ATP-binding cassette (ABC) proteins transport a diverse collection of substrates. It is presumed that these proteins couple ATP hydrolysis to substrate transport, yet ATPase activity has been demonstrated for only a few. To provide direct evidence for such activity in Ste6p, the yeast ABC protein required for the export of a a-factor mating pheromone, we established conditions for purification of Ste6p in biochemical quantities from both yeast and Sf9 insect cells. The basal ATPase activity of purified and reconstituted Ste6p (V max ‫؍‬ 18 nmol/ mg/min; K m for MgATP ‫؍‬ 0.2 mM) compares favorably with several other ABC proteins and was inhibited by orthovanadate in a profile diagnostic of ABC transporters (apparent K I ‫؍‬ 12 M). Modest stimulation (ϳ40%) was observed upon the addition of a a-factor either synthetic or in native form. We also used an 8-azido-[␣-32 P]ATP binding and vanadate-trapping assay to examine the behavior of wild-type Ste6p and two different double mutants (G392V/G1087V and G509D/G1193D) shown previously to be mating-deficient in vivo. Both mutants displayed a diminished ability to hydrolyze ATP, with the latter uncoupled from pheromone transport. We conclude that Ste6p catalyzes ATP hydrolysis coupled to a a-factor transport, which in turn promotes mating.

The cystic fibrosis transmembrane conductance regulator (CFTR) functions in vivo as a cAMPactivated chloride channel. A member of the ATP-binding cassette superfamily of membrane transporters, CFTR contains two transmembrane domains (TMDs), two nucleotide-binding domains (NBDs), and a regulatory (R) domain. It is presumed that CFTR couples ATP hydrolysis to channel gating, and as a first step in addressing this issue directly, we have established conditions for purification of biochemical quantities of human CFTR expressed in Sf9 insect cells. Use of an 8-azido-[α 32 P]-ATP binding and vanadate-trapping assay allowed us to devise conditions to preserve CFTR function during purification of a C-terminal His 10 -tagged variant after solubilization with lyso-phosphatidylglycerol (1%) and diheptanolylphosphatidylcholine (0.3%) in the presence of excess phospholipid. Study of purified and reconstituted CFTR showed that it binds nucleotide with an efficiency comparable to that of P-glycoprotein, and that it hydrolyzes ATP at rates sufficient to account for presumed in vivo activity (V Max of 58 ± 5 nmol/min/mg protein, K M (MgATP) of 0.15 mM). In further work, we found that neither nucleotide binding nor ATPase activity were altered by phosphorylation (using Protein Kinase A) or dephosphorylation (with Protein Phosphatase 2B); we also observed inhibition (∼40%) of ATP hydrolysis by reduced glutathione, but not by DTT. To evaluate CFTR function as an anion channel, we introduced an in vitro macroscopic assay based on the equilibrium exchange of proteoliposome-entrapped radioactive tracers. This revealed a CFTRdependent transport of 125 I that could be inhibited by known chloride channel blockers; no significant CFTR-dependent transport of [α 32 P]-ATP was observed. We conclude that heterologous expression of CFTR in Sf9 cells can support manufacture and purification of fully functional CFTR. This should aid in further biochemical characterization of this important molecule.The Cystic Fibrosis Transmembrane conductance Regulator (CFTR) is the cAMP-activated chloride channel encoded by the gene defective in patients with the disease [1,2]. As a member of the ATP-Binding Cassette (ABC) superfamily of membrane transporters, CFTR shares a conserved architecture consisting of two homologous halves, each containing a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). In CFTR, unlike other ABC transporters, a third domain, termed the regulatory (R) domain, is located between the two half molecules. Current evidence suggests that the TMDs define the CFTR chloride channel, while the NBDs and the R domain mediate channel gating [3][4][5][6][7][8][9][10][11] Although CFTR is glycosylated, there is currently no evidence indicating that the presence of carbohydrate affects CFTR structure or function [12]. Consistent with this presumption, expression of human CFTR in Sf9 insect cells results in appearance of the 140 kD core polypeptide -containing little or no glycosylation -that mediates a newly acquired anion ...

No abstract

The role of the spacer region DNA between the cyclic AMP receptor protein (CRP) site and the RNA polymerase in the lac promoter was examined. We wanted to determine whether the wild-type DNA sequence of this region was an absolute requirement for CRP activation of lac transcription. The sequence of a 9-bp stretch of the spacer, from ؊41 to ؊49 relative to the start of transcription, was randomized, and the effect of randomization on lac expression was investigated in vitro and in vivo. We found that the spacer contains no specific sequence determinants for CRP activation of lac transcription; fewer than 1% of the mutants displayed greater than a 50% decrease in CRP activation of lac transcription.Transcription initiation of the lac operon in Escherichia coli requires activation by the cyclic AMP (cAMP) receptor protein (CRP) (for a review, see the work of Kolb et al. [16]). CRP itself binds cAMP and undergoes a change in conformation. This allosteric change results in its high-affinity binding to a DNA site centered at Ϫ61.5 bp from the lac transcriptional start site. CRP binding to the DNA results in an approximately 50-fold increase of the in vivo transcription of the lac genes over the baseline level observed without functional CRP (10,27). A specific interaction of CRP with an ␣ subunit of RNA polymerase (RNAP) has been inferred from biochemical and genetic experiments (2,5,9,11,16,20,24) and demonstrated directly by cross-linking (8). This CRP-RNAP interaction can be disrupted in a variety of ways: mutations in RNAP (31, 34), truncation of the ␣ subunit of RNAP (15, 34), mutations in CRP (2, 11, 33), and moving the CRP binding site such that it is on a different face of the DNA or too far upstream (13,18,30,32). In each case, CRP activation of transcription is affected, indicating that the protein-protein interaction between CRP and RNAP is an essential feature of CRP activation of lac transcription. However, several experiments have also shown that, while necessary, this protein-protein interaction may not be sufficient for activation and that the spacer DNA in the region between where CRP binds and the lac promoter may play an equally important role. When single-stranded gaps were introduced into this spacer region, CRP activation was drastically reduced or eliminated even though the proteinprotein interaction and the promoter integrity were not affected (26). Situated analogously to this spacer in lac, a DNA sequence called the UP element has been found to act as a third promoter element along with the Ϫ10 and Ϫ35 sites in the P1 promoter of the rrnB operon in E. coli (25). Whereas the latter two sites interact with the subunit of RNAP, the UP element, an AϩT-rich sequence upstream of the Ϫ35 site, specifically interacts with the ␣ subunit of RNAP. By extension, it has been proposed that the interaction between CRP and the ␣ subunit of RNAP in lac causes a direct interaction, as in rrnB, between ␣ and the spacer DNA (4, 6, 31). A possible explanation for the defect in transcription activation by CRP seen...