In eukaryotes, membrane contact sites (MCSs), at which the endoplasmic reticulum (ER) and another organelle come into close proximity, play key roles in lipid homeostasis, but the processes that occur at these sites remain poorly understood at the molecular level. Our approach to better understanding the function of contact sites and the regulation of activities that take place there is to characterize the protein machinery residen at such sites, focusing on contacts between the ER and the plasma membrane (PM). One process recently identified as important for lipid homeostasis as mediated by MCSs is the non- vesicular transport of lipids by lipid transfer proteins. We will combine structural, biochemical, and cell- based studies to characterize lipid transporters at ER-PM contact sites, addressing which proteins participate in lipid transfer, how they are targeted to these sites, which lipids thy transport, how they recognize their lipid ligands, and how transport by these proteins might be regulated.
Aim 1 focuses on the protein TMEM24, present only in metazoans and recently identified as a regulator of insulin secretion. Preliminary data indicate TMEM24 as an ER-PM contact resident with a lipid-binding module that specifically associates with phosphatidylinositol (PI). In probing the mechanisms that underlie TMEM24 activity at contact sites, we will test the hypothesis that TMEM24 transports PI from its place of synthesis in the ER to the PM, where PI can be rapidly converted to phosphatidylinositol-4,5-bisphosphate [PI(4,5)P2]. PI(4,5)P2 is a major PM signaling lipid critical for vesicle docking and priming, and we propose that the role of TMEM24 in secretion is to supply PM PI(4,5)P2.
In Aim 2, we will apply a similar approach to characterize proteins in the lipid transfer proteins anchored at membrane contact sites (LAM) family, newly identified in yeast as key for the transport of sterol, a major PM component. We will expand investigations of the LAM family to the metazoan proteins, identifying which members of this family are targeted to ER- PM contacts, probing whether they play the same critical role as in fungi, and structurally and functionally dissecting the molecular mechanisms by which they are targeted to ER-PM contacts and transport cholesterol to the PM.

Public Health Relevance

These studies will contribute to our understanding of lipid homseostasis, a fundamental process in eukaryotic cells, and as such will have broad medical implications. Many diseases result from a disruption of lipid homeostasis, including cancers and type II diabetes as well as genetic conditions such as Lowe's syndrome, Joubert Syndrome, or Nieman-Pick disease. The studies described here will lay the foundation for understanding these disorders and developing effective therapeutic strategies to treat them.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Membrane Biology and Protein Processing Study Section (MBPP)
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Deatherage, James F
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Yale University
Anatomy/Cell Biology
Schools of Medicine
New Haven
United States
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Horenkamp, Florian A; Valverde, Diana P; Nunnari, Jodi et al. (2018) Molecular basis for sterol transport by StART-like lipid transfer domains. EMBO J 37:
Lees, Joshua A; Messa, Mirko; Sun, Elizabeth Wen et al. (2017) Lipid transport by TMEM24 at ER-plasma membrane contacts regulates pulsatile insulin secretion. Science 355:
Reinisch, Karin M; De Camilli, Pietro (2016) SMP-domain proteins at membrane contact sites: Structure and function. Biochim Biophys Acta 1861:924-927
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Baskin, Jeremy M; Wu, Xudong; Christiano, Romain et al. (2016) The leukodystrophy protein FAM126A (hyccin) regulates PtdIns(4)P synthesis at the plasma membrane. Nat Cell Biol 18:132-8
Horenkamp, Florian A; Kauffman, Karlina J; Kohler, Lara J et al. (2015) The Legionella Anti-autophagy Effector RavZ Targets the Autophagosome via PI3P- and Curvature-Sensing Motifs. Dev Cell 34:569-76
Krishnakumar, Shyam S; Li, Feng; Coleman, Jeff et al. (2015) Re-visiting the trans insertion model for complexin clamping. Elife 4:
Cai, Yiying; Deng, Yongqiang; Horenkamp, Florian et al. (2014) Sac1-Vps74 structure reveals a mechanism to terminate phosphoinositide signaling in the Golgi apparatus. J Cell Biol 206:485-91
Horenkamp, Florian A; Mukherjee, Shaeri; Alix, Eric et al. (2014) Legionella pneumophila subversion of host vesicular transport by SidC effector proteins. Traffic 15:488-99

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