Specific Residues in a Purine Transporter Are Critical for Dimerization, ER Exit, and Function

Area of Science:

• Membrane Biology
• Protein Biochemistry
• Molecular Genetics

Background:

• Transporter proteins mediate solute movement across membranes.
• Nucleobase ascorbate transporter (NAT) family members, like UapA, form functional dimers.
• Phospholipid interactions are vital for UapA dimer stability.

Purpose of the Study:

• Investigate the role of lipid interactions in UapA dimer formation and trafficking.
• Identify genetic suppressors of UapA dimerization and trafficking defects.
• Explore the potential for expressing mammalian transporters in Aspergillus.

Main Methods:

• Genetic screening to identify suppressor mutations.
• Molecular dynamics simulations to analyze protein structure.
• Functional expression of mammalian transporters in Aspergillus nidulans.

Main Results:

• Distinct lipid interactions are essential for UapA's ab initio dimer formation in the ER and subsequent trafficking.
• Suppressor mutations, particularly those in the core domain, restore UapA dimerization by stabilizing interhelical interactions.
• A specific mutation (T401P) restored both dimerization and trafficking, highlighting the importance of core domain stability.
• Equivalent mutations enabled functional expression of a rat NAT (rSNBT1) in Aspergillus.

Conclusions:

• Membrane lipid interactions critically regulate UapA transporter biogenesis and function.
• Stabilizing the transporter's core domain can overcome defects caused by disrupted lipid interactions.
• Aspergillus nidulans serves as a viable system for the functional expression of mammalian transporters.