Role of DUF647 Containing Proteins in Vitamin B6 Homeostasis
By: Melissa Hassmer
Department: Biology
Faculty Advisor: Dr. Zachary He
Vitamin B6 (vitB6) is essential to all organisms, serving as a critical cofactor for many enzymes required in cellular metabolism and signaling. The active form of vitB6, pyridoxal 5’-phosphate (PLP), can be cytotoxic if left unbound in excess levels within the cell. Molecular and genetics studies in our laboratory have identified three genes that play key roles in regulating the PLP homeostasis: Root UV-B Sensitive protein 1 (RUS1), Root UV-B Sensitive protein 2 (RUS2), and Aspartate Aminotransferase protein 2 (ASP2). Mutant plants missing either the RUS1 or the RUS2 gene are developmentally arrested and exhibit phenotypes related to deficiencies in PLP homeostasis. ASP2 has been identified as a key player interacting with both RUS1 and RUS2, possibly forming a complex to regulate PLP homeostasis. RUS1 family proteins contain a Domain of Unknown Function (DUF647) that is conserved throughout the domain Eukaryota. Predicted DUF674 structure is similar to membrane associated channel proteins. I hypothesize that DUF647 domains in RUS1 and RUS2 function as membrane associated channels to regulate vitamin B6 homeostasis. To test this hypothesis, microsomal pellets is prepared via fractionation and ultracentrifugation, followed by western blot. A RUS1-GFP-complemented RUS1 knockout mutant is used to identify expression of GFP and thus RUS1 within the microsomal level via an anti-GFP antibody. Furthermore, antibodies against ASP2 and RUS2-GFP will be used to test the potential co-existence of RUS1, RUS2, and ASP2. We expect to gain important insights into the location of RUS1, RUS2, and ASP2 within the cell, as well as the function of DUF647 in vitamin B6 homeostasis regulation.