Catherine L. Jackson, PhD, Head, Unit on GTPase Regulation of Membrane Traffic
Ting-Kuang Niu, PhD, Postdoctoral Fellow
Sei-Kyoung Park, PhD, Postdoctoral Fellow
Elena Smirnova, PhD, Postdoctoral Fellow
Yi Deng, PhD, Postdoctoral Fellow
Marlene Moskowitz, BS, Predoctoral Fellow
Adam Yadon, BS, Predoctoral Fellow

We study the Sec7 domain guanine nucleotide exchange factors (GEFs) for the Arf family of small GTPases. We are interested in the roles 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 focus 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 pressing questions are how 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 in answering these questions involves defining the roles 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; in collaboration with Brown, Makarova, Segev

The Arf GEFs of the Golgi-localized Gea/GBF and Sec7/BIG subfamilies are large multidomain proteins. A major goal of the 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 remain unknown. 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. One such screen used the Sec7 domain of BIG2, the mammalian homologue of Sec7p; we found a number of partners, of which two are human PI4P 5 kinase beta and the ATGL triglyceride lipase.

The ATGL triglyceride lipase has recently been shown to play 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 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 their 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, these organelles have associated proteins that are involved in membrane trafficking and signaling, such as Rab GTPases. Previous work has 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. Hence 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 carrying out studies to determine the role of the Arf GEFs in both regulating lipid droplet turnover and coordinating the secretory pathway with lipid droplet metabolism.

Given its identification as an effector of Arf, PI4P 5 kinase beta is an interesting potential partner. 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.

Interacting partners and membrane localization of the Golgi-localized Arf GEFs in yeast

Park, Deng; in collaboration with Graham, Rambourg

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. One of the partners is Drs2p, a Golgi-localized amino-phospholipid translocase; another is Gmh1p, a Golgi-localized five-span transmembrane protein of unknown function. As is the case for drs2, cells deleted for GMH1 also show only a mild effect on Gea2p. The double mutant drs2 gmh1 is viable, and, although the 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. Hence, 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 localization of the protein in cells.

We have also identified subunits of two large complexes involved in trafficking in the early secretory pathway in two-hybrid screens with Gea2p. 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 the early steps of membrane fusion reactions before the actual fusion event, and we are now working to understand the role of the Arf GEFs in this process. We have confirmed several interactions by co-immunoprecipitation in yeast, in particular Gea2p and 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.

Dynamics of the GBF1 Arf GEF in mammalian cells

Niu; in collaboration with van Kuppeveld, Lippincott-Schwartz

We are studying GBF1, the mammalian homologue of Gea2p, by using live cell imaging. A YFP-GBF1 fusion protein localizes to the Golgi in mammalian cells, as previously reported. 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 this fusion protein is functional. Remarkably, when cells are treated with BFA, GBF1 is recruited dramatically to Golgi membranes. We 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 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. This result indicates 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 rather than undergoing early recruitment in the pathway and staying associated with membranes through successive steps of the pathway. This observation is consistent with the results described above, namely, that there are numerous transmembrane partners 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 test Arf1 activation in mammalian cells. The assay makes use 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 this effect is completely reversed by overexpressing GBF1 in cells. In collaboration with Frank van Kuppeveld, we found that the enterovirus 3A protein, when expressed in cells, dramatically reduces the level of Arf1-GTP in cells. 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 ER 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 Cell2005;16:1213-22.
Wessels E, Duijsings D, Niu TK, Neumann S, Oorschot VM, de Lange F, Lanke KHW, Klumperman J, Henke A, Jackson CL, Melchers WJG, van Kuppeveld FJM. A viral protein that blocks Arf1-mediated COP-I assembly by inhibiting guanine nucleotide exchange factor GBF1. Dev Cell 2006;11:191-201.

Role of the yeast Arf GEFs in the morphology of the yeast secretory pathway

Park, Yadon

The Arf GEFs are important regulators of organelle structure and protein trafficking in both yeast and mammalian cells. Yeast organelles appear to differ markedly in structure and organization from the organelles of mammalian cells, yet most of the proteins involved in 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 current work aims at determining Golgi structure in yeast by using both live imaging and electron microscopy. Use of 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 a specific organism.

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 them 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 now 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 these mutants at the ultrastructural level, which is yielding a wealth of detailed information, not obtainable by light microscopy, on the structural changes observed in the mutants. All mutants were sequenced to determine which amino acids were altered in the mutant proteins. 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 these domains through suppressor screens aimed at identifying potential interacting partners of these regions of the Arf GEFs. Our studies will provide information on the molecular mechanisms that generate and maintain organelle structure in eukaryotic cells.

COLLABORATORS

William J. Brown, PhD, Cornell University, Ithaca, NY
Todd Graham, PhD, Vanderbilt University, Nashville, TN
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
Frank van Kuppeveld, PhD, Nijmegen Centre for Molecular Life Sciences, Nijmegen, The Netherlands

For further information, contact jackyj@helix.nih.gov.