A simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae: Gap1p as an example

Abstract

We established a simple procedure for protein ubiquitination detection in Saccharomyces cerevisiae. Enhanced green fluorescent protein (EGFP) was split into two parts, an N-terminal (GN) and a C-terminal (GC) region. The fusion fragments GN-UBI3 and multi-cloning site (MCS)-GC were inserted into the vector pY26-TEF/GPD, resulting in pUbDetec16. pUbDetec16 was designed for use in detecting protein ubiquitination. Any gene of interest can be inserted into the MCS and the recombinant plasmid can be transferred into a Δura3 auxotrophic S. cerevisiae strain. Protein ubiquitination can then be detected using a fluorescence microscope. The ubiquitination of a protein can be determined based on a fluorescence signal. To validate the reliability of this procedure, Gap1p, a protein known to be ubiquitinated, was used as a positive control. A triple mutant of Gap1p, Gap1p(K9R,K16R,K76R), which did not contain any ubiquitination site, was used as a negative control. pUbDetec16-GAP1 and pUbDetec16-GAP1(K9R,K16R,K76R) were constructed and transferred into the Δura3 auxotrophic S. cerevisiae strain CEN.PK2-1D. Transformants of pUbDetec16-GAP1 emitted fluorescence, while the pUbDetec16-GAP1(K9R,K16R,K76R) transformants did not. The ubiquitination of Gap1p and Gap1p(K9R, K16R, K76R) was further verified using classical SDS-PAGE analysis. This procedure significantly simplifies manipulation involving ubiquitination detection using the BiFC approach, particularly on a large scale.