Abstract:
This study investigates the structure and function of the purine/H⁺ symporter UapA from Aspergillus nidulans, emphasizing its homodimerization and transport mechanisms. Although substantial information exists on nucleobase-ascorbate transporters (NATs), the role of transporter oligomerization in functionality remains underexplored. This research aims to elucidate the structural basis of UapA dimerization and its impact on substrate specificity and transport activity. Methodologically, a thermostabilized UapA variant was expressed in Saccharomyces cerevisiae, purified using Ni²⁺-affinity chromatography, and crystallized for structural determination through X-ray diffraction. Additionally, transport assays and mutational analyses were conducted to assess dimer functionality. Results reveal that UapA forms stable dimers, with residues at the dimer interface playing critical roles in substrate recognition and transport. Dimerization was shown to be essential for effective substrate translocation, implying that UapA employs an elevator mechanism involving conformational shifts in the core domain. The study’s primary takeaway is that dimerization not only stabilizes UapA structure but also fine-tunes substrate specificity, potentially extending to other NAT family transporters.
Conclusion on the Role of Constant Systems Cell Disruption equipment:
Constant Systems Equipment was pivotal in this research, facilitating the efficient lysis and extraction of yeast cells for membrane protein isolation. This equipment allowed for the high-yield extraction of intact UapA transporter proteins from Saccharomyces cerevisiae, ensuring structural integrity crucial for subsequent purification, crystallization, and functional analysis. Consequently, Constant Systems’ technology was instrumental in the study’s success, supporting detailed structural and functional insights into the UapA transporter’s dimerization and transport mechanisms.
