Facile synthesis of budding yeast a-factor and its use to synchronize cells of α mating type.
The ease with which populations of the budding yeast Saccharomyces cerevisiae can be synchronized using the mating pheromone α-factor has been invaluable for studies of the cell cycle. The α-factor response pathway has also remained an important model to study the molecular mechanism of G-protein coupled receptor signalling. α-Factor is a 13 amino acids long peptide that is readily available by automated peptide synthesis. However, only cells of the a mating type respond to α-factor. Cells of the opposite α mating type respond to a-factor, a farnesylated and C-terminally methylated 12 amino acids peptide. Because of its more difficult chemical synthesis, a-factor is not readily available and consequently the a-factor response is less well understood. Here we describe an improved strategy for producing a-factor, based on solid-phase peptide synthesis, followed by two simple steps in solution that show favourable characteristics and good yield. We demonstrate the successful use of the resulting a-factor to synchronize cell cycle progression of α cells. Notably, the a-factor concentrations required for cell synchronization are an order of magnitude lower than typically used α-factor concentrations. Despite a similar cell cycle response, shmoo formation was less pronounced compared to α-factor-treated a cells. Our protocol makes a-factor widely accessible, extending the ease of cell cycle synchronization to budding yeast cells of both mating types and facilitating the study of a-factor signalling.
Peptide Synthesis Laboratory, Cancer Research UK London Research Institute, UK.
Nicola
N
O'Reilly
Adrian
A
Charbin
Lidia
L
Lopez-Serra
Frank
F
Uhlmann
D002453:Cell Cycle / Q000187:drug effects*; D000072235:Mating Factor; D009172:Mycology / Q000379:methods*; D010455:Peptides / Q000138:chemical synthesis* / Q000302:isolation & purification / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000254:growth & development* / Q000378:metabolism*
D010455:Peptides; D000072235:Mating Factor