Open Collections

Matsell, Eli William

University of British Columbia

ATP8A2 is a P4-ATPase that actively flips phosphatidylserine and to a lesser extent phosphatidylethanolamine across cell membranes to generate and maintain transmembrane phospholipid asymmetry. This transporter is expressed primarily in neuronal tissues, such as the brain, retina and spinal cord. The importance of this flippase is underscored by the findings that loss-of-function mutations in ATP8A2 are known to cause the neurodevelopmental disease known as CAMRQ4 in humans and related neurodegenerative disorders in mice. This thesis focuses on characterizing ATP8A2, investigating its regulatory mechanisms, the impact of disease-associated variants, and its involvement in specific cellular pathways. Little is known about the structural and functional properties of the cytosolic N- and C-terminal segments of this flippase. In addition, there has been uncertainty regarding the methionine start site of ATP8A2 and accordingly the size of the N-terminal segment. Here, we have used mass spectrometry to show that bovine ATP8A2, like its human counterpart has an extended N-terminal segment not apparent in the mouse ortholog. This segment greatly enhances the expression of ATP8A2 without affecting its cellular localization or PS-activated ATPase activity. Using a cleavable C-terminal protein and site-directed mutagenesis, we further show that the conserved GYAFS motif in the C-terminal segment plays a role in autoinhibition as well as efficient folding of ATP8A2 into a functional protein. With regards to understanding the impact of clinical mutations, five variants of ATP8A2 were studied. The expression, localization and ATPase activity of these variants were characterized using a variety of biochemical assays. Using various software programs predicting protein stability, we show that there is a good correlation between the experimental expression of the variants and in silico stability assessments. These findings suggest that such analysis is useful in identifying protein misfolding disease-associated variants. Finally a cellular model was developed to study the role of P4-ATPases in the process of protein trafficking. A P4-ATPase deficient cell line was developed to study the impact on the trafficking of the plasma membrane receptor VLDLR, also associated with CAMRQ. These investigations will help improve our understanding of the function of ATP8A2, and its role in neurodegenerative disease.

Text

2025-04-04

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/90581

Biochemistry and Molecular Biology

University of British Columbia

UBCV

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