consumption (Davies, 1986;Roberts et al., 1992). Dieuaide-Noubhani et al. (1995) compared the labeling pattern of Glu and Ala in oxygenated maize root tips labeled with [1-13 C]Glc, and, using a complex metabolic model, deduced that little malate was converted to pyruvate via ME.In the present study we measured 13 C enrichment in precursors and products f...