APEX2 is a functional tag in S. cerevisiae. (A) Two plasmids were constructed, expressing APEX2-tagged ERG11-V5 or the control construct ERG11-V5. The plasmids were transformed into S. cerevisiae. (B) After overnight growth of the yeast cells in –Leu medium, they were lysed and the proteins were extracted. The protein extracts then were incubated with a DAB solution with H2O2 to detect whether the APEX2 tag fused to Erg11 is expressed and active. Indeed, for the cells expressing ERG11-V5-APEX2, the protein extract was colored brown, indicating that the APEX2 tag retained its peroxidase activity and that the chimeric protein is functionally expressed in S. cerevisiae. As expected, the protein extract of the control cells remained white. (C) Tetrad dissection of IP-S7 cells transformed with the pIP10 plasmid containing ERG11-V5-APEX2 or the pIP12 plasmid containing ERG11-V5, or the empty vector (EV) pBEVY-L as a negative control, shows that the Erg11 proteins expressed from the plasmids are functional, as they can sustain the germination of spores lacking endogenous ERG11. The smaller colonies (indicated by red dots) were shown to lack endogenous ERG11 by PCR. Replating these cells on selective −Leu medium showed that all the smaller colonies retained the pIP10 or pIP12 plasmid.