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REGULATION OF MEMBRANE DYNAMICS AND PROTEIN
TRANSPORT BY THE SEC7-DOMAIN GUANINE NUCLEOTIDE
EXCHANGE FACTORS
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Catherine L. Jackson, PhD, Head, Unit on GTPase Regulation of Membrane Traffic Ting-Kuang Niu, PhD, Postdoctoral Fellow |
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| We study the Sec7-domain guanine nucleotide exchange factors (GEF) 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 the 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 both in 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. Issues of particular interest are the way organelle structure is generated and maintained and how structure correlates with the underlying molecular events of protein sorting and membrane remodeling. An important step toward investigating these issues will be to define the roles of the Arf GEFs at the molecular level through the identification of interacting partners, elucidation of membrane localization mechanisms, and analysis of Arf GEF mutants in vivo. Identification of interacting partners of the Golgi-localized Arf GEFs in yeast and mammalian cells Smirnova, Park 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 different 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; the functions of these homology regions are, however, 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, using the Sec7 domain of the mammalian homolog of Sec7p BIG2. We found a number of partners, two of which are human PI4P 5 kinase beta and a putative lipase. PI4P 5 kinase beta is a particularly interesting potential partner, as it has been identified as an effector of Arf. We carried out a reverse two-hybrid screen to determine the region of the BIG2 Sec7 domain that interacts with PI4P 5 kinase. Interestingly, we found that residues in the C-terminal region of the Sec7 domain abolish interaction with PI4P 5 kinase. We have narrowed 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. We are also mapping the region of Arf interaction in PI4P 5 kinase. Interestingly, it appears to be in a different domain of the protein than the BIG2 Sec7 domain interacting region. It is striking that we have identified three lipid-modifying enzymes as binding partners of different Sec7 domains. It appears that all three proteins bind to the C-terminal region of the Sec7 domain. We know that this region is adjacent to a PH domain in the ARNO subfamily of Arf GEFs, which mediates membrane localization through specific lipid interaction. Using the Gea2p N-terminal region, we have identified Mss4p as an interacting partner; Mss4p is the sole PI4P 5 kinase in yeast. It is intriguing that we identified a human homolog of Mss4p in our two-hybrid screen with the BIG2 Sec7 domain. We have also identified subunits of two large complexes involved in trafficking in the early secretory pathway in two-hybrid screens with Gea2p. With 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. 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 this process. Arnaoutova I, Jackson CL, Al-Awar OS, Donaldson JG, Loh YP. Recycling of Raft-associated pro- hormone sorting receptor, carboxypeptidase E requires interaction with ARF6. Mol Biol Cell 2003;14:4448-4457. Bonifacino JS, Jackson CL. Endosome-specific localization and function of the ARF activator GNOM. Cell 2003;112:141-142. Chantalat S, Courbeyrette R, Senic-Matuglia F, Jackson CL, Goud B, Peyroche A. A novel Golgi localized integral membrane partner of the ARF GEFs Gea1p and Gea2p. Mol Biol Cell 2003;14:2357-2371. Jackson CL. Membrane traffic: Arl GTPases get a GRIP on the Golgi. Current Biol 2003;13:R174-R176. Jackson CL. The Sec7 family of ARF guanine nucleotide exchange factors. In: Kahn RA, ed. The Arf Family of GTPases, Chapter 5. Dordrecht, Netherlands: Kluwer Academic Publishers, 2003; in press. Dynamics of the GBF1 Arf GEF in mammalian cells Niu; in collaboration with, Lippincott-Schwartz, Pfeifer
Using live cell imaging, we are studying GBF1, the mammalian homolog of Gea2p. As 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 in cells overexpressing GBF1, the Golgi remains intact. 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 have tested different mutants in the catalytic domain of GBF1 for their response to BFA. One of these mutations, E694D, resides in the catalytic glutamic acid residue and reduces the rate of exchange 400-fold in vitro. There is 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 being recruited early in the pathway and remaining associated with membranes through successive steps of the pathway. This observation is consistent with the results described above, that is, numerous transmembrane partners have been identified for the GBF/GEA subfamily of Arf GEFs. Role of the yeast Arf GEFs in morphology of the yeast secretory pathway Park, Rainey, Phillips, Thomas The Arf GEFs are important regulators of organelle structure and protein trafficking in both yeast and mammalian cells. Seemingly, yeast organelles differ substantially in structure and organization from those 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 focuses on determining the Golgi structure in yeast by using both live imaging and electron microscopy. The 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 their specific system. We have isolated 75 temperature-sensitive (ts) SEC7 mutants and over 100 GEA2 ts mutants. The mutants were generated in GFP-tagged versions of Sec7p and Gea2p and were screened 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 these 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 providing a wealth of detailed information on the structural changes observed in the mutants not obtained by light microscopy. We sequenced all mutants 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. Chantalat S, Park S-K, Hua Z, Liu K, Gobin R, Peyroche A, Rambourg A, Graham T, Jackson CL. The Arf activator Gea2p and the P-type ATPase Drs2p interact at the Golgi in Saccharomyces cerevisiae. J Cell Sci 2003; in press. COLLABORATORS For further information, contact cathyj@helix.nih.gov |
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