Fei Wang
Postdoctoral Fellow
Harvard University
Department of Molecular and Cellular Biology
FAS Center for Systems Biology
Northwest Building, Room 442.30
phone: (413) 265 8995
Email: feiwang@mcb.harvard.edu
52 Oxford Street
Cambridge MA 02138
EDUCATION AND RESEARCH
2009-Present.
Postdoctoral fellow, Harvard University, MA
Dept. Molecular and Cellular Biology
Advisor: Vladimir Denic, Ph.D.
Research: Tail- anchored protein targeting to ER membrane
I 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 2008
University of Massachusettes, Amherst, MA
Molecular and Cellular Biology Program
Dept. Biochemistry and Molecular Biology
Advisor: Danny J. Schnell, Ph.D.
Research: Protein Import Machinery at Chloroplast Outer Membrane
I 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. 2001
Fudan University, Shanghai, China
Department of Genetics
Advisor: Da-ru Lu, Ph.D.
Research: Gene Therapy of Hemophilia B
I 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. 1997
Fudan University, Shanghai, China
Department of Genetics
TEACHING EXPERIENCES


Teaching assistant, University of Massachusetts-Amherst (2001 - 2002)
Teaching assistant, Fudan University, China (1997 - 2001)
Wang, F. Page 2
AWARDS AND FELLOWSHIPS


Poster 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 IMPACT
Membrane 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 Nterminal 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-Get4Get5 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).
Wang, F. Page 3
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).
PUBLICATIONS

Wang F, Chan C, Weir NR, Denic V. The Get1/2 transmembrane complex is an endoplasmicreticulum 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 endoplasmicreticulum 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 endoplasmicreticulum 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-domaindocking 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 11
Abstract: 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.
Wang, F. Page 4

Stefer S, Reitz S, Wang F, Wild K, Pang YY, Schwarz D, Bomke J, Hein C, Löhr F, Bernhard F,
Denic V, Dötsch 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, ERbound 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, Höfle 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 10

Agne 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.
Wang, F. Page 5

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).
REFERENCE
Danny J Schnell
Vladimir Denic
Professor
Biochemistry and Molecular Biology
University of Massachusetts
LSL N431
Amherst, MA 01003
Phone: (413) 545-4024
Email: dschnell@biochem.umass.edu
Associate Professor
Molecular & Cellular Bio.
Harvard University
52 Oxford Street, NWL 445.30
Cambridge, MA 02138
Phone: (617)-496-6381
Email: vdenic@mcb.harvard.edu
Lab manager (Peter Arvidson) :
arvidson@mcb.harvard.edu
Andrew W Murray
Herschel Smith Professor of Molecular Genetics
Professor of Molecular and Cellular Biology
Director of FAS Center for Systems Biology
Harvard University
52 Oxford Street, NWL 469.20
Cambridge, MA 02138
Phone: (617)-496-1350
Email: amurray@mcb.harvard.edu
Lab manager (Linda Kefalas):
lkefalas@mcb.harvard.edu