We study the Sec7-domain guanine nucleotide exchange factors (GEFs) for the Arf family of small GTPases. We are interested in the role of these proteins in membrane dynamics and protein trafficking. The Arfs and Arf GEFs are important regulators of both organelle structure and protein transport throughout the cell. We are focusing on the large Golgi-localized Arf GEFs involved in transport through the secretory pathway in both budding yeast and mammalian cells. A central question in cell biology is how the elaborate and dynamic structures of membrane systems are maintained in the face of constant trafficking into and out of each organelle, particularly the way organelle structure is generated and maintained and how structure is correlated with the underlying molecular events of protein sorting and membrane remodeling. An important step toward answering these questions is to define the role of the Arf GEFs at the molecular level by identifying interacting partners, elucidating membrane localization mechanisms, and analyzing Arf GEF mutants in vivo.
Identification of interacting partners of the Golgi-localized Arf GEFs in mammalian cells
Smirnova
The Arf GEFs of the Golgi-localized Gea/GBF and Sec7/BIG subfamilies are large multidomain proteins. A major goal of our laboratory is to understand the functions of the domains of the Gea/GBF and Sec7/BIG Arf GEFs. Given that the GEFs are soluble proteins that must be targeted to membranes, identification of their membrane-targeting signals and partners is an important step in understanding their function. Within each subfamily, regions upstream and downstream of the Sec7 catalytic domain are conserved from yeast to humans. However, the functions of these homology regions are not known. We are carrying out two-hybrid screens with the N-terminal and C-terminal regions of the Arf GEFs of the Gea/GBF and Sec7/BIG subfamilies. Specifically, we performed a two-hybrid screen using BIG2, the Sec7 domain of the mammalian homologue of Sec7p, and identified a number of partners, two of which are human PI4P 5 kinase beta and the ATGL triglyceride lipase.
It was recently shown that ATGL triglyceride lipase plays an important role in lipid droplet (LD) degradation in adipose tissue. LDs are found in many eukaryotic cells and are highly upregulated in adipocytes, which are professional lipid storage cells. Given the rapidly spreading obesity epidemic, the mechanism by which LDs and their component neutral lipids are degraded is an important health issue. We have demonstrated that ATGL also functions in non-adipocyte cells and plays an important role in LD degradation in these cells. Overexpression of wild-type ATGL causes a dramatic decrease in LD size, whereas a catalytically inactive mutant retains the ability to localize to LDs but is unable to decrease LD size. Depletion of ATGL by RNA interference leads to a significant increase in the size of LDs. The results demonstrate that ATGL plays an important role in LD/adiposome turnover in mammalian cells.
Several proteomic studies of adiposomes have shown that, in addition to the abundant perilipin/ADRP/TIP47 (PAT) domain proteins and neutral lipid metabolic enzymes, proteins that are involved in membrane trafficking and signaling, such as Rab GTPases, are associated with the organelles. The results of previous work from other labs have suggested that the Arf GTPase is involved in LD formation. The PAT domain protein TIP47 was originally identified as a Rab9-binding protein that is involved in recycling mannose-6-phosphate receptors from endosomes to the trans-Golgi network. Thus, TIP47 is the first example of a growing class of proteins that provide a functional bridge between membrane trafficking pathways and adiposome metabolism. We are currently attempting to determine the role of the Arf GEFs in both regulating lipid droplet turnover and coordinating the secretory pathway with lipid droplet metabolism.
PI4P 5 kinase beta is a potential partner of interest given that it has been identified as an effector of Arf. We have narrowed down the interaction domain of PI4P 5 kinase to a 50–amino acid region in the C-terminal portion of the catalytic domain, just upstream of the activation loop. The activation loop contains the substrate-binding site and is both necessary and sufficient to determine intracellular localization of the kinase. It is striking that we have identified three lipid-modifying enzymes (ATGL, PI4P 5 kinase, and Drs2p; see below) as binding partners of different Sec7 domains. It appears that all three proteins bind to the C-terminal region of the Sec7 domain. Current studies are directed at understanding the role of lipid modification in Arf GEF function.
Cox R, Mason-Gamer RJ, Jackson CL, Segev N. Phylogenetic analysis of Sec7-domain-containing Arf nucleotide exchangers. Mol Biol Cell2004;15:1487-1505.
Smirnova E, Goldberg EB, Makarova K, Lin L, Brown WJ, Jackson CL. The triglyceride lipase ATGL plays a major role in lipid droplet/adiposome degradation in mammalian cells. EMBO Rep 2005 [Epub ahead of print].
Interacting partners and membrane localization of the Golgi-localized Arf GEFs in yeast
Park, Deng
Using different regions of the Gea2p protein, we have identified six transmembrane-domain proteins as potential membrane receptors for the Gea1p and Gea2p proteins in two-hybrid screens. Two of the partners are Drs2p, a Golgi-localized amino-phospholipid translocase, and Gmh1p, a Golgi-localized five-span transmembrane protein of unknown function. As is the case for drs2, cells deleted for GMH1 show only a mild effect on Gea2p. The double-mutant drs2/gmh1 is viable, and although an effect on Gea2p localization is more severe than in either single mutant, some Gea2p is still able to bind to membranes. We are testing the possibility that the other transmembrane-domain proteins identified in two-hybrid screens with Gea2p also contribute to Gea2p localization. Thus, the mechanism by which the large Arf GEFs such as Gea2p are localized to membranes appears to be complex, with several membrane localization determinants each contributing to the steady-state, cellular localization of the protein.
In two-hybrid screens with Gea2p, we have also identified subunits of two large complexes involved in trafficking in the early secretory pathway. Using the N-terminal portion of Gea2p, we identified Cog4p, a member of the eight-subunit COG complex that functions in the Golgi of both yeast and mammalian cells. In the screen with the C-terminus of Gea2p, we identified a component of TRAPP, another Golgi-localized complex conserved in all eukaryotes from yeast to humans. These complexes have been shown to play a role in early steps of membrane fusion reactions before the actual fusion event, and we are now working toward understanding the role of the Arf GEFs in the fusion process. We have confirmed a number of interactions by co-immunoprecipitation in yeast, in particular that of Gea2p and with the coat complex COPI. We are exploring other GEF-coat interactions by two-hybrid screening, GST pulldown, and co-immunoprecipitation in yeast and mammalian cells.
Chantalat S, Park SK, Hua Z, Liu K, Gobin R, Peyroche A, Rambourg A, Graham TR, Jackson CL. The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae. J Cell Sci 2004;117:711-722.
Dynamics of the GBF1 Arf GEF in mammalian cells
Niu
Using live-cell imaging, we are studying GBF1, the mammalian homologue of Gea2p. As others have previously reported, a YFP-GBF1 fusion protein localizes to the Golgi in mammalian cells. Overexpression of GBF1 confers resistance to brefeldin A (BFA), a drug that profoundly affects the structure and functioning of intracellular organelles. Within 10 minutes of treatment, the Golgi apparatus is completely disassembled in a normal cell, whereas it remains intact in cells overexpressing GBF1. YFP-GBF1 also protects cells against the effects of BFA, indicating that the fusion protein is functional. Remarkably, when cells are treated with BFA, GBF1 is recruited dramatically to Golgi membranes. We have tested different mutants in the catalytic domain of GBF1 for their response to BFA. One of the mutations, E694D, is in the catalytic glutamic acid residue and reduces the rate of exchange by 400-fold in vitro. We observed no difference in the dynamics of GBF1-E694D in cells either before or after BFA treatment. In contrast, mutation of a residue known to affect BFA sensitivity/resistance in other Arf GEFs without affecting catalytic activity results in a version of GBF1 that is completely resistant to the effects of BFA. The results indicate that GBF1 is a direct target of BFA at the Golgi in mammalian cells. The very rapid kinetics of association-dissociation from Golgi membranes suggests that GBF1 must interact with localization machinery at each stage of the secretory pathway at which it acts; it is not recruited early in the pathway, and it stays associated with membranes through successive steps of the pathway. This observation is consistent with the results described above; that is, several transmembrane partners have been identified for the GBF/GEA subfamily of Arf GEFs.
As a more direct test of whether GBF1 is a target of BFA, we used an in vivo exchange assay to analyze Arf1 activation in mammalian cells. The assay takes advantage of the tight binding to Arf1-GTP of the Arf effector GGA3. We demonstrated that BFA dramatically reduces the levels of Arf1-GTP in mammalian cells but that the effect is completely reversed by overexpressing GBF1 in such cells. In collaboration with Frank van Kuppeveld, we have found that the enterovirus 3A protein, when expressed in cells, dramatically reduces the level of Arf1-GTP. The 3A protein binds to both Arf1-GDP and GBF1 and causes disassembly of the Golgi apparatus. The enteroviruses include poliovirus and coxsackievirus, and it is known that the 3A protein blocks protein trafficking between the endoplasmic reticulum and Golgi in host cells. Our work demonstrates the mechanism of 3A action in host cells, namely, that it acts like BFA by binding to a GBF1-Arf1-GDP complex, thus blocking the exchange reaction and Arf1 activation.
Niu TK, Pfeifer AC, Lippincott-Schwartz J, Jackson CL. Dynamics of GBF1, a brefeldin A-sensitive Arf1 exchange factor at the Golgi. Mol Biol Cell 2005;16:1213-1222.
Role of the yeast Arf GEFs in the morphology of the yeast secretory pathway
Park, Yadon
Both in yeast and mammalian cells, the Arf GEFs are important regulators of organelle structure and protein trafficking. The structure and organization of yeast organelles appear to differ markedly from those of mammalian cells, yet most of the proteins involved in both organelle structure and trafficking, including the Arf GEFs, are highly conserved. As a first step to identifying the structural features common to yeast and mammalian organelles, our work aims to determine Golgi structure in yeast with both live imaging and electron microscopy. Reliance on both the yeast and mammalian systems will allow us to determine which aspects of Arf GEF function are fundamental to all eukaryotic organisms and which are unique to their specific system.
We have isolated 75 temperature-sensitive (ts) SEC7 mutants and over 100 GEA2 ts mutants. We generated the mutants in GFP-tagged versions of Sec7p and Gea2p and screened all by fluorescence microscopy for localization of the mutant protein at the nonpermissive temperature. We found a category of mutants in each case that showed a largely cytosolic pattern and are examining the mutants to determine whether they have lesions in a membrane-localization domain. Other mutants show abnormal structures at the fluorescence level. We are currently studying the mutants at the ultrastructural level, work that is providing much more detailed information on the structural changes observed in the mutants than can be obtained by light microscopy. We sequenced all mutants to determine which amino acids were altered in the mutant proteins and observed that a subset of the mutants have only one or two amino acid substitutions in domains highly conserved from yeast to humans. We are exploring the functions of the domains through suppressor screens aimed at identifying potential interacting partners of these regions of the Arf GEFs. The studies will provide information on the molecular mechanisms that generate and maintain organelle structure in eukaryotic cells.
Jackson CL. N-terminal acetylation targets GTPases to membranes. Nat Cell Biol 2004;6:379-380.
Park SK, Hartnell LM, Jackson CL. Mutations in a highly conserved region of the Arf1p activator GEA2 block anterograde Golgi transport but not COPI recruitment to membranes. Mol Biol Cell 2005;16:3786-3799.
COLLABORATORS
William J. Brown, PhD, Cornell University, Ithaca, NY
Todd Graham, PhD, Vanderbilt University, Nashville, TN
Frank van Kuppeveld, PhD, Nijmegen Center for Molecular Life Sciences, Nijmegen, The Netherlands
Jennifer Lippincott-Schwartz, PhD, Cell Biology and Metabolism Branch, NICHD, Bethesda, MD
Kira Makarova, PhD, National Center for Biotechnology Information, NLM, Bethesda, MD
Alain Rambourg, PhD, CEA-Saclay, Gif-sur-Yvette, France
Nava Segev, PhD, University of Illinois at Chicago, Chicago, IL
For further information, contact cathyj@helix.nih.gov.