Structural Studies in Membrane Biology
Organelles within a cell differ in terms of the lipid composition of their surrounding membrane, and these differences help to establish organelle identity and thus allow for directional transport of materials between organelles. The focus of the lab and an emerging area of study is to understand the molecular mechanisms by which membrane composition is established and regulated. We are particularly interested in phosphoinositide lipids, which in addition to their role in organelle identity also are critical in signal transduction pathways, and their homeostasis. We are studying the structure and function of the protein complexes involved in making and degrading phosphoinositide lipids as well as lipid transport proteins that modulate membrane composition by transporting lipids between membrane bilayers at so-called membrane contact sites. We use X-ray crystallography, Electron Microscopy, biochemistry and biophysics to understand structure and function, then test hypotheses arising from these studies using cell biology techniques.
A major effort in the laboratory is directed toward understanding phosphatidylinositol 4-phosphate (PI4P) metabolism, and one of our main projects concerns the PI4KA lipid kinase complex. This complex is conserved in eukaryotes and synthesizes PI4P at the plasma membrane (PM). Our structural studies so far have formed the basis for understanding how the kinase is recruited to the PM by the scaffolding proteins EFR3, TTC7, and FAM126 and led to initial insights as to how complex assembly may be regulated to modulate kinase activity there (1,2). Our current effort is directed at understanding how TTC7 and FAM126 stimulate catalysis by PI4KA. We have also obtained insights as to how PI4P levels at the Golgi apparatus are regulated, obtaining a crystal structure of a complex comprising Vps74/GOLPH3 and Sac1. Sac1 is a PI4P phosphatase which resides primarily at the ER, whereas Vps74/GOLPH3 localizes to the Golgi. The structure explains how Sac1 can be recruited to the Golgi by Vps74/GOLPH3 (3) to regulate PI4P levels there.
It is becoming increasingly clear that membrane lipid composition is in part modulated at membrane contact sites, where two organelles are apposed closely enough so that non-vesicular lipid exchange between their membranes is possible. The molecular mechanisms underlying lipid exchange, however, remain unexplored. The Extended-Synaptotagmin proteins were previously characterized as tethers that are localized to and maintain ER-PM contact sites. Our crystal structure of Extended-Synaptotagmin2 (E-Syt2) demonstrated that a protein module of previously unknown function, the SMP domain, within E-Syt2 is a lipid binding module, strongly supporting that lipid exchange at these contact sites is mediated by lipid transfer proteins (4). Because SMP-domains are also found at other contact sites (for example, in the ERMES complex at ER-mitochondrial sites) our findings have broad implications for these sites also.
More recently, we found that another SMP-domain protein at ER-plasma membrane contact sites, TMEM24, plays a critical role in replenishing phosphatidylinositol-4,5-bisphosphate at the PM following glucose stimulation in insulin secreting cells and discovered the mechanism through which this protein regulates calcium pulsatility and insulin exocytosis in these cells (5).
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