Eukaryotic cells are characterized by their internal membrane compartmentalization, which is regulated by two families of molecular switches, Rab and Arf small G proteins. These switches and their regulators contain amphipathic helices that act as membrane sensors to read out information encoded by lipids in cellular membranes.
A major goal of the proposal is to elucidate the mechanisms by which the functions of the two families of small G proteins involved in vesicular trafficking – the Arfs and Rabs – are coordinated. Our project is highly original in that it will demonstrate for the first time a new molecular mechanism linking Arf and Rab signaling pathways, and its function in coordinating protein and lipid trafficking. My group was one of the first to demonstrate a role for Arf1, its activator GBF1 and effector coatomer in lipid droplet (LD) metabolism, in addition to roles in protein trafficking. LDs, composed of a neutral lipid core surrounded by a phospholipid monolayer, are found in most cells but notably in adipocytes. LDs function in energy storage in the form of triglycerides within their neutral lipid core, and in mobilization of these lipids for membrane production during growth and development. Obesity results from overstorage of lipids in LDs, and is a risk factor for a number of serious diseases, including diabetes, cardiovascular disease and cancer. One of our objectives is to develop assays to monitor protein binding to the phospholipid monolayer droplet surface in vitro, and to compare binding of Arf and Rab proteins and their regulators to bilayer liposomes and monolayer droplets. Proteomics and imaging studies will be performed to identify and characterize protein partners involved in regulating localization to LDs and Golgi membranes.
We are using the quantitative cell micropatterning/organelle density map confocal microscopy approach developed by one of our teams (B. Goud) to characterize the roles of Rab and Arf proteins and their regulators and effectors on lipid and protein trafficking pathways in cells. Super-resolution microscopy will be carried out at the IJM imaging core facility, and in collaboration with J. Lippincott-Schwartz (NIH, USA), and correlative light-electron microscopy with J-M. Verbavatz (CBI-MPG, Dresden). We are assaying activity of the domains of the Arf activator GBF1 purified from bacteria, on myristoylated Arf1, in the presence of liposomes, in vitro. We will also express full-length GBF1 in bacculovirus, which has been reported to have in vitro exchange activity on Arf1. We will carry out genome-wide studies using the yeast model system to understand global regulatory circuits involved in coordinating protein and lipid trafficking. Specifically, yeast cells expressing amphipathic helical (AH) bioprobes that exhibit perturbations of membrane trafficking pathways will be analyzed at a genome-wide level.
Affinity purifications of the Arf activator GBF1 have been carried out, and co-purifying proteins identified by mass spectrometry. This approach was a big success. Several proteins were identified, notably a number of Rabs. In the yeast two-hybrid assay, interaction was confirmed for Rab1 and Rab6. These Rabs, in their active form, interacted with the N-terminus of GBF1. In addition, the Rab6 dominant negative mutant form interacted with the catalytic domain of GBF1 and the full-length protein. These results support the conclusion that Rab6 is an important, direct, interacting partner of GBF1. The affinity purificaton-mass spectrometry analysis of GBF1 also identified members of the Arf family, including Arf4, a cis-Golgi localized protein, with functions that overlap with those of Arf1 and GBF1, for example in recruitment of effectors. Two domains of GBF1 localize to lipid droplets (LDs) in cells, but not to the Golgi; full-length GBF1 localizes to both. We are studying the mechanisms of this dual localization of GBF1, in particular the influence of the catalytic Sec7 domain on localization. The LD-localizing domains are direct lipid binding domains in vitro. We have demonstrated that one of them binds to both phospholipid bilayer liposomes and phospholipid monolayer droplets in vitro.
Several proteins in the Arf orbit (activator GBF1, inactivator ArfGAP1 and effectors) are recruited to membranes through lipid-binding domains that form amphipathic helices (AHs). AHs insert into the interfacial region of a phospholipid leaflet, a mode of binding that involves multiple contacts with membrane lipids, such that AHs have an enormous capacity for reading out information encoded by lipids. Recruitment of proteins (such as GBF1) to the lipid droplet phospholipid monolayer frequently inolves AHs. In addition to coordinating trafficking to lipid droplets and the early secretory pathway, Arf1, its activator GBF1 and interacting partner Rab6 may coordinate lipid and protein trafficking at ER-Golgi contact sites, which we will pursue using imaging, biochemical, bioinformatic and genomic studies.
Ellong EN, Soni KG, Bui QT, Sougrat R, Golinelli-Cohen MP, Jackson CL. 2011. Interaction between the triglyceride lipase ATGL and the Arf1 activator GBF1. PLoS One. 6(7):e21889
Donaldson JG, Jackson CL. ARF family G proteins and their regulators: roles in membrane transport, development and disease. Nat Rev Mol Cell Biol. (2011) 12:362-75. Review
The major objective of the proposal is to elucidate the mechanisms by which the functions of the two families of small G proteins involved in vesicular trafficking – the Arf and Rab proteins – are coordinated. Although both families have been investigated intensively, very few studies have addressed functional connections between them. Work from many laboratories has indicated that they have distinct functions in vesicular trafficking: Arf proteins are required for vesicle formation, whereas Rab proteins are necessary for vesicle targeting and fusion. Moreover, Rab proteins show restricted subcellular localization, whereas Arf proteins show less compartment specificity. Strikingly, Rab1-GTP has been reported to recruit the Arf1 GEF GBF1 to early Golgi membranes. Given the specific localization of Rabs, it is an attractive idea that a Rab acts to recruit an Arf GEF to membranes, thus confering specificity to Arf activation. However, this poses a problem in that Rabs act after Arfs in a single vesicle trafficking step.
Many proteins involved in trafficking are transmembrane proteins, such as SNAREs and cargo receptors, and hence must be returned to their donor organelle through vesicle-mediated recycling. It would therefore be reasonable to have a connection between the fusion machinery for one population of vesicles and the vesicle budding machinery for the following recycling step. Traditionally, very few studies have addressed this question. Recently, however, evidence in support of the idea of a close coordination of vesicle budding and tethering/fusion processes has begun to emerge. The recruitment of the Arf1 GEF GBF1 by Rab1 provides a novel molecular mechanism for coordinating anterograde vesicle fusion with budding of vesicles in the subsequent recycling step.
In the current proposal, we will explore the crosstalk between Arf and Rab small G protein networks in vesicular trafficking. We will first focus on GBF1 recruitment to membranes by Rab1, which we refer to as the Rab1-GBF1-Arf1 cascade. The project is divided into three aims. In Aim 1, “Localization and activation of the Arf1 GEF GBF1“, we will study each aspect of the cascade individually: localization of GBF1 in cells either knocked down for or expressing mutant versions of Rab1, biochemical characterization of the Rab1-GBF1 interaction in solution, and development of a GBF1 exchange assay for Arf1 on liposomes. In Aim 2, “Characterization of the Rab1-GBF1-Arf1 cascade in cells and in vitro”, we will reconstitute Rab1 recruitment of GBF1 on artificial membranes, and assay GBF1 exchange activity on Arf1 in the presence of Rab1 and other binding partners. In addition to these mechanistic studies, we will use both live cell imaging and immuno-electron microscopy to determine precisely to what membrane compartments Rab1 recruits GBF1 in cells, and study the functional consequences. In Aim 3, “Parallels between Rab and Arf functions in ER-Golgi and in TGN-endosomal trafficking pathways” we will explore Rab-ArfGEF-Arf1 cascades in TGN-endosomal pathways, and more generally, carry out a systematic study of Rab and Arf interactions in pathways on both the cis and trans sides of the Golgi.
Madame Cathy JACKSON (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B) – email@example.com
The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.
UNIVERSITE DE PARIS SUD XI
CNRS UMR 8621- IGM CNRS UMR 8621-Institut de Génétique et Microbiologie
Institut Curie UMR144 INSTITUT CURIE - SECTION DE RECHERCHE
UNIVERSITE DE PARIS XI [PARIS- SUD]
CNRS UMR 6097 CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE COTE D'AZUR
CNRS UMR 7592 - IJM CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION REGIONALE ILE-DE-FRANCE SECTEUR PARIS B
Help of the ANR 740,000 euros
Beginning and duration of the scientific project: - 36 Months