CURRICULUM VITAE



Fei WangPostdoctoral Fellow52 Oxford StreetHarvard UniversityCambridge MA 02138Department of Molecular and Cellular BiologyFAS Center for Systems BiologyNorthwest Building, Room 442.30phone: (413) 265 8995Email: feiwang@mcb.harvard.eduEDUCATION AND RESEARCH2009-Present. Postdoctoral fellow, Harvard University, MADept. Molecular and Cellular BiologyAdvisor: Vladimir Denic, Ph.D.Research: Tail- anchored protein targeting to ER membraneI studied the biogenesis of tail-anchored (TA) membrane proteins in the budding yeast Saccharomyces cerevisiae. Specifically, I used biochemical and structural approaches to define several key mechanisms in the GET pathway that facilitate endoplasmic reticulum targeting and insertion of TA proteins (Wang et al., 2010; Wang et al., 2011; Stefer et al., 2011; Wang et al., 2014) (The details are described in the research accomplishments section of the research proposal).Ph.D 2008University of Massachusettes, Amherst, MAMolecular and Cellular Biology ProgramDept. Biochemistry and Molecular BiologyAdvisor: Danny J. Schnell, Ph.D.Research: Protein Import Machinery at Chloroplast Outer MembraneI elucidated a GTP hydrolysis mechanism that regulates protein import into chloroplasts of the plant Arabidopsis thaliana. I showed that the GTPase activity of Toc159, a membrane-bound GTPase receptor, is regulating preprotein binding instead of driving membrane translocation (Wang et al., 2008).M.S. 2001Fudan University, Shanghai, ChinaDepartment of GeneticsAdvisor: Da-ru Lu, Ph.D.Research: Gene Therapy of Hemophilia BI worked with adenoviral vector to transfer genes in mammalian cells and mice for expression. This work is part of the group effort in developing a tool for safe delivery and efficient expression of clotting factor IX in human to treat Hemophilia B.B.S. 1997Fudan University, Shanghai, ChinaDepartment of GeneticsTEACHING EXPERIENCESTeaching assistant, University of Massachusetts-Amherst (2001 - 2002)Teaching assistant, Fudan University, China (1997 - 2001)AWARDS AND FELLOWSHIPSPoster Prize, the Molecular and Cellular Biology Program Retreat (April 2008)Poster Prize, the EMBO Meeting (the Endoplasmic Reticulum as a Hub for Organelle Communication, October 2014)Charles A. King Postdoctoral Research Program/Sara Elizabeth O’Brien Trust Postdoctoral Fellowship (July 2013 – June 2015)POSTDOC RESEARCH IMPACTMembrane protein sorting to organelles is a fundamental problem in eukaryotic cell biology. TA proteins are a class of membrane proteins defined by a single C-terminal transmembrane domain (TMD) and an N-terminal domain that faces the cytosol. They are sorted post-translationally for insertion into either the ER or mitochondrial outer membrane. Following insertion into the ER, TA proteins that reside in other compartments of the secretory pathway are delivered there by vesicular traffic. Some prominent examples include most SNAREs, components of ER and mitochondrial protein translocons, and members of the Bcl2 family of apoptosis regulators. Pioneering studies in the 1990’s revealed that the sorting information is encoded in the TMDs. How cells interpret TMD signals, however, had remained mysterious until the discovery that ER-bound TA proteins form complexes with Get3, a cytosolic ATPase, that are recruited for insertion by the Get1/2 receptor in the ER membrane. This paradigm of the Guided Entry of TA proteins (GET) pathway explained how ER-bound TA proteins are targeted to the appropriate membrane once they form a complex with Get3 but left unresolved the critical issue of how these targeting complexes are formed in the first place. Furthermore, for membrane protein insertion to occur, it was unknown how targeting factor Get3 efficiently let go of its substrate upon engaging the appropriate membrane insertion machinery Get1/2. In addition, targeting factor Get3 has to free itself from the insertion machinery following substrate release lest they interfere with the recruitment of new substrates to the membrane. How is the docking/dissociation of Get3 on ER membrane regulated? Lastly, the mechanism of the final TA protein insertion step was missing. As a post-doctoral fellow in the lab of Vladimir Denic at Harvard University, I have made key contributions to elucidating these fundamental mechanisms of the GET pathway in the budding yeast Saccharomyces cerevisiae.First of all, I revealed the composition of a conserved, multi-protein TMD-recognition complex (Sgt2-Get4-Get5 complex) that escorts newly synthesized TA proteins to Get3. Specifically, I showed that Sgt2 is key scaffold component of Sgt2-Get4-Get5 complex that employs a methionine-rich C-terminal domain to recognize diverse ER-bound TMD sequences. Prior to my study, it is unknown that Sgt2 recognizes the targeting determinants of newly synthesized ER-bound TA proteins before Get3 and that it does this using a methionine-rich domain akin to the domain of SRP that binds to signal sequences. My observation enabled finding in higher eukaryotic cell a similar TMD recognition mechanism handled by SgtA, the mammalian homolog of Sgt2. By using a fully reconstituted in vitro system, I demonstrated that Get4/5 activate Get3 for TMD recognition, resulting in ER-bound TA proteins being efficiently transferred from Sgt2-Get4-Get5 complex to Get3 (Wang et al., 2010). This observation has leaded to the substantial biochemical and structural studies of Get3 and Get4-Get5 interactions.In addition, I and others showed that post-translational TA protein insertion can be biochemically reconstituted using just three GET pathway components: the TA targeting factor Get3 in a complex with a substrate and proteoliposomes containing the transmembrane Get1/2 complex. These studies have also shown that the cytosolic domains of the Get1/2 complex interact with Get3 to enable substrate release and insertion. This technical breakthrough allowed us to uncover how the nucleotide cycle of the Get3 ATPase coordinates the targeting and insertion stages of the GET pathway. In brief, ADP bound Get3 in a complex with a substrate targets to ER membrane while ATP binding stimulates Get3 dissociation from the membrane thus freeing Get1 and Get2 for new rounds of substrate recruitment (Wang et al., 2011). Lastly, I have shown that the Get1/2 cytosolic domain interactions with Get3 are necessary but not sufficient to permit Get3 to let go of its substrates; even when these interactions form in proximity to the ER membrane. The additional driving force for substrate release and membrane insertion comes from the transmembrane segments of Get1/2 that assemble into a TMD-docking site embedded in the lipid bilayer. Prior to my work, most researchers believed that the insertion step at the end of the GET pathway is spontaneous. I demonstrated that the lipid bilayer imposes a kinetic barrier to spontaneous insertion, which the Get1/2 complex overcomes by an assisted insertion mechanism (Wang et al., 2014).PUBLICATIONSWang F, Chan C, Weir NR, Denic V. The Get1/2 transmembrane complex is an endoplasmic-reticulum membrane protein insertase. Nature. 2014 Aug 28;512(7515):441-4. Epub 2014 Jul 20.Abstract: Hundreds of tail-anchored proteins, including soluble N-ethylmaleimide-sensitive factor attachment receptors (SNAREs) involved in vesicle fusion, are inserted post-translationally into the endoplasmic reticulum membrane by a dedicated protein-targeting pathway. Before insertion, the carboxy-terminal transmembrane domains of tail-anchored proteins are shielded in the cytosol by the conserved targeting factor Get3 (in yeast; TRC40 in mammals). The Get3 endoplasmic-reticulum receptor comprises the cytosolic domains of the Get1/2 (WRB/CAML) transmembrane complex, which interact individually with the targeting factor to drive a conformational change that enables substrate release and, as a consequence, insertion. Because tail-anchored protein insertion is not associated with significant translocation of hydrophilic protein sequences across the membrane, it remains possible that Get1/2 cytosolic domains are sufficient to place Get3 in proximity with the endoplasmic-reticulum lipid bilayer and permit spontaneous insertion to occur. Here we use cell reporters and biochemical reconstitution to define mutations in the Get1/2 transmembrane domain that disrupt tail-anchored protein insertion without interfering with Get1/2 cytosolic domain function. These mutations reveal a novel Get1/2 insertase function, in the absence of which substrates stay bound to Get3 despite their proximity to the lipid bilayer; as a consequence, the notion of spontaneous transmembrane domain insertion is a non sequitur. Instead, the Get1/2 transmembrane domain helps to release substrates from Get3 by capturing their transmembrane domains, and these transmembrane interactions define a bona fide pre-integrated intermediate along a facilitated route for tail-anchor entry into the lipid bilayer. Our work sheds light on the fundamental point of convergence between co-translational and post-translational endoplasmic-reticulum membrane protein targeting and insertion: a mechanism for reducing the ability of a targeting factor to shield its substrates enables substrate handover to a transmembrane-domain-docking site embedded in the endoplasmic-reticulum membrane.Wang F, Whynot A, Tung M, Denic V.The mechanism of tail-anchored protein insertion into the ER membrane. Mol Cell. 2011 Sep 2;43(5):738-50. Epub 2011 Aug 11Abstract: Tail-anchored (TA) proteins access the secretory pathway via posttranslational insertion of their C-terminal transmembrane domain into the endoplasmic reticulum (ER). Get3 is an ATPase that delivers TA proteins to the ER by interacting with the Get1-Get2 transmembrane complex, but how Get3's nucleotide cycle drives TA protein insertion remains unclear. Here, we establish that nucleotide binding to Get3 promotes Get3-TA protein complex formation by recruiting Get3 to a chaperone that hands over TA proteins to Get3. Biochemical reconstitution and mutagenesis reveal that the Get1-Get2 complex comprises the minimal TA protein insertion machinery with functionally critical cytosolic regions. By engineering a soluble heterodimer of Get1-Get2 cytosolic domains, we uncover the mechanism of TA protein release from Get3: Get2 tethers Get3-TA protein complexes into proximity with the ATPase-dependent, substrate-releasing activity of Get1. Lastly, we show that ATP enhances Get3 dissociation from the membrane, thus freeing Get1-Get2 for new rounds of substrate insertion.Stefer S, Reitz S, Wang F, Wild K, Pang YY, Schwarz D, Bomke J, Hein C, L?hr F, Bernhard F, Denic V, D?tsch V, Sinning I. Structural basis for tail-anchored membrane protein biogenesis by the Get3-receptor complex. Science. 2011 Aug 5;333(6043):758-62. Epub 2011 Jun 30.Wang F, Brown EC, Mak G, Zhuang J, and Denic V. A chaperone cascade sorts proteins for posttranslational membrane insertion into the endoplasmic reticulum. Mol Cell. 2010 Oct 8;40(1):159-71. Epub 2010 Sep 16.Abstract: Tail-anchored (TA) proteins are posttranslationally inserted into either the endoplasmic reticulum (ER) or the mitochondrial outer membrane. The C-terminal transmembrane domains (TMDs) of TA proteins enable their many essential cellular functions by specifying the membrane target, but how cells process these targeting signals is poorly understood. Here, we reveal the composition of a conserved multiprotein TMD recognition complex (TRC) and show that distinct TRC subunits recognize the two types of TMD signals. By engineering mutations in a mitochondrial TMD, we switch over its TRC subunit recognition, thus leading to its misinsertion into the ER. Biochemical reconstitution with purified components demonstrates that TRC tethers and enzymatically activates Get3 to selectively hand off ER-bound TA proteins to Get3. Thus, ER-bound TA proteins are sorted at the top of a TMD chaperone cascade that culminates with the formation of Get3-TA protein complexes, which are recruited to the ER membrane for insertion.Inoue H, Wang F, Inaba T, Schnell DJ. Energetic manipulation of chloroplast protein import and the use of chemical cross-linkers to map protein-protein interactions. Methods Mol Biol. 2011;774:307-20.Oreb M, H?fle A, Koenig P, Sommer MS, Sinning I, Wang F, Tews I, Schnell DJ, Schleiff E. Substrate binding disrupts dimerization and induces nucleotide exchange of the chloroplast GTPase Toc33. Biochem J. 2011 Jun 1;436(2):313-9. Lee J, Wang F, Schnell DJ. Toc receptor dimerization participates in the initiation of membrane translocation during protein import into chloroplasts. J Biol Chem. 2009 Nov 6;284(45):31130-41. Epub 2009 Sep 10Agne B, Infanger S, Wang F, Hofstetter V, Rahim G, Martin M, Lee DW, Hwang I, Schnell D, Kessler F. A toc159 import receptor mutant, defective in hydrolysis of GTP, supports preprotein import into chloroplasts. J Biol Chem. 2009 Mar 27;284(13):8670-9. Epub 2009 Feb 2. Wang F, Agne B, Kessler F and Schnell DJ. 2008. The role of GTP binding and hydrolysis at the atToc159 preprotein receptor during protein import into chloroplasts. J. Cell Biol. 2008 Oct 6;183(1):87-99. Epub 2008 Sep 29.Abstract: The majority of nucleus-encoded chloroplast proteins are targeted to the organelle by direct binding to two membrane-bound GTPase receptors, Toc34 and Toc159. The GTPase activities of the receptors are implicated in two key import activities, preprotein binding and driving membrane translocation, but their precise functions have not been defined. We use a combination of in vivo and in vitro approaches to study the role of the Toc159 receptor in the import reaction. We show that atToc159-A864R, a receptor with reduced GTPase activity, can fully complement a lethal insertion mutation in the ATTOC159 gene. Surprisingly, the atToc159-A864R receptor increases the rate of protein import relative to wild-type receptor in isolated chloroplasts by stabilizing the formation of a GTP-dependent preprotein binding intermediate. These data favor a model in which the atToc159 receptor acts as part of a GTP-regulated switch for preprotein recognition at the TOC translocon.Rounds CM, Wang F, and Schnell DJ. 2007. The Toc Machinery of the Protein Import Apparatus of Chloroplasts. In The Enzymes: Molecular Machines involved in Protein Transport across Cellular Membranes. Vol. 25. F. Tamanoi, R.E. Dalbey, and C.M. Koehler, editors. Academic Press.Smith, MD, Rounds CM, Wang F, Chen K, Afitlhile M, and Schnell DJ. 2004. atToc159 is a selective transit peptide receptor for the import of nucleus-encoded chloroplast proteins. J. Cell Biol. 165:323-334 Yang X, Wang F, Wang Y, Lu D, Qiu X, Xue J. 2001. Constitutive expression of human coagulating factor IX in HeLa cells by homologous recombination of the promoter. Science in China (Series C). 44: 18-24.Gao X, Shi D, Wang F, Lu D, Qiu X, Xue J. 2000. In vitro expression of human clotting factor IX minigene mediated by mini-adenoviral vector. Chinese Journal of Virology. 16 (4): 294-298. (In Chinese).Pan H, Gao XD, Wang F, Yuan HY, Li YY. 2000. Effects of gene copy number and chromosomal position on the expression of a modified HBsAg gene SA-28 in yeast, Sheng Wu Gong Cheng Xue Bao (Chinese Journal of Bioengineering). 16(2):124-8. (In Chinese).REFERENCE33731208255Vladimir DenicAssociate ProfessorMolecular & Cellular Bio.Harvard University52 Oxford Street, NWL 445.30Cambridge, MA 02138Phone: (617)-496-6381Email: vdenic@mcb.harvard.eduLab manager (Peter Arvidson) :arvidson@mcb.harvard.edu4000020000Vladimir DenicAssociate ProfessorMolecular & Cellular Bio.Harvard University52 Oxford Street, NWL 445.30Cambridge, MA 02138Phone: (617)-496-6381Email: vdenic@mcb.harvard.eduLab manager (Peter Arvidson) :arvidson@mcb.harvard.eduDanny J SchnellProfessorBiochemistry and Molecular BiologyUniversity of MassachusettsLSL N431Amherst, MA 01003Phone: (413) 545-4024Email: dschnell@biochem.umass.eduAndrew W MurrayHerschel Smith Professor of Molecular GeneticsProfessor of Molecular and Cellular BiologyDirector of FAS Center for Systems BiologyHarvard University52 Oxford Street, NWL 469.20Cambridge, MA 02138Phone: (617)-496-1350Email: amurray@mcb.harvard.edu Lab manager (Linda Kefalas):lkefalas@mcb.harvard.edu ................
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