The yeast a-factor transporter Ste6p, a member of the ABC superfamily, couples ATP hydrolysis to pheromone export.

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-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 microm). Modest stimulation (approximately 40%) was observed upon the addition of a-factor either synthetic or in native form. We also used an 8-azido-[alpha-(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-factor transport, which in turn promotes mating.

Fluorescence-based protein footprinting using histidine-tagged protein.

We describe a procedure for protein footprinting to identify the region(s) of a protein that interacts with a ligand. The method utilized the affinity of a stretch of histidine residues cloned into the protein to metal-chelated resin. After limited protease digestion, the histidine-tagged end fragments were separated by the resin and labeled with a fluorescein derivative. Resolving the labeled digestion products on a denaturing polyacrylamide gel and visualizing the peptides using a FluorImager provided a way to identify the protease target sites that were protected from digestion because of interaction with DNA. The protection experiments would be applicable not only to detect direct contact sites but also sites allosterically altered by ligand binding.

Analysis of the spacer DNA between the cyclic AMP receptor protein binding site and the lac promoter.

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.

Stabilization of a protein-tyrosine phosphatase mRNA upon mitogenic stimulation of T-lymphocytes.

The expression of a non-receptor type protein-tyrosine phosphatase (the T-cell phosphatase or PTP-S) which shows homology with basic domains of Fos and Jun, was investigated upon mitogenic stimulation of rat splenic T lymphocytes. As studied by Northern blot analysis of total cellular RNA, mitogenic stimulation of T lymphocytes by concanavalin A resulted in an increase in the level of PTP-S mRNA; there was little or no change in the level of mRNA coding for PTP-1 (which is also a non-receptor type tyrosine phosphatase). Maximum increase of about 3-fold in the level of PTP-S mRNA occurred after 72 h of mitogenic stimulation. Mitogenic stimulation did not increase the level of PTP-S transcripts in the nucleus. The half-life of PTP-S mRNA in unstimulated lymphocytes was about 25 min which increased to 5 h after mitogenic stimulation. An inhibitor of protein synthesis, cycloheximide, increased the level of PTP-S transcripts by 6-fold in control lymphocytes but did not increase the level of PTP-1 transcripts. Treatment with cycloheximide increased the half-life of PTP-S transcripts in resting lymphocytes. The PTP-S gene product was identified as a 42 kDa polypeptide by immunoblotting. The level of PTP-S gene product increased upon mitogenic stimulation of lymphocytes by Con A and reached a maximum after 72 h, as determined by immunoblotting. These results suggest that post-transcriptional regulation of mRNA stability is an important factor in controlling the level of this phosphatase mRNA during mitogenic stimulation of T-lymphocytes.

Interaction of the Mnt repressor with 37-base pair synthetic operator DNA fragments. Importance of symmetric GC pairs.

Mnt repressor is indirectly responsible for the maintenance of lysogeny of the phage P22. This repressor interacts with a 21-base pair operator DNA constituting within it a 17-base pair perfect 2-fold symmetric sequence whose bases make a direct contact with the protein. We have synthesized six 37-base pair DNAs consisting of 21 base pair natural operator and its modifications in which certain symmetrically situated GC base pairs were replaced systematically with ATs to understand their importance. The binding interaction studies of Mnt repressor to such natural and modified operator DNAs reported here indicate that the GCs close to the center of symmetry make major contacts with the protein whereas, GCs nearer to the periphery form weak contacts. Methylation protection experiments indicated that when the GCs near the center of symmetry were replaced with AT, the central GC became more accessible for dimethyl sulfate methylation with possible conformational change in DNA. The circular dichroism studies indicated that upon repressor binding conformational changes in DNA takes place with a possible increase in helicity of the repressor protein.