Todd R. Graham, Ph.D.
Stevenson Professor of Biological Sciences
Professor of Biological Sciences
Professor of Cell and Developmental Biology
The research goals of the Graham laboratory are to understand the molecular mechanisms underpinning vesicle-mediated protein transport and membrane biogenesis. Most of our effort is focused on determining how type IV P-type ATPases (P4-ATPases) contribute to the establishment of membrane asymmetry and budding of transport vesicles from organelle membranes using the budding yeast model system.
The P4-ATPases flip specific phospholipid species, such as phosphatidylserine, from the extracellular leaflet of the plasma membrane to the cytosolic leaflet, thus producing an asymmetric membrane structure that is conserved among most eukaryotic cells. This phospholipid asymmetry has a major influence on the localization and activity of many different plasma membrane proteins. Moreover, regulated disruption of membrane asymmetry is a signaling device used in blood clotting reactions and for recognition of apoptotic cells. Humans have 14 P4-ATPases and members of this protein family are implicated in severe liver and neurological disease. Additionally, murine P4-ATPases are implicated in B-cell deficiency, obesity and type 2 diabetes, motor neuron degeneration, defects in bile secretion, and reduced male fertility. A current project in the laboratory is to define the mechanism of substrate recognition and translocation by P4-ATPases using molecular genetic and biochemical approaches. The best characterized P-type ATPases transport small cations across membranes to establish ion gradients and so phospholipid molecules are an unusual substrate for this protein family. Our work is suggesting a novel transport mechanism for the P4-ATPases and is providing insight into how these transporters evolved the ability to transport their “giant substrate”.
In addition to establishing membrane asymmetry, we discovered that P4-ATPases play a crucial role in budding protein transport vesicles from Golgi and endosomal membranes. For example, a P4-ATPase called Drs2 translocates phosphatidylserine across the membrane of the trans-Golgi network and this flippase activity is required to bud AP-1/clathrin-coated vesicles that transport proteins from the Golgi to endosomes. Our work has uncovered both positive and negative regulators of Drs2 activity representing proteins and lipids known to have critical roles in vesicular transport. The ATP-powered, unidirectional translocation of phosphatidylserine to cytosolic leaflet should have a dramatic influence on the biophysical properties of the membrane; enhancing the anionic membrane potential of the cytosolic surface as well as inducing curvature through a bilayer couple mechanism. Another current project in the lab is to determine how these P4-ATPase effects on the membrane are coordinated with the vesicle budding machinery to sort and package cargo proteins into newly forming vesicles. The protein trafficking events dependent on P4-ATPase function have a major influence on the protein composition of the plasma membrane and organelles of the secretory and endocytic pathways.