1. The cell- and organelle-delimiting fluids we call eukaryotic lipid bilayers are unequally
and heterogeneously constituted. Phospholipids are the most populous constituents and
the substrates of my research. There are four principle classes—phosphatidylserine (PS),
phosphatidylethanolamine (PE), phosphatidylcholine (PC), and sphingolipids (SL).
Eukaryotes inequitably enrich these lipids across the endoplasmic or exoplasmic leaflets.
The ‘endo’ leaflet is richest in aminophospholipids like PS and PE, whereas the ‘exo’
leaflet is enriched in the quaternary amine-possessing lipids, PC and SL.
For some of these phospholipids the asymmetry is nearly exclusive. Under basal cell
circumstances, PS is almost perfectly restricted to the ‘endo’ leaflet while the near-
entirety of SL is observed at the ‘exo’ leaflet. This nonrandom distribution implies a
mechanism preventing net movement of phospholipids “down” their concentration
gradients. One explanation is that “flippases” sort these constituents into the appropriate
leaflets by phospholipid-specific vectorial transport. P4-type ATPases are enzyme pumps
that facilitate the movement of certain amphipaths in the inward direction. ATP8a1 is one
such P4-ATPase and PS flippase.
That organellar and plasma membranes tend to asymmetry is compelling in its own right,
but this interleaflet disequilibrium is more significant for its influence on human health
and disease. Consider PS during basal cell circumstances. This GPL concentrates in the
endoplasmic leaflet and reversal of that asymmetry (above a critical threshold) is
sufficient to radically modify cell fate. As a milestone of apoptosis, tumor cells display
PS, which recruits macrophages via the PS-binding receptor (Tim-1) resulting in tumor
cell phagocytosis. After a typical 100-120 days in circulation, erythrocytes display PS
and are targeted for degradation by splenic monocytes. Exoplasmic enrichment of PS is
also implicated in the premature destruction of sickled and thalassemic red cells. For
platelets, the consequences of increased PS mole-fraction in the outer leaflet are cell
activation, cell aggregation, and stimulation of blood coagulation cascades—which
underpins the role of PS exposure in thrombotic balance and, therefore, its relevance to
pulmonary embolism, myocardial infarction, and stroke. For these reasons, PS might be
thought of as a molecular signpost and its transmembrane distribution a matter of
physiological significance.

2. Equally influential as membrane asymmetry are the membrane proteins associated with
its creation, maintenance, and reversal. P4-type ATPases are aminophospholipid
translocases that influence all levels of the secretory pathway. Several have been
implicated in disease. P4’s play a pleiotropic role in select cases of obesity and diabetes,
and ATP8a1 knockout mice show defects in delayed hippocampus-dependent learning.
Genetic studies of families with Progressive Familial Intrahepatic Cholestasis (PFIC)
identify missense mutations in a P4-type ATPase, ATP8b1. PFIC is a severe liver
syndrome of compromised bile flow; onset is juvenile and arrives with a compulsory
indication for transplantation. Cerebellar Ataxia, Mental Retardation, and Disequilibrium
(CAMRQ) syndrome is a rare, congenital, autosomal recessive disorder characterized by
dysarthria, seizures, profound intellectual disability, cerebellar hypoplasia, trunkal ataxia,
and quadrupedal gait. Genome mapping of consanguineous families predisposed to
CAMRQ syndrome pinpoints a missense mutation in ATP8a2, another P4-type ATPase.
This provides a nice survey of the significance of this field and a loose framework for
some of its long-term aspirations. But to understand how these mutations yield these
consequences, we must first understand flippase activity and the catalytic unit itself. As a
membrane-embedded molecular motor, ATP8a1 couples the favorable change in free
energy of hydrolyzing ATP's distal phosphoanhydride bond to the otherwise non-
spontaneous mechanical rearrangements required to bind one molecule of PS on the
exoplasmic leaflet and passage it through to the endoplasmic leaflet. My work focuses
on the nature of ATP8a1’s PS binding site and transport pathway.