A systematic study of yeast sterol biosynthetic protein-protein interactions using the split-ubiquitin system.
Sterol biosynthesis occurs in the ER and most sterol biosynthetic enzymes have transmembrane domains. However, due to difficulties in characterizing membrane protein-protein interactions, the nature of the sterol biosynthetic complex as well as in vivo interactions between various enzymes have not been described. We employed a split-ubiquitin membrane protein yeast two-hybrid system to characterize interactions between sterol biosynthetic proteins. Fourteen bait constructs were co-transformed into a reporter yeast strain with 14 prey constructs representing all sterol enzymatic reactions beginning with the synthesis of squalene. Our results not only confirmed several previous interactions, but also allowed us to identify novel interactions. Based on these results, ergosterol biosynthetic enzymes display specific protein-protein interactions forming a functional complex we designate, the ergosome. In this complex, Erg11p, Erg25p, Erg27p, and Erg28p appear to form a core center that can interact with other enzymes in the pathway. Also Erg24p and Erg2p, two enzymes that are sensitive to morpholine antifungals, appear to interact with one another; however, the profile of protein interaction partners appears to be unique. Erg2p and Erg3p, two enzymes catalyzing sequential reactions also appear to have different interaction partners. Our results provide a working model as to how sterol biosynthetic enzymes are topologically organized not only in yeast but in plant and animal systems that share many of these biosynthetic reactions.
Mo
Caiqing
C
Department of Biology, Indiana University-Purdue University Indianapolis, 723 W. Michigan St. Indianapolis, IN 46202, USA.
Bard
Martin
M
D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D004875:Ergosterol / Q000096:biosynthesis; D005800:Genes, Fungal; D017930:Genes, Reporter; D007763:Lac Operon; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism*; D013261:Sterols / Q000096:biosynthesis*; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism*
D017931:DNA Primers; D004271:DNA, Fungal; D029701:Saccharomyces cerevisiae Proteins; D013261:Sterols; D025801:Ubiquitin; D004875:Ergosterol