Peptide analogues compete with the binding of alpha-factor to its receptor in Saccharomyces cerevisiae.
alpha-Factor, a secreted tridecapeptide pheromone, is required for mating between the a- and alpha-haploid mating types of Saccharomyces cerevisiae. An analogue of alpha-factor, [DHP8,DHP11,Nle12] tridecapeptide (where DHP represents 3,4-dehydro-L-proline and Nle represents norleucine), was catalytically reduced in the presence of 3H gas to produce a radiolabeled pheromone with high specific activity, purity, and biological activity. Association and dissociation kinetics indicated values of 4.9 x 10(4) M-1 s-1 for k1 and 1.1 x 10(-3) s-1 for k-1. Saturation binding studies gave an equilibrium dissociation constant equal to 2.3 x 10(-8) M, which approximated the kinetically derived KD of 2.2 x 10(-8) M. These values compare favorably to the previously determined KD of 6 x 10(-9) M (Jenness, D.D., Burkholder, A.C., and Hartwell, L.H. (1986) Mol. Cell. Biol. 6, 318-320). Scatchard analysis and dissociation in the presence of excess unlabeled ligand indicated interaction with a homogeneous population of noninteracting binding sites (13,000 sites/cell). A number of alpha-factor analogues, previously investigated for their structure-function relationships (Naider, F., and Becker, J.M. (1986) CRC Crit. Rev. Biochem. 21, 225-249), were used to compete with [3H]alpha-factor binding. Four tridecapeptides having conservative amino acid replacements bound strongly to the receptor. In contrast, [Phe3]alpha-factor and 10 des-Trp1-alpha-factor analogues bound to the receptor 1-3 orders of magnitude less effectively than did alpha-factor itself. The binding constants for all active pheromones correlated with biological activity. However, des-Trp1[Phe3]alpha-factor and des-Trp1-[Ala3]alpha-factor, which were not biologically active, still competed with alpha-factor binding, indicating that these analogues fail to induce a secondary signal necessary for biological response to the pheromone. One analogue, des-Trp1-[Cha3,L-Ala9]alpha-factor (where Cha represents cyclohexylalanine), was not biologically active and did not demonstrate binding to the receptor, whereas des-Trp1-[Cha3,D-Ala9]alpha-factor was active and bound to the receptor. This finding suggests that a type II beta-turn is necessary for binding of alpha-factor to its receptor and for subsequent biological activity.
Raths
S K
SK
Department of Microbiology, University of Tennessee, Knoxville 37996.
Naider
F
F
Becker
J M
JM
D001667:Binding, Competitive; D007700:Kinetics; D000072235:Mating Factor; D009842:Oligopeptides / Q000494:pharmacology*; D010455:Peptides / Q000378:metabolism*; D010675:Pheromones / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism*; D014157:Transcription Factors
D009842:Oligopeptides; D010455:Peptides; D010675:Pheromones; D011956:Receptors, Cell Surface; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D000072235:Mating Factor