Functional analysis of the yeast Ran exchange factor Prp20p: in vivo evidence for the RanGTP gradient model.

Numerous cellular processes rely on the movement of macromolecules into and out of the nucleus. The primary regulator of this movement is the small GTPase Ran. Like other small GTPases, the nucleotide-bound state of Ran is regulated by effectors that enhance the rate of nucleotide exchange or hydrolysis. Current models for vectorial nuclear transport suggest that it is the strict compartmentalization of these Ran effector molecules that generates a gradient of RanGTP between the nucleus and the cytoplasm to impart directionality to the transport process. Here we investigate the mechanism by which the Ran exchange factor is targeted to the nucleus, and test the impact of disrupting this nuclear compartmentalization on nucleocytoplasmic transport in vivo. Our results indicate that in Saccharomycces cerevisiae the nucleotide exchange factor Prp20p can be targeted to the nucleus via a classical nuclear localization sequence. This transport mechanism is dependent both on Ran and the receptor that recognizes the nuclear localization sequence, importin alpha. Mutations in the evolutionarily conserved nuclear localization sequence only partially inhibit nuclear import of Prp20p, suggesting the existence of a secondary mechanism for this critical nuclear targeting. In an in vivo test of the RanGTP gradient model, we demonstrate that overexpression of a functional cytoplasmic exchange factor inhibits cell growth and blocks both protein import and RNA export in wild-type cells that contain the endogenous nuclear Prp20 protein. Taken together, our results provide in vivo evidence for the idea that the compartmentalization of the exchange factor serves as a mechanism for establishing directional nuclear transport.

A mutation in a new gene, bglJ, activates the bgl operon in Escherichia coli K-12.

A new mutation, bglJ4, has been characterized that results in the expression of the silent bgl operon. The bgl operon encodes proteins necessary for the transport and utilization of the aromatic beta-glucosides arbutin and salicin. A variety of mutations activate the operon and result in a Bgl+ phenotype. Activating mutations are located upstream of the bgl promoter and in genes located elsewhere on the chromosome. Mutations outside of the bgl operon occur in the genes encoding DNA gyrase and in the gene encoding the nucleoid associated protein H-NS. The mutation described here, bglJ4, has been mapped to a new locus at min 99 on the Escherichia coli K-12 genetic map. The putative protein encoded by the bglJ gene has homolgy to a family of transcriptional activators. Evidence is presented that increased expression of the bglJ product is needed for activation of the bgl operon.

Mutations in a new chromosomal gene of Escherichia coli K-12, pcnB, reduce plasmid copy number of pBR322 and its derivatives.

We describe mutants of Escherichia coli that decrease the plasmid copy number of pBR322 derivatives. One mutant was partially characterized genetically and its mutation, designated pcnB for plasmid copy number, was mapped to approximately 3 min on the E. coli chromosome. This locus is distinct from other genes whose products are known to affect plasmid replication or stable plasmid maintenance. The pcnB mutant strain should be useful for cloning genes into pBR322 that have aberrant or deleterious effects on the cell when present in high copy number.

D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport.

We have isolated mutants defective in high-affinity D-ribose transport. The mutations map in rbsT or rbsB , the structural gene for ribose binding protein. rbsT consists of at least one gene coding for a protein required for high-affinity transport. The high-affinity transport-defective mutants were able to utilize D-ribose, indicating that at least a second, low-affinity transport system for D-ribose is present in Escherichia coli K-12. rbsT and rbsB are located at min 84 on the E. coli genetic map and, together with rbsK , the gene coding for ribokinase , constitute an rbs operon. The order of genes is rbsP /O rbsT rbsB rbsK . The rbs operon is subject to negative control by the product of the rbsR gene. rbsR is located distal to the rbs operon and appears to form a separate transcriptional unit.

Membrane transport of p-nitrophenyl-alpha-galactoside by the melibiose carrier of Escherichia coli.

p-Nitrophenyl-alpha-galactoside (alpha-pNPG) was found to be a substrate for the melibiose transport system of Escherichia coli. This sugar enters induced cells via the carrier and is split by alpha-galactosidase to galactose and p-nitrophenol. In mutant cells lacking the alpha-galactosidase [3H]-alpha-pNPG accumulated to concentrations 15 times higher than the external medium. The transport of alpha-pNPG is inhibited by both Na+ and Li+. Na+ (10 mM) reduced the Km for alpha-pNPG from 0.45 to 0.18 mM and reduced the Vmax from 6.7 nmoles/min/mg dry wt to a value of 3.0.

Effect of lithium ion on melibiose transport in Escherichia coli.

Both Li+ and Na+ stimulated the uptake of thiomethylgalactoside by the melibiose transport system of Escherichia coli. On the other hand, Li+ inhibited the growht of cells on melibiose as a sole source of carbon. This inhibition was specific for melibiose, and Li+ had no effect on growth of cells on glucose, galactose, lactose, or glycerol. The effect of the cation on melibiose transport was investigated in a mutant which cannot utilize glucose. After entry into this cell, melibiose is cleaved into glucose and galactose by alpha-galactosidase, and the resulting glucose is excreted. Since the entry step was found to be rate-limiting, glucose production could be taken as a measure of melibose transport. Li+ inhibited the transport of melibiose, but not the induction of the melibiose operon nor the activity of alpha-galactosidase. Li+ was found to inhibit the entry of p-nitrophenyl-alpha-D-galactoside, but not p-nitrophenyl-beta-D-galactoside entry. Thus, the cation specificity for the melibiose membrane carrier varies different transport substrates.

Role of Na+ and Li+ in thiomethylgalactoside transport by the melibiose transport system of Escherichia coli.

Thiomethyl-beta-galactoside (TMG) accumulation via the melibiose transport system was studied in lactose transport-negative strains of Escherichia coli. TMG uptake by either intact cells or membrane vesicles was markedly stimulated by Na+ or Li+ between pH 5.5 and 8. The Km for uptake of TMG was approximately 0.2 mM at an external Na+ concentration of 5 mM (pH 7). The alpha-galactosides, melibiose, methyl-alpha-galactoside, and o-nitrophenyl-alpha-galactoside had a high affinity for this system whereas lactose, maltose and glucose had none. Evidence is presented for Li+-TMG or Na+-TMG cotransport.