title: 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.
authors.affiliation: School of Biotechnology and Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, 1800 Lihu Road, Wuxi, Jiangsu 214122, China.
authors.forename: Yongkun
authors.initials: Y
authors.lastname: Lv
authors.forename: Xinrui
authors.initials: X
authors.lastname: Zhao
authors.forename: Long
authors.initials: L
authors.lastname: Liu
authors.forename: Guocheng
authors.initials: G
authors.lastname: Du
authors.forename: Jingwen
authors.initials: J
authors.lastname: Zhou
authors.forename: Jian
authors.initials: J
authors.lastname: Chen
mesh_terms: D026905:Amino Acid Transport Systems / Q000378:metabolism*; D017930:Genes, Reporter; D005822:Genetic Vectors; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D008856:Microscopy, Fluorescence; D010957:Plasmids; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D054875:Ubiquitination
chemical_list: D026905:Amino Acid Transport Systems; C491145:GAP1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins