Mutation and cloning of clustered Streptomyces genes essential for sulphate metabolism

Summary A range of mutants auxotrophic for cysteine (cys) and resistant to selenate (sel) were isolated from many Streptomyces strains but chiefly from S. coelicolor A3(2) and S. lividans 66. Two of the classes of sel/cys mutants probably contained simple biochemical lesions of sulphate permease (selC) and ATP sulphurylase (selA) activities, while a further two classes (selD and selE) were pleiotropic and possibly regulatory. Most classes of sel mutations were clustered around the cysD locus of S. coelicolor. Segments of chromosomal DNA cloned from S. coelicolor, S. cattleya and S. clavuligerus and able to complement various sel/cys mutations allowed the relative positions of these mutations and the cysC and cysD mutations of S. coelicolor to be determined. The sel/cys DNA can be used for two-way selection: Cys⁺Sel^sCys^-Sel^r.     

Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p

The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease. Received: 7 May 1998 / Accepted: 16 July 1998     

A simple way for sequential assignment in isotopically enriched proteins using a H(N)CACO correlation

Summary A simplified scheme for sequential assignment in isotopically enriched proteins is presented. It is based on the standard triple resonance experiments HNCO, HN(CO)CA, HNCA and a modified H(N)CACO correlation, in which both of the H^N connectivities to the CA/C pair of residue i and i-1 are observed. The H(N)CACO was tested on uniformly ¹³C/¹⁵N enriched P13 domain of mannose permease (31 kDa).     

Cysteine scanning mutagenesis of putative transmembrane helices IX and X in the lactose permease of Escherichia coli.

Using a functional lactose permease mutant devoid of Cys residues (C-less permease), each amino-acid residue in putative transmembrane helices IX and X and the short intervening loop was systematically replaced with Cys (from Asn-290 to Lys-335). Thirty-four of 46 mutants accumulate lactose to high levels (70-100% or more of C-less), and an additional 7 mutants exhibit lower but highly significant lactose accumulation. As expected (see Kaback, H.R., 1992, Int. Rev. Cytol. 137A, 97-125), Cys substitution for Arg-302, His-322, or Glu-325 results in inactive permease molecules. Although Cys replacement for Lys-319 or Phe-334 also inactivates lactose accumulation, Lys-319 is not essential for active lactose transport (Sahin-Tóth, M., Dunten, R.L., Gonzalez, A., & Kaback, H.R., 1992, Proc. Natl. Acad. Sci. USA 89, 10547-10551), and replacement of Phe-334 with leucine yields permease with considerable activity. All single-Cys mutants except Gly-296 --> Cys are present in the membrane in amounts comparable to C-less permease, as judged by immunological techniques. In contrast, mutant Gly-296 --> Cys is hardly detectable when expressed at a relatively low rate from the lac promoter/operator but present in the membrane in stable form when expressed at a high rate from T7 promoter. Finally, studies with N-ethylmaleimide (NEM) show that only a few mutants are inactivated significantly. Remarkably, the rate of inactivation of Val-315 --> Cys permease is enhanced at least 10-fold in the presence of beta-galactopyranosyl 1-thio-beta-D-galactopyranoside (TDG) or an H+ electrochemical gradient (delta mu-H+). The results demonstrate that only three residues in this region of the permease -Arg-302, His-322, and Glu-325-are essential for active lactose transport. Furthermore, the enhanced reactivity of the Val-315 --> Cys mutant toward NEM in the presence of TDG or delta mu-H+ probably reflects a conformational alteration induced by either substrate binding or delta mu-H+.     

Effect of l-azetidine 2-carboxilic acid on the activity of the general amino-acid permease from Saccharomyces cerevisiae var. ellipsoideus

Addition of the l-proline analogue l-azetidine 2-carboxylic acid to growing cultures of Saccharomyces cerevisiae var. ellipsoideus promoted fast deactivation of the general aminoacid permease, measured as l-valine uptake, without an immediate decrease in the growth rate. Cells preincubated with the analogue for 3 h were unable to restore either growth ability or general aminoacid permease activity in analogue-free medium. Eadie-Hofstee plots of l-valine uptake in the presence of the analogue are consistent with a strong reduction in the number of active molecules of the general amino-acid permease located in the plasma membrane. Inhibitory effects on protein synthesis were seen after preincubations of the yeast with the analogue for 3 h although a 30 min preincubation had no effect.Abbreviations GAP general amino-acid permease - AZC l-azetidine 2-carboxylic acid - YNB yeast nitrogen base - YE Yeast extract     

Some properties of vacuoles isolated from Neurospora crassa slime variant

A method for the isolation of vacuoles based on polybase induced lysis of protoplasts of the cell wall deficient Neurospora crassa slime variant is described. Isolated vacuoles are characterized by 12 to 50 times increased specific activities of several hydrolases as compared with the total homogenate of protoplasts. Total -amino nitrogen, arginine, and polyphosphate are also greatly enriched in these vacuoles. Vacuoles are equipped with a permease for the transport of basic amino acids across the tonoplast.Non-Standard Abbreviation DEAE-dextran diethylaminoethyl-dextran     

Transformation in yeast: development of a hybrid cloning vector and isolation of the CAN1 gene.

We have constructed a plasmid, YEp13, which when used in conjunction with transformation in yeast is a suitable vector for isolating specific yeast genes. The plasmid consists of pBR322, the LEU2 gene of yeast, and a DNA fragment containing a yeast origin of replication from 2 mu circule. We have demonstrated the utility of this cloning system by isolating the yeast gene encoding the arginine permease, CAN1, from a pool of random yeast DNA fragments inserted into YEp13.     

Protein structural similarities predicted by a sequence-structure compatibility method.

A method for protein structure prediction has been developed, which evaluates the compatibility of an amino acid sequence with known 3-dimensional structures and identifies the most likely structure. The method was applied to a large number of sequences in a database, and the structures of the following proteins were predicted: (1) shikimate kinase (SKase), (2) the hydrophilic subunit of mannose permease (IIABMan), (3) rat tyrosine aminotransferase (Tyr AT), and (4) threonine dehydratase (TDH). The functional and evolutionary implications of the predictions are discussed. (1) The structural similarity between SKase and adenylate kinase was predicted. Alignment of their sequences reveals that the ATP-binding type A sequence motif and 2 ATP-binding arginine residues are conserved. The prediction suggests a similarity in their functional mechanisms as well as an evolutionary relationship. (2) The structural similarity between IIABMan and galactose/glucose-binding protein (GGBP) was predicted. The IIA and IIB domains are aligned with the N- and C-terminal domains of GGBP, respectively. The 2 phosphorylated residues, His 10 and His 175, of IIABMan are threaded onto loops located in the substrate-binding cleft of GGBP. The prediction accounts for the phosphoryl transfer from His 10 to His 175, and to the sugar substrate. (3) The structural similarity between rat Tyr AT and Escherichia coli aspartate AT was predicted, as well as (4) the structural similarity between TDH and the tryptophan synthase beta subunit. Predictions (3) and (4) support the previous predictions based on observations of the functional similarities between the proteins.     

Site-directed alkylation of LacY: effect of the proton electrochemical gradient

Previous N-ethylmaleimide-labeling studies show that ligand binding increases the reactivity of single-Cys mutants located predominantly on the periplasmic side of LacY and decreases reactivity of mutants located for the most part of the cytoplasmic side. Thus, sugar binding appears to induce opening of a periplasmic pathway with closing of the cytoplasmic cavity resulting in alternative access of the sugar-binding site to either side of the membrane. Here we describe the use of a fluorescent alkylating reagent that reproduces the previous observations with respect to sugar binding. We then show that generation of an H⁺ electrochemical gradient (Δ_μ¯H+, interior negative) increases the reactivity of single-Cys mutants on the periplasmic side of the sugar-binding site and in the putative hydrophilic pathway. The results suggest that Δ_μ¯H+, like sugar, acts to increase the probability of opening on the periplasmic side of LacY.