Part:BBa_K3384315

Ste5ΔN-CTM

Ste5ΔN-CTM is a highly active Ste5 mutant which can bind to the plasma membrane in the absence of pheromone. Ste5 is an important scaffold protein in the mating pheromone signaling pathway. The 214 amino acids at the N-terminus of Ste5 contain membrane binding sites and mediate the membrane localization of Ste5 under the control of Gβγ. By deleting the N-terminal sequence, the activation of Ste5 is independent of G protein. The C-terminal transmembrane domain of Snc2 was added to the Ste5ΔN C-terminus in order to constitutively recruit Ste5 to the plasma membrane1. As shown in figure 1, the Ste5ΔN-CTM protein can activate the MAPK pathway in the absence of pheromone and promote the expression of pheromone-responsive genes.

Fig.1 The results of the homology and structural modelling protein amajLime.

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Characterization

Ste5ΔN-CTM was integrated into the pgal1 gene expression cassette in the plasmid pYES2. Plate growth experiments were used to verify the function of the protein. Galactose can induce the expression of Ste5ΔN-CTM, which leads to the activation of the pheromone signaling pathway, inhibiting the growth of colonies. As shown in figure 2, it was observed that the recombinant yeast containing Ste5ΔN-CTM grew slower than the wild-type yeast on the solid medium with galactose as the carbon source, indicating that Ste5ΔN-CTM activated the pheromone signal pathway. As shown in figure 3, The growth rate of recombinant yeast containing Ste5ΔN-CTM on the SC solid medium with glucose as the carbon source is no different from that of the wild type.

          <img src="https://2020.igem.org/wiki/images/d/d1/T--NJTech_China--Ste5ΔN-CTM.png" class="imgStyle1" alt="">

Reference

1. Lamson, R. E., Takahashi, S., Winters, M. J., and Pryciak, P. M. (2006) Dual role for membrane localization in yeast MAP kinase cascade activation and its contribution to signaling fidelity, Curr Biol 16, 618-623.

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal EcoRI site found at 365
  Illegal SpeI site found at 1620
  Illegal SpeI site found at 2166
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal EcoRI site found at 365
  Illegal SpeI site found at 1620
  Illegal SpeI site found at 2166
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal EcoRI site found at 365
  Illegal BglII site found at 813
  Illegal BglII site found at 2121
  Illegal XhoI site found at 2058
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal EcoRI site found at 365
  Illegal SpeI site found at 1620
  Illegal SpeI site found at 2166
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal EcoRI site found at 365
  Illegal SpeI site found at 1620
  Illegal SpeI site found at 2166
  
• 1000
  COMPATIBLE WITH RFC[1000]