ORIGINAL PAPER

TMEM166, a novel transmembrane protein, regulates cellautophagy and apoptosis

Lan Wang Æ Chuanfei Yu Æ Yang Lu Æ Pengfei He ÆJinhai Guo Æ Chenying Zhang Æ Quansheng Song ÆDalong Ma Æ Taiping Shi Æ Yingyu Chen

Published online: 11 May 2007

� Springer Science+Business Media, LLC 2007

Abstract Programmed cell death can be divided into

apoptosis and autophagic cell death. We describe the bio-

logical activities of TMEM166 (transmembrane protein

166, also known as FLJ13391), which is a novel lysosome

and endoplasmic reticulum-associated membrane protein

containing a putative TM domain. Overexpression of

TMEM166 markedly inhibited colony formation in HeLa

cells. Simultaneously, typical morphological characteris-

tics consistent with autophagy were observed by trans-

mission electron microscopy, including extensive

autophagic vacuolization and enclosure of cell organelles

by double-membrane structures. Further experiments con-

firmed that the overexpression of TMEM166 increased the

punctate distribution of MDC staining and GFP-LC3 in

HeLa cells, as well as the LC3-II/LC3-I proportion. On the

other hand, TMEM166-transfected HeLa and 293T cells

succumbed to cell death with hallmarks of apoptosis

including phosphatidylserine externalization, loss of mito-

chondrial transmembrane potential, caspase activation and

chromatin condensation. Kinetic analysis revealed that the

appearance of autophagy-related biochemical parameters

preceded the nuclear changes typical of apoptosis in

TMEM166-transfected HeLa cells. Suppression of

TMEM166 expression by small interference RNA inhibited

starvation-induced autophagy in HeLa cells. These findings

show for the first time that TMEM166 is a novel regulator

involved in both autophagy and apoptosis.

Keywords Autophagy � Apoptosis � Programmed cell

death � TMEM166 � FLJ13391

Introduction

Autophagy in eukaryotic cells constitutes a degradative

mechanism for removal and turnover of bulk cytoplasmic

constituents via the endosomal–lysosomal system. Early

studies revealed that autophagy is an adaptive response of

cells to nutrient deprivation, to ensure upkeep of minimal

housekeeping functions. Recently, studies have revealed

that the function of autophagy is much more complex. It is

involved in physiological processes as diverse as biosyn-

thesis, regulation of metabolism through elimination of

specific enzymes, morphogenesis, cellular differentiation,

tissue remodeling, aging and cellular defense [1–3].

Overall, autophagy constitutes a fundamental survival

strategy of cells. On the other hand, extensive autophagy

causes cell death. Thus, the study of autophagy has initi-

ated a controversial discussion on how cell suicide may be

considered a cellular survival function [2, 4–7]. Nonethe-

less, the biochemical mechanisms involved in autophagy

remain largely unexplored in type II cell death. It has been

difficult to establish whether autophagy plays a causal role

in Type II cell death or represents a failed attempt at cell

survival in cells undergoing programmed cell death [8].

L. Wang � C. Yu � Q. Song � D. Ma � Y. Chen

Laboratory of Medical Immunology, School of Basic Medical

Science, Peking University Health Science Center, 38# Xueyuan

Road, Beijing 100083, P. R. China

L. Wang � C. Yu � Q. Song � D. Ma � Y. Chen (&)

Peking University Center for Human Disease Genomics, 38#

Xueyuan Road, Beijing 100083, P. R. China

e-mail: yingyu_chen@bjmu.edu.cn

Y. Lu � P. He � J. Guo � C. Zhang � T. Shi (&)

Chinese National Human Genome Center, #3-707 North

YongChang Road BDA, Beijing 100176, P. R. China

e-mail: taiping_shi@yahoo.com.cn

123

Apoptosis (2007) 12:1489–1502

DOI 10.1007/s10495-007-0073-9

Meanwhile, the mutual relationship between apoptotic and

autophagic cell death is currently under debate [9, 10].

In our human functional genomics project, we have

cloned hundreds of functionally unknown human open

reading frames (ORFs) by searching the human Refseq and

expressed sequence tag (EST) databases in GenBank.

Using a cell-based high-throughput assay [11], we identi-

fied several novel genes associated with cell viability,

including TMEM166 (transmembrane protein 166, previ-

ously identified as FLJ13391). Here, we report the identi-

fication and characterization of the TMEM166 protein. We

find that TMEM166 is a lysosomal and endoplasmic

reticulum-associated protein that can induce HeLa cell

death. Interestingly, cell death triggered by TMEM166

exhibits both autophagic and apoptotic characteristics.

Suppression of TMEM166 by small interfering RNA

(siRNA) inhibits starvation-induced autophagy in HeLa

cells. Therefore, TMEM166 appears to be a novel regulator

of programmed cell death, facilitating autophagy and

apoptosis.

Materials and methods

Cell lines and reagents

HeLa and 293T cell lines (a kind gift from T. Matsuda,

Japan) were maintained in Dulbecco’s modified Eagle

medium (Life Technologies, USA) supplemented with

10% fetal bovine serum (Hyclone, USA) and 2 mM

L-glutamine. All experiments were performed on logarith-

mically growing cells. Earle’s Balanced Salt Solution

(EBSS), DAPI, monodansylcadaverine (MDC) and a

monoclonal antibody against b-actin were obtained from

Sigma (USA). Calcein-AM [4¢5¢-bis (N¢, N¢-bis (carb-

oxymethyl) aminomethyl fluorescein acetoxymethyl ester),

ethidium homodimer-1 (EthD-1), 3, 3¢-dihexyloxacarbo-

cyanine iodide [DiOC6 (3)], Lyso Tracker Red, ER

Tracker Blue were from Molecular Probes (USA).

Ac-DEVD-AMC and z-VAD-fmk were purchased from

Pharmingen (BD Biosciences Europe, Brussels, Belgium).

A polyclonal antibody against PARP was from Cell Sig-

naling Technology, and an IRDye 800-conjugated affinity

purified anti-Green Fluorescent Protein (GFP) antibody

was obtained from Rockland (USA). IRDye 800-conju-

gated secondary antibodies against mouse and rabbit IgG

were purchased from LI-COR Bioscience (USA). The

GFP-LC3 plasmid was kindly provided by Professor Zhe-

nyu Yue (Mount Sinai School of Medicine, New York,

USA). The rabbit anti-LC3 polyclonal antibody was pre-

pared by using recombinant rat LC3 protein expressed in

E. coli and purified in our lab. It was validated by ELISA

and Western blot.

Constructs and transfections

Full-length cDNA of TMEM166 (GenBank accesion no.

BC016157) was amplified from a human kidney cDNA

library (Clontech, USA) by PCR using the forward primer

P1 (5¢-tgtcccatgaggctgccc-3¢) and reverse primer P2 (5¢-tccctaatagtagcgattcaggctc-3¢). The N-terminal truncated

mutant, TMEM166-DTM, was amplified by P3 (5¢-cac-

catgacagactgc-3¢) and P2. The purified PCR product was

ligated into the pGEM-T Easy vector (Promega, USA). The

insert was released by EcoRI and subcloned into the EcoRI

site of pcDNA.3.1/myc-His (–) B (Invitrogen, USA) to

construct pcDB/TMEM166 and pcDB/TMEM166-DTM.

To construct the TMEM166-GFP and TMEM166-DTM-

GFP plasmids, the reverse primer P4 (5¢-cgcggatccgca-

tagtagcg-3¢) was used to create a fusion with a C-terminal

GFP tag in the pEGFP-N1 vector (Clontech) by BamHI.

All plasmids were confirmed by DNA sequencing. DNA

transfection was performed using VigoFect (Vigorous,

China), a non-liposomal cationic formula, according to

manufacturer’s instructions. In some experiments, cells

were electroporated for 20 ms at 120 V, with 10 lg plas-

mid per 106 cells in 2 mm gap cuvettes using an ECM 830

Square Wave Electroporation System (BTX, USA).

Northern blot and RT-PCR assay

A 468-bp TMEM166 PCR product amplified by primers P1

and P2 was purified and labeled with fluorescein using a

Gene Images Random Prime Labeling Kit (Amersham

Biosciences, Uppsala, Sweden) according to the manufac-

turer’s instructions. Total RNA of TMEM166 was ex-

tracted from human adult tissues using TRIzol reagent

(Invitrogen, USA). Samples (20 lg of each tissue speci-

men) were separated by electrophoresis and transferred

onto a nylon membrane (Amersham Biosciences, Uppsala,

Sweden), which was subsequently hybridized with the

probe at 65�C overnight. After washing with SSC buffer,

the membrane was incubated with anti-fluorescein-AP

conjugate and images were developed using a Gene Images

CDP-Star Detection Module (Amersham Biosciences). RT-

PCR was performed with ThermoScript RT-PCR System

(Invitrogen, USA) using primers P1, P2 and GAPDH.

Clonogenic assay

Long-term survival of TMEM166-transfected HeLa cells

was examined using a clonogenic assay. Briefly, cells were

transfected with different constructs by electroporation as

described above. At 48 h post-transfection, cells were

trypsinized and seeded into 100 mm dishes (at 1000 cells

per dish) and selection medium with G418 (600–800 lg/

ml) was added. 15 days later, cells were fixed and stained

1490 Apoptosis (2007) 12:1489–1502

123

with crystal violet, and colonies with a diameter of larger

than 0.5 mm were counted. Each group was assayed in

triplicate dishes, and each experiment was repeated twice.

Fluorescence and confocal microscopy

Two fluorescent dyes, calcein-AM (green fluorescence,

indicating live cells) and EthD-1 (red fluorescence, indi-

cating dead cells) were used to validate cell viability.

Transfected cells were stained with Calcein-AM (1 lM)

and EthD-1 (2 lM) and incubated at 37�C for 30 min.

Subsequently, digital images of cells were obtained using

an inverted fluorescent microscope (Olympus, Japan).

Cells transfected with GFP-LC3 plasmids were observed

using fluorescence microscopy. Percentages of punctuate

distribution of GFP-LC3 were counted in five non-over-

lapping fields and the statistical data were obtained from

three repeated experiments. Cell nuclei were stained with

DAPI (0.5 lg/ml).

Autophagic vacuoles were labeled with 0.05 mM MDC

by incubating cells grown on the coverslips at 37�C for 1 h.

After incubation, cells were washed and fixed with 4%

paraformaldehyde and observed under a Leica TCS SP2

Confocal System (Heidelberg, Germany) equipped with the

UV filter (360-nm excitation/457-nm emission).

Transiently transfected HeLa cells expressing

TMEM166-GFP or TMEM166-DTM-GFP were cultured

on the coverslips, stained with either 200 nM Lyso Tracker

Red or 100 nM ER Tracker Blue for 15 min at 37�C, and

observed by fluorescence confocal microscopy.

Transmission electron microscopy

For transmission electron microscopy (TEM), cells were

initially fixed in 0.1 M sodium phosphate buffer containing

2.5% glutaraldehyde, pH 7.4. Next, cells were fixed in

0.1 M sodium phosphate buffer containing 1% OsO4, pH

7.2 for 2 h at 4�C, and dehydrated in a graded series of

ethanol. Cells were then embedded into Ultracut (Leica,

Germany) and sliced into 60 nm sections. Ultrathin sec-

tions were stained with uranyl acetate and lead citrate, and

examined with a JEM-1230 transmission electron micro-

scope (JEOL, Japan).

Flow cytometry

PS externalization analysis was performed as described

previously [12]. Briefly, transfected cells (2 · 105) were

trypsinized, washed twice with PBS, and resuspended in

200 ll binding buffer (10 mM HEPES, pH 7.4, 140 mM

NaCl, 1 mM MgCl2, 5 mM KCl, 2.5 mM CaCl2). FITC-

conjugated Annexin V was added to a final concentration

of 0.5 lg/ml. After incubation for 20 min at room tem-

perature in the dark, PI was added at 1 lg/ml, and the

samples were immediately analyzed on a FACSCalibur

flow cytometer (Becton Dickinson, USA).

DiOC6(3) was used to evaluate changes of mitochon-

drial membrane potential (DWm). Transfected cells

(2 · 105) were harvested as described above and resus-

pended in 400 ll PBS containing 20 nM DiOC6(3). After

incubation for 15 min at 37�C in the dark, the samples

were analyzed by a FACSCalibur flow cytometer. Results

were expressed as the proportion of cells exhibiting low

mitochondrial membrane potential estimated by the re-

duced DiOC6 (3) uptake.

Assay for caspase-3 activity

Caspase-3 activity was measured using a caspase-3 fluor-

ogenic substrate Ac-DEVD-AMC Protease Assay Kit

(PharMingen, San Diego, CA, USA). All procedures were

carried out according to the manufacturer’s instructions.

Briefly, the transfected cells were lysed in whole cell lysis

buffer (10 mM Tris–HCl, pH 7.5, 130 mM NaCl, 1%

Triton-X100, 1 mM PMSF), and equal amounts of total

cell lysates were mixed with caspase-3 assay buffer

(25 mM HEPES, pH 7.5, 1 mM EDTA, 0.1% CHAPS,

100 mM NaCl, 10 mM DTT) containing 20 lM Ac-

DEVD-AMC in a 96-well plate in triplicate. Caspase-

3 mediated cleavage of Ac-DEVD-AMC into free AMC

was measured using a FLUOStar fluorometer (BMG Lab-

technologies, Germany) with an excitation filter of 380 nm

and emission filter of 460 nm. Results were calculated as a

proportion of the control over 120 min (T120 to T0).

Western blot analysis

Treated cells were pelleted by centrifugation and lysed in

lysis buffer (10 mM HEPES pH 7.4, 0.15 M NaCl, 1 mM

EDTA, 1 mM EGTA, 1% Triton X-100, 0.5% NP-40, with

freshly added proteinase inhibitor cocktail) for 30 min on

ice. Cell lysates were centrifuged at 18,000 · g for 10 min

at 4�C and the supernatant was measured using the BCA

protein assay reagent (Pierce, USA). Equal amounts of

protein were separated by 15% SDS-PAGE and transferred

onto nitrocellulose membranes (Amersham Pharmacia,

UK). Membranes were blocked in Tris-buffered saline

containing 0.1% Tween-20 (TBS-T) and 5% non-fat milk

for 2 h and incubated overnight at 4�C with the appropriate

primary antibody. After washing in TBS-T buffer, mem-

branes were incubated for 1 h in the dark with the appro-

priate IRDye 800-conjugated secondary antibodies. Signals

were detected on an Odyssey Infrared Imager (LI-COR

Bioscience, USA) after washing in TBS-T buffer.

Apoptosis (2007) 12:1489–1502 1491

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TMEM166 siRNAs synthesis and Real-time

quantitative PCR

Specific siRNA against TMEM166 with targeting se-

quence 5¢-TGATAAGGATCTCTTGCCA-3¢ (si-

TMEM166) was designed, chemically synthesized and

PAGE purified according to manufacturer’s instructions,

free of RNase contamination by Genechem Corporation

(Shanghai, China). Non-silencing siRNA that had no se-

quence homology to any known human genes was used as

the control. All siRNAs were dissolved at a concentration

of 20 lM in buffer containing 20 mM KCl, 6 mM HEPES,

pH 7.5, 0.2 mM MgCl2. Cells were fed with fresh culture

medium prior to experiments. HeLa cells were transfected

by electroporation as described above. To verify the effect

of TMEM166 siRNA, quantitative real-time PCR assay

was carried out in an ABI Sequence Detection System

(Applied Biosystems, USA) with specific primers for

TMEM166 and GAPDH. Preliminary reactions were run to

optimize the concentration and ratio of each primer set. All

the cDNA templates were diluted 100 times and 8 ll of

each diluted cDNA template was used in a 20 ll real-time

PCR amplification system of SYBR Green PCR Master

Mix Kit as the manufacturer directed. The expression level

of wild type HeLa cells (control) was treated as the

baseline. The experiments were repeated twice with con-

sistent results.

Results

Cloning and bioinformatics analysis of human

TMEM166

The full-length human TMEM166 cDNA clone (GenBank

accesion no. BC016157) was directly isolated from a hu-

man kidney cDNA library. It is 1639 base pairs long with

an in-frame stop codon upstream of the putative ATG start

codon and a 3¢-poly (A) tail. The open reading frame en-

codes 152 amino acids with a predicted molecular mass of

17.5 kDa and an isoelectric point of 6.5. The full length

cDNA and predicted amino acid sequences of TMEM166

are shown in Fig. 1A. Human TMEM166 is located on

chromosome 2p12, and encompasses 4 exons and 3 introns

(Fig. 1B). TMEM166 is conserved in human, chimpanzee,

rat, mouse and dog (Fig. 1C), but shares no obvious

homology to any known genes or proteins. Transmembrane

analysis (http://www.cbs.dtu.dk/services/TMHMM-2.0/)

[13] suggests that there is a conserved TM domain near the

N-terminus of the protein (Fig. 1D). To our knowledge, no

functional studies have been performed on this hypotheti-

cal gene.

Expression profiles of TMEM166

Northern blot analysis was used to confirm the existence

of TMEM166 mRNA in human adult tissues. As shown

in Fig. 2A, a band of ~1.7 kb was detected in lung tis-

sue, which was consistent with the bioinformatics anal-

ysis (1639 bp). RT-PCR analysis reveals that TMEM166

is expressed in a variety of normal tissues, including

kidney, liver, lung, pancreas, placenta, but not in heart

and skeletal muscle (Fig. 2B). TMEM166 is broadly

expressed in all tumor tissues examined (Fig. 2C).

TMEM166 mRNA is also detected in various cell lines

including HeLa, MDA, HEK-293, A549, PC3, U937,

Jurkat and 293T (Fig. 2D).

TMEM166 protein localizes to lysosome and

endoplasmic reticulum

To examine the subcellular localization of TMEM166,

we constructed a TMEM166-GFP fusion plasmid. After

transient transfection, TMEM166-GFP exhibited a punc-

tate cytoplasmic distribution, concentrated in the peri-

nuclear region. Confocal microscopic analysis revealed

that TMEM166-GFP co-localized with the lysosome-

specific fluorescent dye Lyso Tracker and the endoplas-

mic reticulum (ER)-specific fluorescent dye ER Tracker

(Fig. 3A). We did not observe the co-localization of the

mitochondria-specific fluorescent dye Mito Tracker (data

not shown). The same distribution pattern of TMEM166

was observed in 293T cells (data not shown).

TMEM166 induces cell death both in 293T and HeLa

cells

To investigate the biological functions of TMEM166, we

constructed eukaryotic vectors expressing TMEM166 and

screened a platform based on cell viability, which had been

previously established in our laboratory [11] We observed

morphological changes in 293T cells transiently transfect-

ed with the TMEM166 expression vector. Typical apop-

totic changes, similar to Bax-transfected cells as a positive

control were noted in these cells, including marked

rounding, shrinkage, blebbing, and detachment from the

culture dish. No such changes were detected in empty

vector-transfected cells (mock, Fig. 4A). The two-color

fluorescence cell viability assay comparing the red fluo-

rescence in the 293T cells transfected with the TMEM166

plasmid and the empty vector showed that dead cells were

clearly evident (Fig. 4B).

Next, we analyzed the long-term survival of HeLa cells

expressing TMEM166 using a clonogenic assay. As shown

in Fig. 4C, a significant decrease in the number of survival

1492 Apoptosis (2007) 12:1489–1502

123

colonies were observed in the TMEM166-transfected cells

compared with the mock-transfected cells. Taken together,

overexpression of TMEM166 appears to inhibit cell growth

and induce cell death.

Overexpression of TMEM166 induces autophagic

characteristics in the early stage of cell death

To explore the mechanism of cell death induced by

TMEM166 overexpression, we examined whether

TMEM166 is able to induce cell autophagy. We examined

the ultrastructure of TMEM166-transfected HeLa cells by

using TEM. At 20 h after transfection, >80% of cells were

alive. TMEM166-transfected HeLa cells showed extensive

cytoplasm vacuolization. At higher magnifications, most

vacuoles contained electron dense material and degraded

organelles. Chromatin condensation and nuclear fragmen-

tation were absent in these cells. In contrast, empty vector

transfected cells displayed normal cell morphology

(Fig. 5A).

Fig. 1 Identification and

sequence analysis of

TMEM166. (A) Nucleotide

sequence and predicted amino

acid sequences of human

TMEM166. Primers used to

amplify the ORF are underlined,

and the start and stop codons are

italicized. The poly (A) signal

sequence is shown in shadow.

The putative TM domain

(amino acid residues from 34 to

56) is indicated with a broken

line. (B) The sketch map of the

TMEM166 gene and cDNA

structure. The boxes show the

exons with their relative size

and the positions in the

TMEM166 gene. (C)

Phylogenetic analysis of

TMEM166. (D) Schematic

representation of human

TMEM166 and constructs used

in this study

Apoptosis (2007) 12:1489–1502 1493

123

Mitchener et al. reported that starved HeLa cells dis-

played extensive autophagy [14, 15]. In our study, Earle’s

Balanced Salt Solution (EBSS) was used to induce auto-

phagy in HeLa cells. To further confirm the effects of

TMEM166 overexpression on cell autophagy, we detected

other biochemical parameters for monitoring autophagy,

such as monodansylcadaverine (MDC) staining and GFP-

LC3 distribution [9, 16]. Confocal and fluorescence

microscopy showed extensive punctate MDC staining

(Figure 5B) and GFP-LC3 localization (Fig. 5C) in

TMEM166-transfected and starved HeLa cells, in contrast

to the diffuse pattern in control cells (mock). We quantified

the distribution of GFP-LC3 in mock, TMEM166-trans-

fected, and starved HeLa cells. Data from three indepen-

dent experiments showed that compared with control cells

(mock), TMEM166 overexpression increased punctate

LC3, similar to starved cells (Fig. 5D). Immunoblots using

anti-LC3 and anti-GFP antibodies showed that the mem-

brane-bound LC3-phospholipid conjugate LC3-II was in-

creased in TMEM166-transfected and starved cells,

compared with control cells (Fig. 5E). Therefore, our re-

sults suggested that TMEM166 can induce hallmarks of

autophagy early after transfection.

TMEM166 induced cell death involves hallmarks of

apoptosis

At 40 h after transfection, we found that TMEM166-

overexpressing 293T cells exhibited chromatin condensa-

tion, detected with the nuclear stain DAPI (Fig. 6A). A key

biochemical hallmark of apoptotic cell death is the trans-

location of PS from the cytoplasmic surface of the cell

membrane to the external cell surface [17]. Exposure of PS

on the surface of apoptotic cells can be easily identified by

flow cytometry using fluorescence-labeled Annexin V,

which specifically binds PS [18]. Using this assay, we

Fig. 2 Expression profiles of

human TMEM166. (A)

Northern blot analysis of

TMEM166 expression in adult

human tissues. The position of

the TMEM166 transcript is

indicated. TMEM166 mRNA

expression was also analyzed by

RT-PCR in human normal

tissues (B), human tumor tissues

(C) and human cell lines (D).

GAPDH expression was

amplified as an internal control

Fig. 3 Localization of TMEM166 and TMEM166-DTM. (A)

TMEM166 is localized to the lysosome and endoplasmic reticulum

(ER). HeLa cells were transiently transfected with TMEM166-GFP

expression vectors, and two-color confocal microscopy analysis of

TMEM166-GFP fusion protein (green) and lysosome (Lyso Tracker

staining, red) or endoplasmic reticulum (ER Tracker staining, blue)

was performed after 24 h transfection. (B) TMEM166-DTM failed to

localize to the lysosome and ER, but displayed a diffuse cytoplasmic

expression pattern

1494 Apoptosis (2007) 12:1489–1502

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detected PS on the surface of cells by FITC/Annexin-V

staining at 40 h after transfection with TMEM166. Plasma

membrane integrity was simultaneously assessed by PI

exclusion using two-color fluorescence-activated cell sort-

ing (FACS) analysis. As shown in Fig. 6B, in 293T and

HeLa cells transfected with TMEM166, the proportion of

Annexin V and/or PI positive cells is increased compared

with mock-transfected cells.

Activation of effector caspases, such as caspases 3 and

7, is responsible for the proteolytic cleavage of a diverse

range of structural and regulatory proteins in apoptosis

[19]. To determine whether PS externalization is related to

caspase-3-mediated proteolysis, we analyzed the activation

of caspase-3 in mock-, TMEM166- and Bax-transfected

cells approximately 40 h after transfection. Caspase-3-like

activity is quantitatively measured using a DEVD cleavage

assay [20]. DEVD cleavage activity in lysates of

TMEM166- or Bax-transfected 293T cells exhibited in-

creases (nearly 9 times higher in TMEM166-transfected

cells and 25 times higher in Bax-transfected cells) relative

to mock-transfected cells, as shown in Fig 6C. Because

proteolytic cleavage of specific substrates by activated

caspases is responsible for cellular dysfunction and struc-

tural destruction [21], we analyzed PARP by western blot.

Cleaved PARP was observed in TMEM166- or Bax-

transfected cells, but was rarely detected in mock cells

(Fig. 6D). These findings suggest that the caspase cascade

is activated during TMEM166-induced cell death, but the

level of activation is lower than that in Bax-transfected

cells. We therefore wondered whether inhibition of casp-

ases with the pan-caspase inhibitor, Z-VAD-fmk, would

prevent cell death. The data obtained from flow cytometry

showed that Z-VAD-fmk partially reduced the cell mor-

tality (approximately 50% less) (Fig. 6E). These results

indicate that the caspase cascade is involved in TMEM166-

induced apoptosis, but it is not the only pathway triggering

cell death.

Overexpression of TMEM166 disrupts mitochondrial

transmembrane potential

An overwhelming amount of evidence shows that mito-

chondria play central roles in the regulation of both

apoptotic and non-apoptotic cell death [9, 22, 23]. Among

the sequence of events taking place in mitochondria during

the course of cell death, loss of the mitochondrial mem-

brane potential (DWm) appears to be a major event closely

associated with cell death [24]. At 36 h after transfection,

we examined the involvement of mitochondria in

TMEM166-induced cell death by monitoring DWm. Loss

of mitochondrial membrane potential was observed in both

TMEM166- or Bax-transfected 293T cells (Fig. 7), sug-

Fig. 4 TMEM166 induces cell

death both in 293T and HeLa

cells. (A) Light microscopy

images of transfected 293T

cells. Classic characteristics of

apoptosis, including cell

shrinkage, marked rounding,

and nuclear condensation were

observed in TMEM166- and

Bax-transfected cells 40 h post-

transfection. (B) Fluorescence

microscopy of transfected 293T

cells. Cells were stained with

calcein-AM and EthD-1 and

observed under an inverted

fluorescence microscope 40 h

post-transfection. Separate

digital images of red (dead) and

green (living) cells were taken

and overlaid in this composite

image. All pictures were taken

at 10· magnification. (C)

Colony formation assay was

used to determine the long-term

survival of TMEM166 on HeLa

cells. Data are presented as

means ± standard deviation

(s.d.) from triplicate plates

Apoptosis (2007) 12:1489–1502 1495

123

gesting that mitochondria are involved in the regulation of

TMEM166-induced cell death.

Accumulation of autophagic vacuoles precedes

apoptotic cell death

Our findings described above indicate that the accumula-

tion of autophagic vacuoles induced by TMEM166 over-

expression in HeLa cells was followed by the appearance

of apoptotic characteristics. Kinetic experiments revealed

that the punctate distribution of GFP-LC3 appeared in

TMEM166-transfected HeLa cells before nuclear apoptosis

(Fig. 8A, B). Therefore, we used transmission electron

microscopy to determine whether TMEM166-transfected

HeLa cells underwent both type I and type II cell death.

The observed morphological characteristics suggested that

TMEM166 induced both autophagic and apoptotic cell

death at 40 h post-transfection. As shown in Fig. 8C,

Fig. 5 TMEM166 induces autophagy of HeLa cells. (A) Represen-

tative electron microscopic images obtained from transfected HeLa

cells. Extensive cytoplasmic vacuolization was seen in TMEM166-

transfected HeLa cells (b, c). The high magnification image showed

that the autophagic vacuoles contained electron dense material and

degraded subcellular organelles (c). A micrograph of empty vector-

transfected HeLa cells provided the normal cell control (a). N,

nucleus; AV, autophagic vacuole; M, mitochondria. Scale bars, 2 lM

(a,b), 1 lM (c). (B) MDC staining and (C) GFP-LC3 localization.

Cells transfected with GFP-LC3 and an empty vector were subjected

to nutrient starvation for 3 h. HeLa cells transfected with TMEM166

or nutrient-starved cells showed punctate distribution of MDC and

GFP-LC3, in contrast to the diffuse pattern in mock and TMEM166-

DTM transfected cells. Representative fluorescence microphotographs

are shown in B and C, and the frequency of cells exhibiting the

accumulation of GFP-LC3 in vacuoles was quantified (means ± sd.,

n = 3) in D. (E) Immunoblot analyses of accumulating LC3-II protein

in mock, TMEM166-transfected and 3 h nutrient-starved cells

1496 Apoptosis (2007) 12:1489–1502

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Fig. 6 TMEM166 induces

apoptosis in both 293T and

HeLa cells. 293T and HeLa

cells were transiently

transfected with the indicated

plasmids. (A) Nuclei were

stained with DAPI and

examined under a fluorescence

microscope. Transfected cells

(B, F) or transfected cells

treated with z-VAD-fmk (E)

were harvested and stained with

Annexin V/PI. The percentage

of Annexin V and/or PI positive

are shown. (C) DEVD activity

assay. Cell lysates were

extracted after 40 h

transfection. Data are presented

as means ± sd. (n = 3). (D)

Western blot analysis the

cleavage of PARP in 293T cells

at 40 h post-transfection

Apoptosis (2007) 12:1489–1502 1497

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TMEM166-transfected HeLa cells revealed typical apop-

totic features, including condensed chromatin with mar-

gination at the nuclear periphery (Fig. 8C, c), similar to

Bax-transfected cells (Fig. 8C, b). Empty vector trans-

fected cells had normal cell phenotype (Fig. 8C, a).

Meanwhile, typical autophagic features were also observed

in TMEM166-transfected cells (Fig. 8C, d-f). These char-

acteristic changes included distorted nuclei (Fig. 8C, d),

extensive autophagic vacuoles in the cytoplasm, and

membrane-bound compartments containing subcellular

organelles, such as mitochondria (Fig. 8C, e). In some

cells, most mitochondria have already been degraded

(Fig. 8C, f) and the destruction of these essential cellular

structures probably leads to the point of no return for

autophagic cell death.

The N-terminal TM domain of TMEM166 is critical for

its specific localization and biological function

TMEM166 contains a putative TM domain near the N-

terminus (amino acid residues 34–56). To test whether

this domain is required for localization to the lysosome

and ER, a truncated mutant of TMEM166 was generated

in which 60 N-terminal amino acids were deleted. The

expression vectors pcDB/TMEM166-DTM and

TMEM166-DTM-GFP were constructed (Fig. 1D) and

used to evaluate the subcellular localization and func-

tions of truncated TMEM166. The TMEM166 mutant

failed to localize to the lysosome and ER, and displayed

a diffuse cytoplasmic expression pattern (Fig. 3B), indi-

cating that the N-terminal TM domain of TMEM166 is

required for its specific localization. Next, we transfected

the TMEM166-DTM plasmid into HeLa cells and ana-

lyzed both the GFP-LC3 distribution and PS external-

ization. Compared to the wild-type TMEM166, the TM

deletion mutant failed to induce autophagic and apoptotic

changes in HeLa cells (Figs. 5C and 6F). These findings

suggest that the putative N-terminal TM domain is

necessary for the localization and biological activity of

TMEM166.

Suppression of TMEM166 expression protects cells

from autophagy

To further determine the role of TMEM166 in autophagy

under physiological conditions, siRNA was designed to

silence the expression of TMEM166 in HeLa cells. Non-

silencing siRNA or siRNA against TMEM166 (si-

TMEM166) was transfected into HeLa cells alone or

combined with the TMEM166-GFP vector. At 48 h after

transfection, TMEM166 mRNA and protein levels were

significantly decreased in cells transfected with si-

TMEM166, as assessed by quantitative real-time RT-PCR

(Fig. 9A, B) and fluorescence microscopy (Fig. 9C). Next,

we evaluated whether si-TMEM166 treatment could inhibit

starvation-induced autophagy. HeLa cells transfected with

non-silencing siRNA or si-TMEM166 were induced by

starvation for 2 h to promote autophagy. As illustrated in

Fig. 9D, si-TMEM166 inhibited the starvation-increased

LC3-II levels. Confocal microscopy shows decreased MDC

staining in si-TMEM166-transfected HeLa cells, both in

number and fluorescence intensity, compared with non-

silencing RNAi-transfected cells (Fig. 9E). These data

suggest that TMEM166 might play a key role in the reg-

ulation of cell autophagy.

Discussion

In the current study, we cloned the entire ORF of a human

gene FLJ13391 and named it TMEM166 (transmembrane

protein 166). Sequence analysis reveals that TMEM166 is

conserved in human, chimpanzee, rat, mouse and dog,

indicating that it may have important functions in verte-

brate animals. TMEM166 shares no obvious homology to

any known gene or protein in the GenBank databases.

Bioinformatic analysis suggests that TMEM166 has a

conserved TM domain near the N-terminus of the protein.

Confocal microscopy analysis showed that TMEM166

localized to the lysosome and endoplasmic reticulum (ER),

while the TM domain deletion mutant, TMEM166-DTM,

Fig. 7 Mitochondrial changes in TMEM166-transfected 293T cells.

Transfected cells were subjected to mitochondrial transmembrane

potential assessment at 36 h post-transfection using DiOC6(3)

potentiometric dye. The proportion of cells with reduced DiOC6(3)

staining are shown. Experiments were performed in triplicate with

similar results

1498 Apoptosis (2007) 12:1489–1502

123

lost its specific localization and displayed a diffuse

expression pattern. Based on the characteristics and func-

tion of the TM domain, we postulate that TMEM166 may

act as an adaptor protein, recruiting or binding other spe-

cific proteins in the lysosome or ER.

Autophagy is an evolutionarily-conserved process that

was first defined genetically in yeast [25]. TMEM166 is

also evolutionarily conserved with orthologs in mammalian

species. In this study, we provide evidences that

TMEM166 may be an important regulator of autophagy.

The typical morphological characteristic of autophagy,

(autophagic vacuolization in cytoplasm) was confirmed in

TMEM166-overexpressing HeLa cells (Fig. 5A). Other

phenotypic markers of autophagy including the increase of

MDC staining, punctate distribution of GFP-LC3, and the

increased LC3-II/LC3-I proportion were also observed

Fig. 8 Accumulation of

autophagic vacuoles precedes

apoptotic cell death. (A, B)

Distribution of GFP-LC3-,

mock- and TMEM166-

transfected HeLa cells were

observed for the indicated

times. Representative cells are

shown in panel A, and the

frequency (means ± sd., n = 5)

of cells with clear vacuolar

distribution of GFP-LC3 (GFP-

LC3Vac) or apoptotic nuclei

(arrows) was scored. (C)

Electron microscopy of

TMEM166-induced cell death.

(a) The micrographs of empty

vector-transfected HeLa cells

provided normal control,

whereas Bax-(b) or TMEM166-

(c) transfected HeLa cells

displayed typical characteristics

of apoptosis, including

chromatin condensation and

margination. TMEM166-

transfected cells also show

typical characteristics of

autophagic cell death such as

distorted nuclei and cytoplasmic

autophagic vacuoles (d).

Higher-magnification images of

the vacuolization suggested that

some vacuoles contained

cellular organelles, such as

mitochondria (e). Furthermore,

in some cells, most

mitochondria have been

degraded (f)

Apoptosis (2007) 12:1489–1502 1499

123

(Fig. 5B–E). Secondly, suppression of TMEM166 expres-

sion could inhibit starvation-induced autophagy, implying

that TMEM166 might be involved in autophagic regulation

under physiological conditions (Fig. 9D, E). Thirdly,

compared to the wild-type TMEM166, the TM deletion

mutant failed to induce cell autophagy and apoptosis,

indicating that membrane localization of TMEM166 is

required for its function. Thus, we speculate that

TMEM166 may directly participate in the formation of the

autophagosome in the autophagic process.

An interesting observation from our investigation is the

ultimate result of cell death triggered by TMEM166. Our

kinetic studies demonstrated that TMEM166-overexpres-

sed HeLa cells began to show autophagic characteristics

about 20 h after TMEM166-transfection, and remained in

the autophagic state, whereas obvious nuclear changes

associated with apoptosis were observed at a later stage,

(i.e. about 36 h later). Other hallmarks of apoptosis were

also detected in this period, including chromatin conden-

sation and rupture, PS externalization, activation of cas-

pase-3, cleavage of PARP, as well as the depolarization of

the mitochondrial membrane potential. Ultrastructural

study using TEM after 40 h transfection, revealed that

TMEM166 expression was correlated to autophagic and

apoptotic cell death in HeLa cells (Fig. 8C). Taken to-

gether with previous reports, the present results indicate

that TMEM166 is involved in the induction of autophagy.

Autophagy may have an adaptive function to degrade

nonessential and dysfunctional organelles and proteins to

produce amino acids, which are reused during functional

recovery and thus contribute to the continued survival of

cells. However, when TMEM166 is overexpressed or

undergoes sustained cellular expression, autophagy may

not be able to reverse the damage and may contribute to

Fig. 9 Silencing of TMEM166 inhibits starvation-induced auto-

phagy. (A) RT-PCR results for TMEM166 mRNA expression. Si-

TMEM166 showed strong inhibitory effect for TMEM166 mRNA

expression, whereas TMEM166 mRNA expression was not inhibited

by non-silencing siRNA compared with the wild type HeLa cells

(control). (B) The expression level of TMEM166 detected by real-

time PCR. The expression level in wild type HeLa cells (control) was

treated as 1. (C) Effects of si-TMEM166 on the expression of

TMEM166-GFP fusion protein. HeLa cells were transfected with

TMEM166-GFP alone or cotransfected with TMEM166-GFP and

non-silencing siRNA, si-TMEM166, respectively. At 24 h after

transfection, cells were observed with fluorescence microscopy. Si-

TMEM166 shows much fainter fluorescence than cells transfected

with non-silencing siRNA. (D) Immunoblot analysis of LC3-II level

after si-TMEM166 transfection. Cells were transfected with non-

silencing siRNA and si-TMEM166. At 48 h after transfection, cells

were starved for 2 h. Western blot indicated that si-TMEM166

inhibited the increased LC3-II level induced by starvation. Similar

results were obtained from three independent experiments. (E) MDC

staining. Confocal microscopy shows decreased punctate distribution

of MDC staining in si-TMEM166-transfected HeLa cells, both in

number and fluorescence intensity, compared with non-silencing

siRNA-transfected cells

1500 Apoptosis (2007) 12:1489–1502

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severe cell dysfunction resulting in type II programmed

cell death with or without type I programmed cell death.

Increasing evidence suggests that complex interrela-

tionships exist between the autophagic and the apoptotic

cell death pathways [26]. Several regulators of apoptosis

also play a role in the process of autophagy, including

DRAM [18], Ca2+/calmodulin-regulated death kinases

DAPk and DRP-1 [27, 28], PTEN [29, 30], steroid-induc-

ible gene E93 [31], signaling molecules Akt/PKB and

mTOR [32], Bcl-2 family proteins [33], TRAIL [34], and

beclin 1 [10, 35]. Previously, it has been shown that genetic

inhibition of autophagy can activate apoptotic death in

nutrient-starved mammalian cells [9], suggesting that

autophagy activation can function to prevent apoptosis.

Conversely, it has also been suggested that autophagy

activation may lead to apoptosis. Now Espert et al. dem-

onstrate that siRNAs specific for both beclin 1 and ATG7

can completely block apoptosis triggered by CXCR4

engagement by the HIV envelope glycoprotein [36]. This

result suggested that autophagy acts upstream of signaling

transduction events leading to apoptotic cell death. Our

results also suggest that TMEM166-induced autophagy

occurs earlier than apoptosis. Lysosomes are important

mediators of PCD [37–39]. Due to the lysosomal locali-

zation of TMEM166, we postulate that TMEM166 may

regulate the lysosomal-mitochondrial pathway of pro-

grammed cell death. TMEM166 may mediate lysosomal

membrane permeabilization (LMP) resulting in the release

of proteolytic enzymes such as cathepsins that may damage

mitochondrial membrane potential (Fig. 7) triggering a

series of autophagic and apoptotic events. Nevertheless, the

molecular mechanism through which TMEM166 can acti-

vate cell autophagy and apoptosis has remained elusive.

Further research on the mechanism of TMEM166-induced

cell death and molecules that interact with TMEM166 may

provide new information about signal pathways existing

between the processes of apoptotic and autophagic-pro-

grammed cell death.

Conclusion

We have described the preliminary functions of

TMEM166, a novel lysosome and endoplasmic reticulum

associated protein, which regulates the autophagic pathway

and contributes to autophagic and apoptotic cell death. Our

findings provide more evidence that there exists an

important cross talk between autophagy and apoptosis.

More importantly, the involvement of TMEM166 in both

type I and type II programmed cell death implies that it

may have an essential role in proper cell growth and cell

death, and might have potential applications in the treat-

ment and diagnosis of disease.

Acknowledgements We thank Dr. Zhenyu Yue for providing the

GFP-LC3 plasmid. We thank Dr. Zhendong Zhao for the gift of the

recombinant LC3 protein. Dr. Lan Yuan is acknowledged for help

with the confocal laser scanning microscope. We are also grateful to

Dr. Zhao for Northern blot assistance. This work was supported by

grants from the National High Technology Research and Develop-

ment Program of China (2002BA711A01)

References

1. Gozuacik D, Kimchi A (2004) Autophagy as a cell death and

tumor suppressor mechanism. Oncogene 23:2891–2906

2. Lum JJ, DeBerardinis R, Thompson CB (2005) Autophagy in

metazoans: cell survival in the land of plenty. Nat Rev Mol Cell

Biol 6:439–448

3. Mizushima N (2005) The pleiotropic role of autophagy: from

protein metabolism to bactericide. Cell Death Differ 12 (Suppl

2):1535–1541

4. Lockshin RA, Zakeri Z (2004) Apoptosis, autophagy, and more.

Int J Biochem Cell Biol 36:2405–2419

5. Bursch W (2001) The autophagosomal-lysosomal compartment

in programmed cell death. Cell Death Differ 8:569–581

6. Edinger AL, Thompson CB (2004) Death by design: apoptosis,

necrosis and autophagy. Curr Opin Cell Biol 16:663–669

7. Levine B (2005) Eating oneself and uninvited guests: autophagy-

related pathways in cellular defense. Cell 120:159–162

8. Scott RC, Juhasz G, Neufeld TP (2007) Direct induction of

autophagy by Atg1 inhibits cell growth and induces apoptotic cell

death. Curr Biol 17:1–11

9. Boya P, Gonzalez-Polo RA, Casares N et al (2005) Inhibition

of macroautophagy triggers apoptosis. Mol Cell Biol 25:1025–

1040

10. Furuya D, Tsuji N, Yagihashi A, Watanabe N (2005) Beclin 1

augmented cis-diamminedichloroplatinum induced apoptosis via

enhancing caspase-9 activity. Exp Cell Res 307:26–40

11. Wang L, Gao X, Gao P et al (2006) Cell-based screening and

validation of human novel genes associated with cell viability. J

Biomol Screen 11:369–376

12. Wang Y, Li X, Wang L et al (2004) An alternative form of

paraptosis-like cell death, triggered by TAJ/TROY and enhanced

by PDCD5 overexpression. J Cell Sci 117:1525–1532

13. Sonnhammer EL, von Heijne G, Krogh A (1998) A hidden

Markov model for predicting transmembrane helices in protein

sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182

14. Mitchener JS, Shelburne JD, Bradford WD, Hawkins HK (1976)

Cellular autophagocytosis induced by deprivation of serum and

amino acids in HeLa cells. Am J Pathol 83:485–491

15. Simonsen A, Birkeland HC, Gillooly DJ et al (2004) Alfy, a

novel FYVE-domain-containing protein associated with protein

granules and autophagic membranes. J Cell Sci 117:4239–4251

16. Munafo DB, Colombo MI (2001) A novel assay to study auto-

phagy: regulation of autophagosome vacuole size by amino acid

deprivation. J Cell Sci 114:3619–3629

17. Fadok VA, Bratton DL, Frasch SC, Warner ML, Henson PM

(1998) The role of phosphatidylserine in recognition of apoptotic

cells by phagocytes. Cell Death Differ 5:551–562

18. Crighton D, Wilkinson S, O’Prey J et al (2006) DRAM, a p53-

induced modulator of autophagy, is critical for apoptosis. Cell

126:121–134

19. Salvesen GS, Dixit VM (1997) Caspases: intracellular signaling

by proteolysis. Cell 91:443–446

20. Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn

JH (2002) Single-cell fluorescence resonance energy transfer

analysis demonstrates that caspase activation during apoptosis is

Apoptosis (2007) 12:1489–1502 1501

123

a rapid process. Role of caspase-3. J Biol Chem 277:24506–

24514

21. Thornberry NA, Lazebnik Y (1998) Caspases: enemies within.

Science 281:1312–1316

22. Green DR, Reed JC (1998) Mitochondria and apoptosis. Science

281:1309–1312

23. Sperandio S, de Belle I, Bredesen DE (2000) An alternative,

nonapoptotic form of programmed cell death. Proc Natl Acad Sci

U S A 97:14376–14381

24. Susin SA, Zamzami N, Castedo M et al (1996) Bcl-2 inhibits the

mitochondrial release of an apoptogenic protease. J Exp Med

184:1331–1341

25. Klionsky DJ, Emr SD (2000) Autophagy as a regulated pathway

of cellular degradation. Science 290:1717–1721

26. Levine B, Yuan J (2005) Autophagy in cell death: an innocent

convict? J Clin Invest 115:2679–2688

27. Shohat G, Shani G, Eisenstein M, Kimchi A (2002) The DAP-

kinase family of proteins: study of a novel group of calcium-

regulated death-promoting kinases. Biochim Biophys Acta

1600:45–50

28. Inbal B, Bialik S, Sabanay I, Shani G, Kimchi A (2002) DAP

kinase and DRP-1 mediate membrane blebbing and the formation

of autophagic vesicles during programmed cell death. J Cell Biol

157:455–468

29. Yamada KM, Araki M (2001) Tumor suppressor PTEN: modu-

lator of cell signaling, growth, migration and apoptosis. J Cell Sci

114:2375–2382

30. Arico S, Petiot A, Bauvy C et al (2001) The tumor suppressor

PTEN positively regulates macroautophagy by inhibiting the

phosphatidylinositol 3-kinase/protein kinase B pathway. J Biol

Chem 276:35243–35246

31. Thummel CS (2001) Steroid-triggered death by autophagy. Bi-

oessays 23:677–682

32. Paglin S, Lee NY, Nakar C et al (2005) Rapamycin-sensitive

pathway regulates mitochondrial membrane potential, autophagy,

and survival in irradiated MCF-7 cells. Cancer Res 65:11061–

11070

33. Pattingre S, Levine B (2006) Bcl-2 inhibition of autophagy: a

new route to cancer? Cancer Res 66:2885–2888

34. Mills KR, Reginato M, Debnath J, Queenan B, Brugge JS (2004)

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)

is required for induction of autophagy during lumen formation

in vitro. Proc Natl Acad Sci USA 101:3438–3443

35. Zeng X, Overmeyer JH, Maltese WA (2006) Functional speci-

ficity of the mammalian Beclin-Vps34 PI 3-kinase complex in

macroautophagy versus endocytosis and lysosomal enzyme traf-

ficking. J Cell Sci 119:259–270

36. Espert L, Denizot M, Grimaldi M et al (2006) Autophagy is

involved in T cell death after binding of HIV-1 envelope proteins

to CXCR4. J Clin Invest 116:2161–2172

37. Chwieralski CE, Welte T, Buhling F (2006) Cathepsin-regulated

apoptosis. Apoptosis 11:143–149

38. Persson HL, Kurz T, Eaton JW, Brunk UT (2005) Radiation-

induced cell death: importance of lysosomal destabilization.

Biochem J 389:877–884

39. Boya P, Andreau K, Poncet D et al (2003) Lysosomal membrane

permeabilization induces cell death in a mitochondrion-depen-

dent fashion. J Exp Med 197:1323–1334

1502 Apoptosis (2007) 12:1489–1502

123