Ternary Complex Formation of Ino2p-Ino4p Transcription Factors and Apl2p Adaptin β Subunit in Yeast

Abstract

Yeast Ino2p-Ino4p heterodimeric complex is well known as a transcriptional activator for the genes regulated by inositol and choline, such as the INO1 gene. Apl2p is a large subunit of the yeast adaptin complex, an adaptor complex required for the clathrin coat to bind to the membrane. We found that Ino2p, Ino4p, and Apl2p form a ternary complex. This interaction was initially observed in a yeast two-hybrid study and subsequently verified by co-immunoprecipitation. Ino2p and Ino4p bind to Apl2p in the same region of Apl2p, viz., at the middle part and the C-terminal part. Ino2p and Ino4p bind to Apl2p independently, but more strongly when both are present. Furthermore, a disruption of APL2 together with INO2 or INO4 rendered yeast cells sensitive to oxidative stress. INO2-APL2 double disruptants also showed growth inability in nonfermentable carbon sources, such as glycerol. These results indicate a genetic interaction between APL2, INO2 and INO4 and uncovere novel functions of the Ino2p-Ino4p-Apl2p complex in yeast.

Metabolic Engineering of Saccharomyces cerevisiae for Astaxanthin Production and Oxidative Stress Tolerance

“…The following 2-based high-copy-number plasmids were used for the construction of expression vectors in this study: pTV3, pUV2, and pHV1, containing the S. cerevisiae TRP1, URA3, and HIS3 selection markers, respectively (25), and pAD4 (supplied by J. Nikawa [Kyushu Institute of Technology, Fukuoka, Japan]) (20), which contains S. cerevisiae LEU2 as a selection marker and the ADH1 promoter/terminator. To eliminate one of the BamHI sites of pHV1, pTV3 and pHV1 were digested with PstI and a small fragment derived from pHV1 and a large fragment from pTV3 were ligated and named pHV3.…”

Characterization of a New Saccharomyces cerevisiae Isolated From Hibiscus Flower and Its Mutant With L-Leucine Accumulation for Awamori Brewing

“…The centromere-based low-copy-number plasmid pYC130 containing the G418 resistance gene (KanMX4) (supplied by National Research Institute of Brewing, Hiroshima, Japan) (Calera et al, 2000) and the 2 μ-based high-copy-number plasmid pAD4 containing the ADH1 promoter and terminator (supplied by J. Nikawa, Kyushu Institute of Technology, Fukuoka, Japan) (Nikawa et al, 2006) were used to subclone and express the LEU4 gene, respectively. Escherichia coli strain DH5α [F - λ - Φ 80lacZΔM15 Δ(lacZYA argF)U169 deoR recA1 endA1 hsdR17 ( r k + m k - ) supE44 thi-1 gyrA96 ] was used to construct plasmids.…”