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P4-ATPase structure-function relationships : mechanism and roles of ATP8A2-CDC50A in aminophospholipid transport, protein trafficking, and visual disorders

University of British Columbia

P₄-ATPases are a family of membrane transporters which have been implicated in the energy-dependent transport of aminophospholipids from the exocytoplasmic to cytoplasmic surface of biological membranes. This thesis investigation examined the structure-function relationships of ATP8A2, a novel member of the P₄-ATPase family initially discovered in a proteomic study of photoreceptor outer segments. Photoreceptor outer segments are organelles which consist of stacks of membraneous discs containing visual pigment molecules. ATP8A2 is shown to be present in photoreceptor outer segment discs and preferentially transports phosphatidylserine towards the cytosolic leaflet, providing the first direct demonstration of lipid transport by a purified mammalian P₄-ATPase. CDC50A, the β-subunit of ATP8A2 was discovered using mass spectrometry and Western blotting. Subunit interactions are mediated through the extracellular and membrane domains of CDC50A. The N-terminal domain of CDC50A appears to play a role in pump modulation. ATP8A2 forms a phosphoenzyme intermediate at Asp⁴¹⁶ and phosphatidylserine appears to be transported in a similar manner to that of other cation-transporting P-type ATPases. The phosphoenzyme exists in two distinct conformations: E₁P and E₂P. The E₂P form interacts with aminophospholipids. Lys⁸⁷³ in transmembrane segment M5 located in a region known to be important for cation binding for P-type ATPases is critical for phosphatidylserine binding. Glu¹⁹⁸ in the DGET motif is essential for E₂P dephosphorylation. ATP8A2 gene expression was disrupted in mice using a neo cassette. Knockout mice develop short outer segments and visual function is impaired. Modification of transbilayer asymmetry and composition appear to be responsible for reduced visual function rather than structural defects. The decrease in outer segment length suggests that ATP8A2 is involved in vesicular trafficking of proteins to the outer segment by regulating the step of vesicle budding possibly from the trans-Golgi network. We speculate that ATP8A2 plays a similar role in other neuronal cells and thus provide insight into the phenotype of human disorders caused by mutations in ATP8A2. In summary, this study has identified for the first time the transported substrate of a mammalian P₄-ATPase, discovered protein-protein interactions regulating function, elucidated the mechanism of lipid transport, and illuminated the function of ATP8A2 in photoreceptor and neuronal biology.

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2013-03-28

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/44073

Biochemistry and Molecular Biology

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/