No abstract

We describe the construction and use of two sets of vectors for the over-expression and purification of protein from E. coli. The set of pTEV plasmids (pTEV3, 4, 5) directs the synthesis of a recombinant protein with a N-terminal hexahistidine (His 6 ) tag that is removable by the tobacco etch virus (TEV) protease. The set of pKLD plasmids (pKLD66, 116) directs the synthesis of a recombinant protein that contains a N-terminal His 6 and maltose-binding protein tags in tandem, which can also be removed with TEV protease. The usefulness of these plasmids is illustrated by the rapid, high-yield purification of the 2-methylcitrate dehydratase (PrpD) protein of Salmonella enterica, and the 2-methylaconitate isomerase (PrpF) protein of Shewanella oneidensis, two enzymes involved in the catabolism of propionate to pyruvate via the 2-methylcitric acid cycle.

A reverse genetic strategy was used to isolate Arabidopsis plants containing "knockout" mutations in AKT1 and AKT2, two members of a K ϩ channel gene family. Comparative studies of growth and membrane properties in wild-type and mutant seedlings were performed to investigate the physiological functions of these two related channels. The growth rates of plants supplied with rate-limiting concentrations of K ϩ depended on the presence of AKT1 but not AKT2 channels. This result indicates that AKT1 but not AKT2 mediates growth-sustaining uptake of K ϩ into roots, consistent with the expression patterns of these two genes. K ϩ -induced membrane depolarizations were measured with microelectrodes to assess the contribution each channel makes to the K ϩ permeability of the plasma membrane in three different organs. In apical root cells, AKT1 but not AKT2 contributed to the K ϩ permeability of the plasma membrane. In cotyledons, AKT1 was also the principal contributor to the K ϩ permeability. However, in the mesophyll cells of leaves, AKT2 accounted for approximately 50% of the K ϩ permeability, whereas AKT1 unexpectedly accounted for the remainder. The approximately equal contributions of AKT1 and AKT2 in leaves detected by the in vivo functional assay employed here are not in agreement with previous RNA blots and promoter activity studies, which showed AKT2 expression to be much higher than AKT1 expression in leaves. This work demonstrates that comparative functional studies of specific mutants can quantify the relative contributions of particular members of a gene family, and that expression studies alone may not reliably map out distribution of gene functions.The most abundant inorganic solute in plant cells is K ϩ . The transport in and out of cells of this essential element is a highly regulated process mediated by specific transporters within the plasma membrane (Maathuis et al., 1997; Chrispeels et al., 1999). Present at concentrations on the order of 100 mm, K ϩ serves as an osmoticum important to turgor pressure, and may act as an essential cofactor for certain enzymes. Its abundance contributes to the electrolyte character of cytoplasm and affects electrostatic interactions between charged entities such as proteins and other biopolymers. The transport of K ϩ helps set the electric potential difference across the plasma membrane, which powers the transport of other substances. Because K ϩ serves such fundamental functions throughout the plant, understanding the molecular mechanisms of its uptake and redistribution is an important goal. Progress in this regard may also spawn novel strategies for improving plant mineral nutrition and fertilizer application in the field.The first isolation of Arabidopsis genes encoding plasma membrane K ϩ channels by complementation of yeast K ϩ uptake mutants marked a major step toward this goal (Anderson et al., 1992;Sentenac et al., 1992). The transport properties displayed by AKT1 and KAT1 channels expressed in heterologous systems (Schachtman et al., 1992; Bertl et al., 1995 ...

A reverse genetic strategy was used to isolate Arabidopsis plants containing "knockout" mutations in AKT1 and AKT2, two members of a K ϩ channel gene family. Comparative studies of growth and membrane properties in wild-type and mutant seedlings were performed to investigate the physiological functions of these two related channels. The growth rates of plants supplied with rate-limiting concentrations of K ϩ depended on the presence of AKT1 but not AKT2 channels. This result indicates that AKT1 but not AKT2 mediates growth-sustaining uptake of K ϩ into roots, consistent with the expression patterns of these two genes. K ϩ -induced membrane depolarizations were measured with microelectrodes to assess the contribution each channel makes to the K ϩ permeability of the plasma membrane in three different organs. In apical root cells, AKT1 but not AKT2 contributed to the K ϩ permeability of the plasma membrane. In cotyledons, AKT1 was also the principal contributor to the K ϩ permeability. However, in the mesophyll cells of leaves, AKT2 accounted for approximately 50% of the K ϩ permeability, whereas AKT1 unexpectedly accounted for the remainder. The approximately equal contributions of AKT1 and AKT2 in leaves detected by the in vivo functional assay employed here are not in agreement with previous RNA blots and promoter activity studies, which showed AKT2 expression to be much higher than AKT1 expression in leaves. This work demonstrates that comparative functional studies of specific mutants can quantify the relative contributions of particular members of a gene family, and that expression studies alone may not reliably map out distribution of gene functions.The most abundant inorganic solute in plant cells is K ϩ . The transport in and out of cells of this essential element is a highly regulated process mediated by specific transporters within the plasma membrane (Maathuis et al., 1997; Chrispeels et al., 1999). Present at concentrations on the order of 100 mm, K ϩ serves as an osmoticum important to turgor pressure, and may act as an essential cofactor for certain enzymes. Its abundance contributes to the electrolyte character of cytoplasm and affects electrostatic interactions between charged entities such as proteins and other biopolymers. The transport of K ϩ helps set the electric potential difference across the plasma membrane, which powers the transport of other substances. Because K ϩ serves such fundamental functions throughout the plant, understanding the molecular mechanisms of its uptake and redistribution is an important goal. Progress in this regard may also spawn novel strategies for improving plant mineral nutrition and fertilizer application in the field.The first isolation of Arabidopsis genes encoding plasma membrane K ϩ channels by complementation of yeast K ϩ uptake mutants marked a major step toward this goal (Anderson et al., 1992;Sentenac et al., 1992). The transport properties displayed by AKT1 and KAT1 channels expressed in heterologous systems (Schachtman et al., 1992; Bertl et al., 1995 ...