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Studies on the expression and function of the human TMEM56 protein

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Studies on the expression and function of the human TMEM56 protein

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STUDIES ON THE EXPRESSION AND FUNCTION OF
THE HUMAN TMEM56 PROTEIN

by

Hsiao-Fan Wei

A Thesis Presented to the
FACULTY OF THE USC GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
MASTER OF SCIENCE
(BIOCHEMISTRY AND MOLECULAR BIOLOGY)

August 2011

Copyright 2011 Hsiao-Fan Wei
ii

TABLE OF CONTENTS
LIST OF FIGURES
ABBREVIATIONS
ABSTRACT
CHAPTER ONE: INTRODUCTION
CHAPTER TWO:
METHODS
2.1 Cell Culture Conditions
2.2 Plasmids and TMEM56 siRNA cDNA
2.3 Cell Transient Transfection
2.4 Cell Lysate Preparation and affinity purification
2.5 Western Blot for expression of TMEM56
2.6 Acetone precipitation and in solution digestion
2.7 Immunostaining
2.8 Mitochondria isolation and ATP-synthase enzyme
activity assay
CHAPTER THREE:
RESULTS
3.1 TMEM56 expression on Western Blotting
3.2 Purification of TMEM56 and Identification of interacting proteins
3.2.1 T able 1
3.3 Subcellular Localization of TMEM56
3.4 siRNA Expression Analysis and Mitochondria Isolation
3.5 ATP-synthase activity assay

CHAPTER FOUR: CONCLUSION AND DISCUSSION
BIBLIOGRAPHY

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LIST OF FIGURES
Figure 1. Fluid mosaic model of cell membrane

Figure 2. Mitochondria
Figure 3. ATP-Synthase (F
1
F
o
–type ATPase)
Figure 4. TMEM56 molecular features
Figure 5. Western Blotting for expression of the TMEM56 protein in HEK
293T cell extracts by unmodified procedure
Figure 6. Western Blotting for expression of the TMEM56 protein in HEK
293T cell extracts by unboiled procedure
Figure 7. Western Blotting of the immunoprecipitated TMEM56 protein
in HEK 293T cell extracts
Figure 8. Immunofluorescence images for localization of TMEM56
protein
Figure 9. Western Blot for confirmation of purified proteins in
mitochondria isolation fractions
Figure 10. ATP-synthase activity assay

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ABBREVIATIONS
ADP
ATP
BCL-2
BSA
DNA
DNP
DTT
HEK 293T
IgG
IKK
LDAO
LTQ-ETD
NaCl
NADH
PBS
pCMV
SDS-PAGE
siRNA
TMEM56
Adenosine-5'-triphospha
Adenosine diphosphate
B-cell lymphoma-2
Bovine serum albumin
Deoxyribonucleic acid
2, 4-Dinitrophenol
Dithiothreitol
Human Embryonic Kidney 293 cell
Immunoglobulin G
IκB kinase
Lauryldimethylamine oxide
Linear trap quadrupole-Electron Transfer Dissociation
Sodium chloride
Nicotinamide adenine dinucleotide
Phosphate buffered saline
Cytomegalovirus
Sodium dodecyl sulfate polyacrylamide gel
electrophoresis
Small interfering RNA
Transmembrane Protein 56

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ABSTRACT
Transmembrane 56 (TMEM56) is a 30 KDa protein and it is highly conserved in most
species. The protein was identified as an IKKβ-interacting protein by mass spectrometry
in Dr. Zandi’s Lab. Initially, abnormal aggregation of TMEM56 was observed, which was
determined to be due to the boiling step prior to SDS-PAGE. This procedure precluded
the detection of protein by Western blotting. To investigate the function of TMEM56, a
FLAG-tagged version of it was expressed in human 293T kidney cells, and interacting
proteins were identified by co-immunoprecipitation and mass spectrometry. The
predominant TMEM56-interacting proteins are mitochondrial F
1
components, ATP
synthase α and β subunits. TMEM56 and ATP synthase α subunit were shown to be
co-localized in the mitochondria. Expression of FLAG-TMEM56 in 293T cells resulted
in an increased ATP synthase activity using isolated mitochondria in vitro. On the other
hand, siRNA-mediated knock down of TMEM56 decreased the ATP synthase activity in
isolated mitochondria. The data presented in this study indicated that TMEM56 is
localized in the mitochondria and may play a role in regulating ATP synthesis by
interacting with ATP synthase α, and perhaps, the β subunits of the F
1
complex.
1

CHAPTER ONE: INTRODUCTION
Key components of biological membranes are the membrane proteins. Membranes are
composed of lipids, proteins, and attached carbohydrates in varying combinations to each
species, cell types, and organelles. The fluid mosaic model describes the biological
membrane structure (Lehniger et al, 2008 ;Singer SJ et al., 1972). The lipid bilayer is the
basic structural unit, and different types of membrane proteins associate with the lipid
bilayer in several means. A number of membrane proteins are peripheral proteins that are
covalently linked to lipids; several are integral proteins that float in the lipid and are held
by hydrophobic interactions with their non–polar amino acid side chains. Integral
membrane proteins, also called transmembrane proteins, are mostly categorized into (i)
one transmembrane helix, (ii) multiple transmembrane helices in one single polypeptide,
(iii) several different polypeptides assembling to form a channel, (iv) β barrel, and others.
According to UniProKB/Swiss–Prot (Universal Protein Resource Knowledgebase,
reviewed) (Tusnády GE et al., 2004), as of April 2011, an approximate 5,660
transmembrane proteins have been identified in humans, the function of more than 1,000
of these transmembrane proteins have yet to be revealed. Due to the difficulties with
experimental techniques, their properties and functions are hard to characterize.
2

Figure 1. Fluid mosaic model of cell membrane
Ref. http://buffonescience9.wikispaces.com
Transmembrane proteins play roles in numerous cellular processes, such as cell–cell
interaction, adhesion, transporters, and ion channels. Integral membrane proteins might
be receptors for hormones, neurotransmitters, and growth factors. In the immune system,
they are responsible for cell-to-cell and antigen-to-cell recognition. These proteins play
important role in the process of membrane fusion, including exocytosis, endocytosis, and
the entry of numerous types of viruses into host cells. Aside from these essential roles of
transmembrane proteins, scientists have discovered more functions or diseases related to
these proteins than before. For instance, transmembrane proteins might be anticancer
agents because recent research indicates that mitochondrial integral membrane proteins
3

(e.g., BCL–2 protein family) participate in regulating the mitochondrial pathways of cell
death (Youle RJ. et al., 2008; Scorrano L. et al., 2005); TMEM 16 protein family may be
a new class of chloride channels (Galietta et al., 2009; Haung F.. et al., 2009); TMEM
70 mutations cause isolated ATP-synthase deficiency and neonatal mitochondrial
encephalocardiomyopathy (Cízková A. et al, 2008; Houstek J. et al., 2008; Hejzlarova K.
et al., 2011). Thus, the transmembrane proteins participate in a diversity of cellular
functions including energy metabolism and cell survival.

Figure 2. Mitochondria
Ref. http://biology.about.com/od/cellanatomy/ss/mitochondria.htm
Mitochondria are essential organelles involved in signaling, cellular differentiation, cell
death as well as the control of the cell cycle and cell growth. Adenosine triphosphate
(ATP) is required for biological endoergonic processes. The enzyme for producing ATP
4

is called ATP-synthase, and is an integral membrane protein. In mammals, the
mitochondrial ATP-synthase (also called F
1
F
o
–type ATPase) is located in the inner
mitochondrial membrane. The enzyme participates in the last step of the electron
transport chain, and uses the electron proton gradient provided by the electron transport
chain to convert ADPs to high-energy ATPs (Lehniger et al., 2008; Houstek J. et al.,
2008; Hejzlarova K. et al., 2011; Boyer PD, 1997; Kucharczyk R. et al., 2009; Loscher
HR., 1984; Mitchell P., et al., 1967).

Figure 3. ATP-Synthase (F
1
F
o
–type ATPase)

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The mitochondrial ATP-synthase is a large multiprotein complex, with an approximate
550 kDa in molecule weight, and formed by one transmembrane channel (F
o
) and the
synthase domain (F
1
). The synthase domain (catalytic complex) is composed of three α,
three β, γ, δ, and ε subunits, one for each enzyme (Lehniger et al., 2008; Boyer PD, 1997;
Kucharczyk R. et al., 2009; Abrahams JP. et al., 1994; Gibbons C. et al., 2000; Stock D.
et al., 1999; Rubinstein JL. et al., 2003). The subunits have deliberate functions in the
ATP regenerating system. In 1994, the crystal structure of the F
1
complex by John E.
Walker and his colleagues revealed that each of the three β subunits has a catalytic site
for ATP synthesis, and γ subunit is associated with the αβ pairs as a center shaft
(Abrahams JP. et al., 1994). Research further indicates that γ and ε subunits act as the
central stalk to sustain structure, and the δ subunit acts as a peripheral stalk. The
peripheral stalk functions as a stator that prevents the α
3
β
3
subcomplex from following
the rotation of the central stalk. The γ
1
δ
1
ε
1
subunits complex interacts together with the F
o
domain (Gibbons C. et al., 2000; Stock D. et al., 1999; Rubinstein JL. et al., 2003).
Numerous scientists contribute to the research of ATP-synthase. Paul Boyer proposed a
mechanism in which the three αβ

sites of F
1
take turns catalyzing ATP synthesis

in 1997
(Boyer PD., 1997). The β subunits have three different conformations – one binds to ADP,
6

another binds to ATP, and the other is empty conformation. ATP synthesis is catalyzed by
the rotation of the γ subunit and the binding – change of α/β subunits. ATP-synthase
activity is another significant subject to study. ATP synthesis is driven by a
proton–motive force and coupled by NADH oxidation, according to the chemiosmotic
theory proposed by Peter Mitchell (Mitchell P., et al., 1967). In the 1960s, Efraim Racker
and his colleagues purified the mitochondrial ATP-synthase enzyme and determined the
ATP-synthase activity by the measurement of released inorganic phosphate (P
i
). They
also showed that the addition of 2, 4–Dinitrophenol (DNP) can stimulate ATP hydrolysis.
Isolated F
1
, without association with the F
o
domain, catalyzes ATP to ADP and phosphate
(Pullman ME. et al., 1960). Moreover, Paul Boyer indicated that ATP synthesis and ATP
hydrolysis are reversible on the surface of the F
1
domain via isotope exchange
experiments (Hackney DD. et al., 1979). Therefore, the enzymatic ATP regenerating
system of ATP-synthase is established by coupling ATP hydrolysis to NADH oxidation,
regenerating ATP from ADP. The ATP-synthase activity can be monitored by observing
the decrease in NADH absorption at a wavelength of 340 nm because the changes in the
rate of ATP hydrolysis represent ATP-synthase activity. Uncouplers, which only inhibit
ATP synthesis and do not affect the following electron transfer from NADH to O
2
, are
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usually added during the experiments for measuring ATP-synthase activity. Furthermore,
the addition of the amphipathic detergent lauryldimethylamine oxide (LDAO) can
enhance ATP hydrolytic activity (Lotscher HR., 1984). Therefore, LDAO is often used to
measure ATP-synthase activity. Other than studying the properties of ATP-synthase,
understanding the defects and diseases caused or related by this enzyme is also necessary.
Oxidative damage of the α subunits of the mitochondrial ATP-synthase results in the loss
of activity, consequently leading to OXPHOS diseases (Beal MF., 1995; Terni B. et al.,
2010). Defects in energy metabolism, excitotoxicity, and oxidative damage are involved
in normal aging and neurodegenerative diseases, such as Alzheimer’s disease,
Huntington’s disease, and Parkinson’s disease (Beal MF., 1995). Recent studies show that
oxidative damage occurred in the early stages of AD pathology (Terni B. et al., 2010).
Previous studies not only show that the deficiency in ATP–synthase is responsible for the
activity disorder, but also that ATP-synthase activity is related to other transmembrane
proteins (Scorrano L., 2005; Beal, 1995; Terni B. et al., 2010; Cízková A. et al, 2008 ).
According to the aforementioned description, a number of transmembrane protein
families with unknown functions or localization exist. There are several technical
8

difficulties associated with investigating membrane proteins. Specifically,
transmembrane proteins are frequently difficult to purify and characterize. For example, a
large number of transmembrance proteins may aggregate at the surface of SDS-Page gel
(Fig. 5A) during electrophoresis, causing greater difficulty in crystallization and
determining the structure of membrance proteins, compared to water-soluble proteins.
Transmembrane protein 56 (TMEM56), the subject of this study was identified as an
IKK-interacting protein in Dr. Zandi’s lab. It is a 263 AA protein with an estimated
molecular weight of 300,41 Da (Fig. 4). According to UniProtKB/Swiss–Prot, TMEM56
is predicted to have 6 helical transmembrane regions every 21 amino acids, starting from
the
7
Lys and one TLC domain, though the function and properties are not well known.
Furthermore, this protein is conserved in humans, mice, frogs, zebras, fishes, and molds.
Prior to this study, there were no experimental data available for TMEM56. .Initially,
abnormal aggregation of TMEM56, which was determined to be due to the boiling step
prior to SDS-PAGE, precluded the detection of protein by western blotting. To investigate
the function of TMEM56, a FLAG-tagged version of it was expressed in human 293T
kidney cells, and interacting proteins were identified by co-immunoprecipitation and
9

mass spectrometry. The predominant TMEM56-interacting proteins are mitochondrial F
1
components, ATP synthase α and β subunits. TMEM56 and ATP synthase α subunit were
shown to be co-localized in the mitochondria. Expression of FLAG-TMEM56 in 293T
cells resulted in an increased ATP synthase activity in isolated mitochondria in vitro. On
the other hand, siRNA-mediated knock down of TMEM56 decreased the ATP synthase
activity in isolated mitochondria. The data presented in this study indicated that
TMEM56 is localized in the mitochondria and may play role in regulating ATP synthesis
by interacting with ATP synthase α, and perhaps, the β subunits of the F
1
complex.

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Human TMEM 56 (Transmembrane protein 56)
A. Protein Sequence
MEINTKLLIS VTCISFFTFQ LLFYFVSYWF SAKVSPGFNS LSFKKKIEWN SRVVSTCHSL
VVGIFGLYIF LFDEATKADP LWGGPSLANV NIAIASGYLI SDLSIIILYW KVIGDKFFIM
HHCASLYAYY LVLKNGVLAY IGNFRLLAEL SSPFVNQRWF FEALKYPKFS KAIVINGILM
TVVFFIVRIA SMLPHYGFMY SVYGTEPYIR LGVLIQLSWV ISCVVLDVMN VMWMIKISKG
CIKVISHIRQ EKAKNSLQNG KLD

B. Predicted transmembrane region from UniprotKB database

Figure 4. TMEM56 molecular features
A. TMEM56 sequence data from NCBI Reference Sequence: NP_689700.1 and UniProt ID:
Q96MV1. The bold regions of Amino Acid sequences indicate the transmembrane regions.
B. The graph shows the predicted transmembrane region from the UniprotKB database by
entering TMEM56 protein ID Q96MV1
(Ref: http://www.uniprot.org/uniprot/Q96MV1).

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CHAPTER TWO: METHODS
2.1 Cell Culture Conditions –
HEK 293T cells were maintained in Dulbecco’s Modification of Eagle’s Medium
(Cellgro, Manassas, VA) supplemented with 10% fetal bovine serum, 2 mM L–glutamine
(Invitrogen, Carlsbad, CA), 100 units/ml penicillin (Invitrogen, Carlsbad, CA), and 100
μg/ml streptomycin (Invitrogen, Carlsbad, CA) at 37 °C in the presence of 5% CO
2

atmosphere. The cells were trypsinized and split every 3 to 4 days.
2.2 Plasmids and TMEM56 siRNA cDNA –
For the expression plasmid for, transmembrane protein 56 (Ref. ID: NM_152487) was
purchased from OriGene. The plasmid is a cytomegalovirus (pCMV) vector and
TMEM56 is expressed with a C–terminal Myc–Flag
®
tag. Specific siRNAs for TMEM56
knock down were designed using BLOCK–iT
TM
RNAi Designer (Invitrogen, Carlsbad,
CA). The sequences of 21–mers oligonucleotides used in the study are as follows:
TMEM56 siRNA (siRNA-TM56-2), sense strand
(5'–GCGGAUUGCCUCAAUGCUUTT–3'), and antisense strand
(5'–AAGCAUUGAGGCAAUCCGCTT–3'); TMEM56 control siRNA (siRNA-TM56-c),
sense strand (5'–GGUCUAUACACCGUGACAUTT–3'), and antisense strand
(5'–AUGUCACRRURUAUAGACCTT–3’). Single–stranded siRNA oligonucleotides
were annealed before transfection experiments. The annealing process comprised each
RNA oligo being aliquot and diluted by DEPC–treated water to a concentration of 50 µM,
12

and combining each oligo solution with an 5X annealing buffer in a 2:1 ratio. The
concentration of the 5X annealing buffer is 50 mM Tris, pH 7.5–8.0, and 100 mM NaCl
in DEPC–treated water. The siRNA duplex (20 µM) was incubated for 1 min at 90 °C
water before being cooled down to room temperature slowly. Once annealed, the duplex
siRNAs were stored and frozen at –20 °C until usage.
2.3 Cell Transient Transfection –
HEK 293T cells were transfected at 50–75% confluence using Lipofectamine
TM
(Invitrogen, Carlsbad, CA) and PLUS
TM
reagent (Invitrogen, Carlsbad, CA), in accordance
with the manufacturer's protocol. Briefly, 1.0 µg plasmid DNA and 10 µl PLUS
TM
Regent
were diluted and mixed in serum–free medium Opti–MEM
®
I (Invitrogen, Carlsbad, CA),
and incubated for 15 min at room temperature. The DNA–PLUS
TM
complexes were then
mixed with 8 µl prediluted Lipofectamine

and incubated for another 15 min at room
temperature. Concurrently, the old cell growth medium was replaced with Opti–MEM
®
I medium. The DNA–PLUS
TM
–Lipofectamine complexes were added on the cells
drop–by–drop and incubated for 5 h at 37 °C in a CO
2
incubator. After incubation, the
plates were placed in regular growth medium containing twice the normal concentration
of serum (FBS), without removing the transfection mixture for 16–18 h or 43 h.
For transient knockdown of TMEM56, the cells, at approximately 50% confluency, were
transfected with siRNA or control siRNA using Oligofectamine
TM
Reagent (Invitrogen,
Carlsbad, CA), as described by the manufacturer’s protocol.
13

Generally, 8 µl Oligofectamine
TM
Reagent were diluted with Opti–MEM
®
I Reduced
Serum Medium for 10 min at room temperature. 20nM siRNA were also mixed with
Opti–MEM
®
I in other tubes. The combination of siRNA–Oligofectamine
TM
complexes
were added on the cells drop–by–drop after 20 min incubation at room temperature, and
put in a CO
2
incubator at 37 °C for 4 hours. Following the plates were then added with
regular growth medium containing three times the normal concentration of the serum,
without removing the transient knockdown mixture for 43 h.
2.4 Cell Lysate Preparation and affinity purification –
Transfected HEK 293T cells were lysed in regular lysis buffer containing 10x APB, 5M
NaCl, 10 % triton, and supplemented with 1mM DTT and protease inhibitors (1mM
leupeptin, 1mM pepstatin, 1mM bestatin, and 1mM phenylmethylsulfonyl fluoride) on
ice for 10 minutes. Lysed cells were centrifuged at 14000 rpm for 20 min at 4 °C and
determined the protein concentration of supernatant using a colorimetric assay kit
(Bio–Rad, Hercules, CA) before storing at –80 °C for purification by
Immunoprecipitation.
For the Western Blot experiments, the lysates were purified by Anti–Flag
®
M2–Agarose
beads (SIGMA, St. Louis, MO) for 4 hours at 4 °C, and eluted by elution buffer (regular
lysis buffer with 6M urea and 0.1% SDS) for 30 minutes at room temperature. Thereafter,
the eluants were transferred to clean tubes, mixed with 6X SDS loading buffer, and
placed at room temperature for 30 min. Meanwhile, the beads were added 1X SDS
14

loading buffer plus β–mercaptoethanol, and placed at room temperature for 30 minutes.
The samples were then loaded onto the SDS–PAGE for gel electrophoresis.
For in solution digestion experiments, the samples were first purified by Protein
G-Sepharose beads (GE Healthcare, United Kingdom) twice, for 1 hour at 4 °C. The
supernatants were then secondly purified by Anti–Flag M2–Agarose beads for 4 hours at
4 °C, and eluted by elution buffer for 30 minutes at room temperature. The eluants were
mixed with four volumes of cold 100% acetone at –20 °C overnight for acetone
precipitation and in solution digestion steps for Mass spectrometry analysis.
2.5 Western Blot for expression of TMEM56 –
The samples were treated with an SDS loading buffer for 30 minutes at room temperature,
instead of being boiled for 10 min, which is the usual method, before being loaded and
resolved by 10% or 12% SDS–PAGE. After electrophoresis, the gels were transferred to
polyvinylidene difluoride (PVDF) membranes (Bio–Rad, Hercules, CA) and probed with
indicated antibodies. FLAG antibody (1:1000 dilution) was purchased from Sigma
®
.
Anti–ATPase α antibody (1:10,000 dilution) was purchased from BD Transduction
Laboratories

(Franklin Lakes, NJ). The membranes were developed and imaged using the
Fluor–S max quantification system (Bio–Rad, Hercules, CA).
2.6 Acetone precipitation and in solution digestion –
The eluted samples from Immunopercipitation were added with four volumes of cold
100% acetone at –20 °C overnight before being spun down at 15,000g at 4 °C for 20
15

minutes and carefully removing the supernatants. The pellets were dried out using
SpeedVac for 5 minutes. After the acetone precipitation procedure, the proteins were
digested with trypsin directly in the solution.
Briefly describing the in solution digestion steps, the protein samples were added with
10μl 6M urea and heated at 37°C for 5 minutes. Then the mixtures were added with 2μl
of 50mM Dithiothreitol (DTT) at room temperature and 2μl of 13.5mM iodoacetamide at
room temperature for 30 minutes sequentially. Thereafter, 50μl of 50mM ammonium
bicarbonate and 2.4μl of 50mM CaCl
2
were added, to dilute the urea concentration to 1M. The
samples were digested by adding 1μg trypsin at 37°C overnight. The samples were
continuously trypsinized at 37°C overnight once more before being mixed with 1μl of
formic acid prior to storage at –80 °C. After C–18 beads clean–up, the samples were
washed twice with 5% acetonitrile, eluted with 70% acetonitrile, dried with SpeedVac for
approximately 2 hours and resuspended in 5% acetonitrile plus 1% formic acid, the
samples were then analyzed using an LTQ–ETD mass spectrometer (Thermo, Waltham,
MA).
2.7 Immunostaining —
HEK 293T cells were grown on cover glasses overnight and transfected with TMEM56
and pCMV–RC. After 24 hours, cells were fixed with 2.5% formaldehyde in PBS for 15
minutes at room temperature and permeabilized with ice–cold 100% methanol for 10
minutes at room temperature. Cover glasses were rinsed three times in PBS for 5 minutes
per time. Cells were blocked with 5% goat serum in immunostaining wash buffer (1%
16

BSA, 0.3% Triton, and 1x PBS) for 1 hour before being incubated with the anti–FLAG
®

antibody and/or anti–ATPase α antibody (BD Transduction Laboratories
,
, Franklin Lakes,
NJ), both diluted 1:1000 in an immunostaining wash buffer for 1 hour at room
temperature. Cover glasses were washed three times with PBS for 5 minutes per time,
followed by incubation with a secondary goat anti–mouse IgG
2a
antibody (Invitrogen

,
Carlsbad, CA, Cat.#A21136) and a goat anti–mouse IgG
1
antibody (Invitrogen, Carlsbad,
CA, Cat.#A21121), diluted together at 1:200 in immunostaining wash buffer for 1 hour at
room temperature in the dark. In the same dark condition, cells were incubated with
2μg/ml Hoechst 33342 for 15 minutes at room temperature before drop mounting the
solution onto cover glasses and placing the glasses on slides, for storage at 4
o
C in the
dark. The transfected HEK 293T cells were analyzed via fluorescence microscopy
(Olympus
®
AX70 research system microscopy, Tokyo, Japan).
2.8 Mitochondria isolation and ATP-synthase enzyme activity assay–
The ATP-synthase enzyme activity was assayed by the protocol according to the
manufacturer’s manual (MitoSciences, Eugene, Oregon, Cat. MS451). Isolation of the
crude mitochondria from HEK 293T cells is described below. The transfected cells were
scraped from the culture plates to tubes using PBS, and centrifuged at 300g for 10 min.
The pellets were resuspended with cold Mitochondria Isolation buffer (containing 0.3M
mannitol, 0.1% BSA, 10mM 4–(2–hydroxyethyl)–1–piperazineethanesulfonic acid
17

(HEPES), pH 7.4 and 0.2mM ethylenediaminetetraacetic acid) once the supernatants
were removed. The cells were transferred to 2–ml glass dounce homogenizers, and
homogenized by 30 times dounce strokes on ice. The homogenized whole cell extracts
were transferred to clean tubes and centrifuged at 1000g for 10 minutes at 4 °C, and the
resulting supernatants were collected. Following the supernatants centrifuged once more
at 12,000g (max speed) for 15 minutes at 4 °C, the cell extracts were separated into
cytosolic fractions and mitochondrial fractions. The resulting pellets were the
mitochondrial fractions. Next, the pellets were washed twice using a Mitochondria
Isolation buffer containing a protease inhibitors cocktail, at 12,000g for 15 minutes at
4 °C, resuspended in 500μl Mitochondria Isolation buffer containing protease inhibitors
cocktail, and frozen overnight at –80 °C. The cytosolic fractions were saved for Western
Blots, as well as the 50μl of homogenized whole cell extracts, and approximately 1 μg of
the mitochondrial fractions.
After the isolated mitochondria with different transfected conditions were collected and
frozen in tubes, the ATP–synthase enzyme activity microplate assay was performed
according to the manual. Shortly after, the mitochondria samples were thawed and
18

pelleted at 16,000rpm for 10 minutes at 4°C. Then the pelleted samples were resuspended
with 50μl SOLUTION 1 (MitoScience, Eugene, Oregon), and upon measuring protein
concentration, 5μl DETERGENT (MitoScience, Eugene, Oregon) was added. Each
sample was mixed immediately, and incubated on ice for 30 min before being spun at
16,000rpm for 20 minutes at room temperature. The resulting supernatants were saved as
purified isolated mitochondria. Before loading onto the 96–well pre–coated plate, the
samples were diluted in 1:4 ratio with SOLUTION 1. The loaded microplate was then
incubated for 3 h at room temperature, followed by the addition of 40μl of Lipid Mix
(MitoScience, Eugene, Oregon) to each well used, and incubated for 45 minutes at room
temperature. Immediately prior to measuring the plate using the 96–well microplate
reader (TECAN, Switzerland) in the absorbance at OD 340 nm at 30 °C, the wells used
were added with 200 μl of REAGENT MIX (MitoScience, Eugene, Oregon). The activity
rate of ATP–synthase is based on the decreases in absorbance at 340 nm per minute.

19

CHAPTER THREE: RESULTS
The Transmembrane Protein 56 (TMEM56) was identified as an IKK complex
interacting protein by mass spectrometry in Dr. Ebrahim Zandi’s Lab. The objective of
this study was to investigate the function(s) of TMEM56 in cultured cells.
3.1 TMEM56 expression on Western Blotting –
To express human TMEM56 protein in HEK 293T cells, the expression plasmid
containing a C–terminal Myc–FLAG tagged cDNA of transmembrane protein 56 was
purchased. Transfected total HEK 293T cell lysates were analyzed by Western Blotting,
expecting a band at approximately 30 kDa when the blots were probed with an
anti–FLAG antibody. To render the results comparable, IL17RB transfected HEK 293T
cell lysates were used as positive control to verify the anti–FLAG antibody probing
ability and the empty vector transfected HEK 293T cell lysates as negative control to
confirm TMEM56 protein existence. However, observing bands at the desired expression
region in the first few times of the experiment was not possible (data not shown). After
modifying and rechecking the transfection procedures back and forth, a reasonable
explanation could not be found. Therefore, to satisfy curiosity, the stacking gel was not
20

cut out for electrophoresis as per usual, instead directly transferring the whole gel to a
PVDF membrane before probing with an anti–FLAG antibody. Interestingly, high
molecular weight protein aggregated at the stacking/running gel interface (upper arrow,
Fig. 5A). This observation showed the aggregation behavior of TMEM56 protein on the
SDS–PAGE when carrying the total cell lysates of expressed TMEM56 in HEK 293T
cells as a sample. In order to verify the reason for protein aggregation, TMEM56 was first
purified via Immunoprecipitation. Both samples, the vector-transfected cell lysates and
the TMEM56-transfected cell lysates, were then immunoprecipitated with anti–FLAG
M2–Agarose beads and eluted via regular lysis buffer. As the general Western Blotting
method, the eluants were solubilized in a SDS loading buffer and boiled for 5 min before
being loaded onto the SDS–PAGE. Though the samples were purified for excluding
potential reasons that several interactions might occur between the target proteins and
other proteins, the TMEM56 protein expression still cannot be observed at approximately
30 kDa (Fig. 5B).

21

A

B

Figure 5. Western Blotting for expression of the TMEM56 protein in HEK 293T
cell extracts by unmodified procedure
A. HEK 293T cells were transfected with expression vectors for empty vector (pCMV) or
FLAG-TMEM56 and FLAG-IL17RB. Samples of cell lysates subjected to 10 % SDS–PAGE
were solubilized in SDS loading buffer and heated for 5 min at 100℃ as a general western
Blotting method. Western blot was probed with an anti–FLAG antibody. High molecule weight
aggregation of the TMEM56 proteins occurred at the interface of the stacking and running gel.
Expression of IL17RB protein was used as a positive control (PC).
B. Transfected HEK 293T cells were harvested and immunoprecipitated using anti–FLAG
antibody. Samples subjected to 10 % SDS–PAGE were solubilized in the SDS loading buffer and
heated for 5 min at 100℃ as a general western Blotting method. These experiments were repeated,
with the same results.
22

Western Blotting is an essential and basic experiment for confirming successful transient
transfection of specific proteins in cells. If the expected proteins are unable to be seen on
the blots, this signifies that the transfection could be invalid because the cells might
produce undesired proteins, or not produce it at all. However, the results of the high
molecule weight aggregate shown in Fig. 5A should not be ignored. For this reason, a
step was modified in the Western Blotting technique. Instead of denaturing the proteins
by adding the SDS loading buffer and boiling the mixture for 5 minutes in the final step
before loading the samples onto the SDS–PAGE, the mixed samples were placed at room
temperature for 30 minutes. After 30 minutes, electrophoresis, transferring, blocking, and
detection procedures were performed, as usual. Surprisingly, a band of approximately 26
kDa was detected, and there was no high molecule weight aggregation at the interface of
the gels (Fig. 6). This indicated that heating TMEM56 resulted in its aggregation.
3.2 Purification of TMEM56 and Identification of interacting proteins –
Upon solving the problem of the TMEM56 protein on Western blots with a minor
alteration in the methods, the HEK 293T cell lysates that had been expressed with
TMEM56 or empty pCMV vector were further purified for following experiments. Fig. 7
23

shows Western Blotting for the empty vector and TMEM56, processed after
immunoprecipitation. The lysates were precleared with Protein G–Sepharose beads twice
before being immunoprecipitated with Anti–Flag M2 Agarose beads. Preclearing the
lysates can help reduce non–specific binding of proteins to beads by using an irrelevant
antibody to remove proteins that bind immunoglobulins non–specifically. This
experiment showed that TMEM56 was successfully immunoprecipitated and eluated.
To identify the TMEM65-interacting proteins, the eluants were concentrated using
Acetone precipitation. After digestion by trypsin, the peptides were analyzed on a linear
ion trap LTQ mass spectrometer.
24

Figure 6. Western Blotting for expression of the TMEM56 protein in HEK 293T cell
extracts by unboiled procedure
HEK 293T cells were transfected with expression vectors for empty vector (pCMV) or
FLAG-TMEM56 (lines 1 to 4) and FLAG-IL17RB. Samples of whole cell lysates subjected to 10
% SDS–PAGE were solubilized in SDS loading buffer and placed at room temperature for 30
minutes before loading on gels. Western blot was probed with an anti–FLAG antibody. TMEM56
protein was expressed ~26 kDa and no high molecule weight aggregation behaviour of at the
interface of gels.

25

Figure 7. Western Blotting of the immunoprecipitated TMEM56 protein in HEK
293T cell extracts
Transfected HEK 293T cells were harvested and purified by immunoprecipitation using Protein
G–Sepharose beads to preclear first before taking the supernatants for immunoprecipitation with
the Anti–Flag M2 Agarose beads. Samples were eluted via regular lysis buffer with 6 M urea and
0.1 % SDS (also elution buffer). Western blot was probed with an anti–FLAG antibody.
TMEM56 protein was abundantly expressed in the eluants and several remained on the beads.
Expression of IL17RB protein was as a positive control (PC). L, whole cell lysates; S,
supernatants; W, wash buffer; B, beads; Elu, eluants.

26

The result of the top four proteins that co-immunoprecipiatetd with TMEM56 is listed in
Table 1. The experiments were repeated three times, yielding the finding that the
TMEM56 protein was most likely to interact with the ATP-synthase subunit α and
ATP-synthase subunit β.

Table 1. Proteins interacting with TMEM56 in three experiments
Proteins were identified with LTQ–ETD mass spectrometry and ordered by probability (P). The
Xcorr score was calculated using SEQUEST. The three experiments were performed using three
different samples that were in the digested solution.

27

3.3 Subcellular Localization of TMEM56 –
Since the ATP-synthase (which may interact with TMEM56 protein in HEK 293T cells)
is localized on the membrane of the mitochondria, this study investigated the subcellular
localization of TMEM56 via immunofluorescent experiments. Fig. 8A shows
immunostaining with an anti–FLAG antibody in cells transfected with either empty
vector or TMEM56 expression vector for 48 hours. The transfected HEK 293T cells were
co-stained with the anti–ATPase α antibody and an anti–FLAG antibody. The
anti–ATPase α antibody was used for detecting the ATP-synthase α on the mitochondrial
membrane, and the anti–FLAG antibody was used for detecting TMEM56 protein. These
antibodies were stained in turn with rhodamine-tagged (red for anti FLAG), and
fluorescein -tagged (green for anti ATPase a). The nuclei were visualized with Hoeschst
blue staining, which indicated the position of the cells. Immunoflourescence was
analyzed via confocal microscopy (Fig. 8B). The overexpression of TMEM56 was
detected as several irregular red dots shape in the cytoplasm. , The confocal microscopy
merged image (Fig. 8B, center) shows in yellow that A TPase α and TMEM56 are

28

A B

Figure 8. Immunofluorescence images for localization of TMEM56 protein
A. HEK 293T cells transfected with control vector (pCMV) or TMEM56 were immunostained
with an anti–FLAG antibody for TMEM56 protein.
B. Confocal microscopy images obtained from the same cells immunostained with the
anti–ATPase α and an anti–FLAG antibody, showing the TMEM56 localized on the
mitochondria.

29

co–localized mostly on the mitochondria. Altogether, these results indicate that TMEM56
colocalize with A TP-synthase α on the mitochondria in HEK 293T cells.
3.4 siRNA Expression Analysis and Mitochondria Isolation –
To further examine the subcellular localization and functional interaction of TMEM56
with ATP-synthase α, TMEM56 was knocked down by transfecting the 21–mer siRNA of
TMEM56 (siRNA–TM56-2) into HEK 293T cells. The siRNA is used to reduce the
endogenous protein level. The negative control siRNA of TMEM56 (siRNA–TM56–c)
was also used, which did not target any gene products. According to the aforementioned
results, to separate whole cell lysates into cytosolic and mitochondrial fractions is a
compelling method for analyzing TMEM56 subcellular localization and interacting
proteins. Western Blotting images (Fig. 9) show the total cell lysates, cytosolic fractions,
and mitochondrial fractions of HEK 293T cells in four different transfection experiments,
which were subsequently transfected with empty vector, TMEM56, siRNA–TM56-2, and
siRNA–TM56–c. In conclusion, ATP-synthase α was specifically purified in the
mitochondria fractions (Fig. 9A), indicating that the performance of Mitochondria

30

A

B

Figure 9. Western Blot for confirmation of purified proteins in mitochondria
isolation fractions
HEK 293T cells were transfected with control vector (pCMV), TMEM56, siRNA of TMEM56
and control siRNA of TMEM56, as described in the Methods and Material section. Cells were
harvested as lysates, and separated into cytosolic fractions and mitochondria fractions via
mitochondria isolation assay. V, vector; T, TMEM56; 2, siRNA of TMEM56; C, control siRNA
of TMEM56.
A. Western blot was probed with anti–ATPase α antibody.
B. Western blot was probed with an anti–FLAG antibody. The TMEM56 proteins were expressed
in the mitochondria fractions.
31

Isolation was successfully accomplished. Furthermore, TMEM56 protein expression
occurred specifically in the mitochondria (Fig. 9B). This result is consistent with the mass
spectrometry findings and the immunofluorescent presentations.
3.5 ATP-synthase activity assay –
To see whether the expression of TMEM56 in HEK 293T cells affects ATP-synthase α
activity, the microplate assay kit was used to investigate ATP-synthase enzyme activity
(detailed in the Methods section). The activity is coupled to the conversion of NADH to
NAD
+
, measured as a decrease in absorbance at OD 340 nm. Fig. 10 shows the
decreasing absorbance at 340 nm during one hour period of mitochondrial samples,
which were isolated from transfected HEK 293T cell lysates with empty vector,
FLAG-TMEM56, and siRNA–TM56-2. The mitochondria expressing FLAG-TMEM56
showed an increasing in ATP-synthase enzyme activity compared to vector transfected
cells. Meanwhile, the mitochondria from cells in which TMEM56 was knocked down
have lower ATPase activities. The data indicate that increased expression of TMEM56
increases ATPase activity, while decreasing it expression reduces ATPase activity.

32

Figure 10. ATP-synthase activity assay
The x–axis indicates time (minute) and the y–axis (OD) displays the absorbance at 340 nm of
NAD
+
. The ATP-synthase activity rate is based on absorbance decreasing. The graph shows that
TMEM56 may increase the activity of the ATP-synthase.

33

CHAPTER FOUR: CONCLUSION AND DISCUSSION
This study establishes, for the first time, that TMEM56 is physically associated with
mitochondria via ATPase, and it regulates mitochondrial ATP synthesis. Experimental
conditions for expression and western blot analysis of TMEM56 were established in this
study. Co-immunoprecipitation of FLAG-TMEM56 with the ATPase α subunit is the
basis for the findings presented in this study. Cell fractionation and immunoflourescence
experiments confirmed the interaction between TMEM56 and ATPase. Changes in the
ATP-synthase activity of mitochondria as a function of increased or decreased expression
of TMEM65 further establishes the functional relationship of these two proteins.
As stated earlier, mitochondria are essential organelles for the life and death of a cell.
ATP is required for biological endoergonic processes, and the enzyme responsible for
producing ATP is called ATP-synthase, which is also an integral membrane protein.
Understanding the defects and diseases caused by or related to this enzyme is also
necessary. Oxidative damage of the α subunits of the mitochondrial ATP-synthase results
in the loss of activity, consequently leading to OXPHOS diseases. Defects in energy
metabolism, excitotoxicity, and oxidative damage are involved in normal aging and
34

neurodegenerative diseases, such as Alzheimer’s disease, Huntington’s disease, and
Parkinson’s disease (Beal MF, 1995). Previous studies not only show that the deficiency
in ATP–synthase is responsible for the activity disorder, but also that ATP-synthase
activity is related to other transmembrane proteins. Therefore, TMEM56 could be a
regulator of ATP-synthase activity, and hence it is possible that regulating the expression
level of TMEM56 would directly regulate mitochondrial function. In this case, TMEM56
expression could be a marker, and potentially, a therapy target for mitochondrial related
diseases. Clearly more studies are required to confirm the data presented in this study,
for example, via luminescent ATP assays or extracellular flux to further investigating the
ATP-synthase activity assay of TMEM56 proteins interfering. With the further studies in
the future, in vivo regulation of mitochondrial ATP-synthase and/or other aspects of
mitochondrial biogenesis by Transmembrane protein 56 would be established.

35

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Abstract (if available)

Abstract Transmembrane 56 (TMEM56) is a 30 KDa protein and it is highly conserved in most species. The protein was identified as an IKKβ-interacting protein by mass spectrometry in Dr. Zandi’s Lab. Initially, abnormal aggregation of TMEM56 was observed, which was determined to be due to the boiling step prior to SDS-PAGE. This procedure precluded the detection of protein by Western blotting. To investigate the function of TMEM56, a FLAG-tagged version of it was expressed in human 293T kidney cells, and interacting proteins were identified by co-immunoprecipitation and mass spectrometry. The predominant TMEM56-interacting proteins are mitochondrial F1 components, ATP synthase α and β subunits. TMEM56 and ATP synthase α subunit were shown to be co-localized in the mitochondria. Expression of FLAG-TMEM56 in 293T cells resulted in an increased ATP synthase activity using isolated mitochondria in vitro. On the other hand, siRNA-mediated knock down of TMEM56 decreased the ATP synthase activity in isolated mitochondria. The data presented in this study indicated that TMEM56 is localized in the mitochondria and may play a role in regulating ATP synthesis by interacting with ATP synthase α, and perhaps, the β subunits of the F1 complex.

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Asset Metadata

Creator Wei, Hsiao-Fan (author)

Core Title Studies on the expression and function of the human TMEM56 protein

School Keck School of Medicine

Degree Master of Science

Degree Program Biochemistry and Molecular Biology

Publication Date 06/03/2011

Defense Date 05/12/2011

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag ATP synthease enzyme activity assay,LTQ-ETD,mitochondria,OAI-PMH Harvest,TMEM56,transmembrane protein,western blotting

Language English

Contributor Electronically uploaded by the author (provenance)

Advisor Tokes, Zoltan A. (committee chair), Kalra, Vijay K. (committee member), Zandi, Ebrahim (committee member)

Creator Email hsiaofaw@usc.edu,sumstarwei@gmail.com

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c127-615287

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