Abstract
STRA6 is a plasma membrane protein that mediates the transport of vitamin A, or retinol, from plasma retinol binding protein (RBP) into the cell. Mutations in human STRA6 are associated with Matthew-Wood syndrome, which is characterized by severe developmental defects. Despite the obvious importance of this protein to human health, little is known about its structure and mechanism of action. To overcome the difficulties frequently encountered with the production of membrane proteins for structural determination, STRA6 has been expressed in Pichia pastoris as a fusion to green fluorescent protein (GFP), a strategy which has been a critical first step in solving the crystal structures of several membrane proteins. STRA6-GFP was correctly targeted to the cell surface where it bound RBP. Here we report the large-scale expression, purification and characterisation of STRA6-GFP. One litre of culture, corresponding to 175 g cells, yielded about 1.5 mg of pure protein. The interaction between purified STRA6 and its ligand RBP was studied by surface plasmon resonance-based binding analysis. The interaction between STRA6 and RBP was not retinol-dependent and the binding data were consistent with a transient interaction of 1 mole RBP/mole STRA6.
Highlights
The vitamin A transporter/retinol binding protein (RBP) receptor, alternatively called STRA6 (Uniprot: Q9BX79), is an integral plasma membrane protein that mediates the bidirectional transfer of retinol, or vitamin A, between plasma RBP (Uniprot: P02753) and the intracellular retinoid-handling protein machinery [1, 2]
We describe the expression and purification of the RBP receptor and vitamin A transporter, STRA6
A STRA6-green fluorescent protein (GFP) fusion protein was expressed in Pichia pastoris and was correctly targeted to the cell surface where it was able to bind its ligand, RBP
Summary
The vitamin A transporter/retinol binding protein (RBP) receptor, alternatively called STRA6 (Uniprot: Q9BX79), is an integral plasma membrane protein that mediates the bidirectional transfer of retinol, or vitamin A, between plasma RBP (Uniprot: P02753) and the intracellular retinoid-handling protein machinery [1, 2]. RBP circulates in a non-covalent complex with transthyretin (TTR), which stabilizes the binding of retinol to RBP [3, 4]. On binding of RBP to STRA6, retinol is transported into the cell, but the RBP is not internalised [1, 5, 6]. Seen in bacteria [7, 8], this type of double-function as a receptor and transporter is unique in eukaryotic systems. Mutations in human STRA6 can cause Matthew-Wood syndrome, which is characterized by variable combinations of severe developmental defects such
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