Investigation of HsGOT1 Through Yeast Two Hybridization

Undergraduate #58
Discipline: Biological Sciences
Subcategory: Cell and Molecular Biology

Stacey Phan - San Francisco State University
Co-Author(s): Zheng-Hui He, Hong-Yun Tong, Elias Michael Duarte, and Cinthya Ibarra, San Francisco State University, SF, CA

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Vitamin B6 functions as a crucial coenzyme for over 140 enzymes. Homeostasis of vitamin B6 is critical for cellular functions for all organisms, but how vitamin B6 homeostasis is regulated and maintained is unknown. Genetic and molecular studies in Arabidopsis have shown that aspartate aminotransferase 2 (ASP2) plays a role in vitamin B6 homeostasis signaling (Leasure et al., 2011). ASP2 covalently binds to the active form of vitamin B6, pyridoxal 5’-phosphate (PLP), and specific mutations in the PLP binding pocket of ASP2 suppress phenotypes in a vitamin B6 signaling mutant (Tong et al., 2008). Genetic complementation studies in Arabidopsis suggested that the human ASP2 ortholog, HsGOT1, may play similar roles in vitamin B6 homeostasis regulation. HsGOT1 is an important clinical indicator utilized ubiquitously to detect abnormalities in human liver health. How HsGOT1 may function in vitamin B6 homeostasis signaling is totally unknown. We hypothesize that HsGOT1 and its interactive partners play a role in vitamin B6 homeostasis signaling. HsGOT1 protein variants that carry the same mutations in the PLP binding pocket as those in ASP2 protein will be tested for their protein-protein interactions via yeast two-hybrid assays. Two constructs carrying specific residue substitutions (G387A and HsGOT S1-1) were generated and confirmed by sequencing. These constructs were subcloned into the two yeast two-hybrid assay vectors (AD, GAL4 transcription active domain and BD, GAL4 DNA-binding domain), all of which were subsequently confirmed by sequencing. Yeast competent cells were prepared and the transformed cells carrying HsGOT1 various alleles were successfully screened on selective dropout media. Our results confirmed that we have successfully engineered yeast two-hybrid constructs that carry three alleles (HsGOT1, HsGOT 387, and HsGOT S1-1), each of which was fused with either the BD or the AD domain. Our initial test suggested that HsGOT1 interacts with itself, indicating that HsGOT1 forms a homodimer. In conclusion, yeast two-hybrid assay can be effectively used to test protein-protein interaction for HsGOT1 and its variants. We are in the process of testing protein-protein interactions among HsGOT1 and HsGOT1 variants. Our studies are expected to provide insight into how aminotransferases in general interact with vitamin B6 and other proteins to function in vitamin B6 homeostasis signaling.

Funder Acknowledgement(s): National Science Foundation, National Institutes of Health

Faculty Advisor: Zheng-Hui He, zhe@sfsu.edu

Role: HsGOT1 subcloning into BD and AD vectors separately. Yeast transformation of HsGOT1 AD and BD vectors simultaneously into dropout plates.