Cellular metabolic energy is stored in the form of neutral lipids, particularly triacylglycerols (TGs), which are packaged in cytoplasmic lipid droplets (LDs). LDs also contain sterol esters (SEs), which are required for membrane biogenesis. Excessive accumulation of LDs occurs during the progression of certain diseases, including obesity, metabolic syndrome and atherosclerosis. LDs are also required for hepatitis C virus replication, and a number of viral proteins specifically interact with this organelle. Despite their significance to human health, surprisingly little is known about basic LD cell biology, especially in terms of protein targeting. The hydrophobic core of LDs consisting of TGs and SEs is bounded by a phospholipid monolayer, which harbors a set of largely-unidentified proteins. Most functions of LDs, including TG synthesis, TG storage, and energy mobilization, are executed and regulated by these surface proteins. Among organelles, LDs are unique because their surface is an apposition of a hydrophobic phase (the LD core) and an aqueous phase (the cytoplasm). This monolayer is thus not configured to accommodate typical transmembrane proteins with globular domains flanking transmembrane segments. Therefore, the targeting of particular proteins to LDs must involve unique mechanisms, which are the subject of the research proposed here. To elucidate these mechanisms, we must first determine a high-confidence LD proteome, which we can accomplish via our state-of-the-art quantitative mass spectrometry-based proteomics methods. We will then proceed to determine how proteins are targeted to LDs from the cytoplasm, focusing on a set of model proteins and applying a variety of cell biological and biochemical methods we have established in our laboratory. We will subsequently use our unbiased proteomics approach to define the scope of proteins that share the pathways defined for these models. In a second line of research, we propose to determine how a distinct set of proteins that contain sequences predicting multiple trans-membrane domains are targeted to LDs or closely- associated membranes. For each part of the project, we will test the functional consequences of LD protein targeting as well as evolutionary conservation of the mechanisms we discover. Although the research proposed here is basic, our determination of the fundamental cellular mechanism that drives lipid droplet protein targeting will facilitate the development of therapeutic strategies to combat diseases that involve lipid droplets, as well as propel further research into the cell biology of these organelles.

Public Health Relevance

Lipid Droplets function in energy storage, metabolism and homeostasis. They are bounded by a unique phospholipid monolayer that contains specific proteins with functions in these processes. We propose here to establish the LD proteome, to determine the features of these proteins that enable their specialized targeting, to identify factors required for targeting to occur, and to characterize how targeting participates in regulating the functions of LDs. Our results will define an unstudied fundamental cellular process, provide a basis for exploring speculated LD functions, and establish an intervention site for the treatment of various metabolic and viral diseases.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
3R01GM097194-03S1
Application #
8738798
Study Section
Membrane Biology and Protein Processing (MBPP)
Program Officer
Chin, Jean
Project Start
2011-09-30
Project End
2016-08-31
Budget Start
2013-09-01
Budget End
2014-08-31
Support Year
3
Fiscal Year
2013
Total Cost
$63,164
Indirect Cost
Name
Yale University
Department
Anatomy/Cell Biology
Type
Schools of Medicine
DUNS #
043207562
City
New Haven
State
CT
Country
United States
Zip Code
06520
Wilfling, Florian; Haas, Joel T; Walther, Tobias C et al. (2014) Lipid droplet biogenesis. Curr Opin Cell Biol 29:39-45
Bazzini, Ariel A; Johnstone, Timothy G; Christiano, Romain et al. (2014) Identification of small ORFs in vertebrates using ribosome footprinting and evolutionary conservation. EMBO J 33:981-93
Thiam, Abdou Rachid; Antonny, Bruno; Wang, Jing et al. (2013) COPI buds 60-nm lipid droplets from reconstituted water-phospholipid-triacylglyceride interfaces, suggesting a tension clamp function. Proc Natl Acad Sci U S A 110:13244-9
Currie, Erin; Schulze, Almut; Zechner, Rudolf et al. (2013) Cellular fatty acid metabolism and cancer. Cell Metab 18:153-61
Nguyen, Andrew D; Nguyen, Thi A; Cenik, Basar et al. (2013) Secreted progranulin is a homodimer and is not a component of high density lipoproteins (HDL). J Biol Chem 288:8627-35
Wilfling, Florian; Wang, Huajin; Haas, Joel T et al. (2013) Triacylglycerol synthesis enzymes mediate lipid droplet growth by relocalizing from the ER to lipid droplets. Dev Cell 24:384-99
Krahmer, Natalie; Hilger, Maximiliane; Kory, Nora et al. (2013) Protein correlation profiles identify lipid droplet proteins with high confidence. Mol Cell Proteomics 12:1115-26
Frohlich, Florian; Christiano, Romain; Walther, Tobias C (2013) Native SILAC: metabolic labeling of proteins in prototroph microorganisms based on lysine synthesis regulation. Mol Cell Proteomics 12:1995-2005
Walther, Tobias C; Farese Jr, Robert V (2012) Lipid droplets and cellular lipid metabolism. Annu Rev Biochem 81:687-714