Significant differences in lipid distribution are maintained between intracellular organelles. Cholesterol comprises ~30% of the lipid molecules in the plasma membrane and is also enriched in the endocytic recycling compartment (ERC). In the endoplasmic reticulum (ER) cholesterol accounts for 5% of the lipid molecules. Cholesterol can move between membranes by vesicular and non-vesicular transport mechanisms. However, only a small fraction of membrane components internalized from the plasma membrane reach the ER, indicating that cholesterol sensing in the ER would be very slow and inefficient if it depended on vesicle transport. There is substantial evidence for high rates of no-vesicular sterol transport in cells. Since cholesterol is very poorly soluble in water, non-vesiculr transport requires binding to carrier proteins. The steroidogenic acute regulator-related lipid-transfer (START) domain containing proteins are involved in several pathways of non-vesicular trafficking of sterols. Among the soluble START proteins, STARD4 has been shown to increase cholesteryl ester accumulation in lipid droplets, in an acyl-CoA:cholesterol acyl-transferase dependent manner, and is controlled at the transcriptional level by cholesterol. However, the precise molecular mechanisms that mediate STARD4 membrane targeting, interaction and sterol extraction are unknown. This proposal will address the mechanisms that facilitate StARD4 activity and distribution required to maintain cholesterol homeostasis.
Aim 1 will evaluate the mechanism of STARD4 membrane interaction and identify regions mediating interaction. Previous molecular dynamic simulations of STAR domains in complex with cholesterol have suggested that movement of the Omega-1 loop is required for sterol absorption and release. We will utilize nuclear magnetic resonance and x-ray crystallographic techniques to investigate these processes of sterol-protein complex formation as well as determine the structure of the sterol-STARD4 complex.
Aim 2 we will analyze the lipid specificity of STARD4 sterol transfer in vitro. In preliminary studies, we have identified two organelle-specific anionic lipids, PI(4,5)P2 and PI(3,5)2, which modulate STARD4 localization and activity. Additionally, we will identify the rate limiting step in sterol transfer of STARD4 using fluorescence resonance energy transfer sterol transfer assays and stop-flow kinetic analysis. To determine the cellular factors required for the maintenance of cholesterol homeostasis by STARD4, we will develop a sterol transport kinetic model to evaluate the role of STARD4 sterol transport between the plasma membrane and the ERC. Additionally, we will analyze the role of specific phosphatidylinositols phosphates in targeting STARD4 to specific membranes to facilitate sterol transfer using fluorescent microscopy techniques in Aim 3.

Public Health Relevance

Cholesterol accounts for ~30% of the lipid molecules in the plasma membrane while the endoplasmic reticulum (ER), which is responsible for the storage and biosynthesis of cholesterol, is relatively sterol poor. Numerous studies indicate that cholesterol trafficking is maintained by non- vesicular carrier-mediated transport mechanisms. This proposal will address the biophysical and cellular mechanisms that facilitate STARD4, a soluble sterol transfer protein, activity and distribution required to maintain cholesterol homeostasis.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Predoctoral Individual National Research Service Award (F31)
Project #
1F31DK104631-01
Application #
8838431
Study Section
Special Emphasis Panel (ZDK1-GRB-R (O1))
Program Officer
Mcbryde, Kevin D
Project Start
2015-05-01
Project End
2017-04-30
Budget Start
2015-05-01
Budget End
2016-04-30
Support Year
1
Fiscal Year
2015
Total Cost
$43,120
Indirect Cost
Name
Weill Medical College of Cornell University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
060217502
City
New York
State
NY
Country
United States
Zip Code
10065
Iaea, David B; Maxfield, Frederick R (2017) Membrane order in the plasma membrane and endocytic recycling compartment. PLoS One 12:e0188041
Jena, Prakrit V; Roxbury, Daniel; Galassi, Thomas V et al. (2017) A Carbon Nanotube Optical Reporter Maps Endolysosomal Lipid Flux. ACS Nano 11:10689-10703
Iaea, David B; Mao, Shu; Lund, Frederik W et al. (2017) Role of STARD4 in sterol transport between the endocytic recycling compartment and the plasma membrane. Mol Biol Cell 28:1111-1122
Iaea, David B; Dikiy, Igor; Kiburu, Irene et al. (2015) STARD4 Membrane Interactions and Sterol Binding. Biochemistry 54:4623-36
Iaea, David B; Gale, Sarah E; Bielska, Agata A et al. (2015) A novel intrinsically fluorescent probe for study of uptake and trafficking of 25-hydroxycholesterol. J Lipid Res 56:2408-19