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Innate immunity signalling and membrane trafficking
Tomohiko Taguchi1,2 and Kojiro Mukai1
The mammalian innate immune system serves as the front line
of the host to eliminate invading pathogens. The receptors that
sense invading pathogens or the pathogen-associated
molecules localized at various membrane compartments that
include the plasma membrane, endosomes, and the
endoplasmic reticulum. Intriguingly, growing evidence
indicates that the sites of pathogen detection do not always
represent the site where innate immune signal is triggered.
Rather, pathogen detection often induces translocation of the
receptors by membrane trafficking. Furthermore, dysregulated
membrane trafficking of the receptors renders the host
susceptible to infection or prone to autoinflammatory diseases.
These findings underscore the critical role of membrane
trafficking in the innate immunity. In this review, we highlight
emerging issues regarding PRRs and membrane trafficking,
with the particular focus on STING and TLR4, the activity of
which is tightly regulated by membrane trafficking.
Addresses
1
Laboratory of Organelle Pathophysiology, Department of Integrative
Life Sciences, Graduate School of Life Sciences, Tohoku University,
Aobayama, Aoba-ku, Sendai, Miyagi, 980-8578, Japan
2
AMED-PRIME, Japan Agency for Medical Research and Development,
1-7-1, Otemachi, Chiyoda-ku, Tokyo, 100-0004, Japan
Corresponding author: Taguchi, Tomohiko (tomohiko.taguchi.
b8@tohoku.ac.jp)
Current Opinion in Cell Biology 2019, 59:1–7
This review comes from a themed issue on Membrane Trafficking
Edited by Akihiko Nakano and Peter Cullen
https://doi.org/10.1016/j.ceb.2019.02.002
0955-0674/ã 2018 Elsevier Ltd. All rights reserved.
Introduction
Vertebrates have evolved biological systems to combat
invading pathogens. As the first line of host defense, the
innate immune system recognizes microbial pathogens
with host germline-encoded pattern recognition receptors
(PRRs) that bind unique pathogen-associated molecular
patterns (PAMPs) [1,2]. Activated PRRs triggers intracel-
lular signalling cascades, which lead to transcriptional
expression of proinflammatory cytokines, type I interfer-
ons, and other antiviral proteins that all coordinate the
elimination of pathogens and infected cells. PRRs
include Toll-like receptors (TLRs) [3], RIG-I-like
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receptors [4], nucleotide-binding domain and leucine-
rich repeat-containing receptors (NLRs) [5], C-type lec-
tin receptors (CLRs) [6], cyclic GMP-AMP (cGAMP)
synthase [7], and stimulator of interferon genes (STING)
[8].
Several PRRs, such as TLRs, CLRs, and STING, are
transmembrane proteins and distribute at various mem-
brane compartments, such as the plasma membrane
(PM), early endosomes, late endosomes, recycling endo-
somes (REs), and the endoplasmic reticulum (ER). Some
PRRs, upon binding to PAMPs, translocate to other
compartments by membrane trafficking and triggers
the innate immunity signalling there. Not surprisingly,
dysregulated membrane trafficking of PRRs often under-
lies the pathogenesis of autoinflammatory diseases. This
article is not intended as a comprehensive review of the
innate immunity signalling and PRRs. Rather, we aim to
highlight recent publications and emerging issues regard-
ing PRRs and membrane trafficking, with the particular
focus on STING and TLR4, the activity of which is
revealed to be tightly coupled with membrane trafficking.
Exocytic trafficking of STING from the ER
DNA is normally present in the nucleus and mitochondria
of eukaryotic cells, and the presence of DNA in aberrant
locations, such as cytosol, is believed to trigger immune
response [9]. Indeed, recent studies have identified two
central PRRs, cGAMP synthase (cGAS) [7] and STING
[8], which are required for the immune response against
cytosolic DNA. DNA viruses and bacteria can release
their genomic dsDNA to the host cytoplasm during
infection. The cytosolic dsDNA activates cGAS, leading
to the generation of cGAMP, a cyclic dinucleotide
(CDN), from GTP and ATP [10]. cGAMP then activates
STING to trigger the type I interferon responses [11].
Other CDNs including cyclic di-GMP [12] and cyclic di-
AMP [13] are secreted during intracellular bacterial infec-
tion, and they directly activate STING [11,14].
STING [8], also known as MITA [15], ERIS [16], MPYS
[17], or TMEM173, is an ER-localized transmembrane
protein (Figure 1). Mechanistically, STING serves as a
scaffold for TBK1 kinase and the transcription factor
interferon regulatory factor 3 (IRF3) [18,19]. IRF3 is
phosphorylated by TBK1, after which it translocates into
the nucleus to stimulate the transcription of type I inter-
ferons such as interferon b [20]. Interestingly, after the
binding of STING to CDNs, STING immediately trans-
locates from the ER to perinuclear compartments that
include the Golgi, endosomes, and autophagy-related
compartments [21,22]. The exocytic membrane
Current Opinion in Cell Biology 2019, 59:1–7
2 Membrane Trafficking

Figure 1

DNA virus Cell membrane

ATP Genomic DNA

+ cGAS
GTP Mitochondrial DNA
cGAMP
(cyclic GMP-AMP)
P P
Bacteria IRF3 TBK1
STING STING STING STING
CDNs Endoplasmic
(cyclic dinucleotides) reticulum
Palmitoylation
Nucleus
Cys88/91

P IFNβ Golgi Recycling endosomes Lysosomes

IRF3

Antiviral response
Autoinflammatory diseases

Current Opinion in Cell Biology

Regulation of the cGAS-STING signalling pathway by membrane trafficking.

The cytosolic dsDNA derived from DNA virus, host genome, or mitochondria activates cGAS, leading to the generation of cGAMP. cGAMP directly
binds to STING and induces translocation of STING from the ER to the Golgi. STING undergoes palmitoylation at the Golgi which is essential for
the activation of STING. After exit from the Golgi, STING further moves to REs and then to the p62-positive compartments/lysosomes.

trafficking of STING may be essential for the STING-
dependent signalling events, because treatment with
brefeldin A (BFA) or expression of Shigella effector
IpaJ, which blocks ER-to-Golgi traffic, abolished
phosphorylation of IRF3 and induction of interferon b
[21,23

,24,25]. More direct evidence was provided by the
recent observation that knockdown of Sar1, a small
GTPase that regulates coat protein complex II (COP-
II)-mediated ER-to-Golgi traffic, suppressed phosphory-
lation of IRF3 [26
].
Structural insight of STING activation
Several crystal structures of the STING cytoplasmic
domain and its complex with CDNs were elucidated.
Three independent crystal structures of STING bound to
cyclic di-GMP did not reveal any major structural changes
following c-di-GMP binding [27–29]. However, these
studies used a rare human STING variant, R232H, which
is partially defective in stimulating type I interferons.
Other structural studies, which used wild-type STING
(R232), revealed a conformational change induced by
c-di-GMP binding [30,31] or cGAMP binding [32,33].
In brief, cGAMP binding induced the STING dimer to
form a closed conformation, resulting in a deeper cGAMP
binding pocket that is covered with a b sheet lid.
These structural analyses provided a detailed picture of
how STING recognizes CDNs. However, it is still not
clear how the conformational change associated with the
CDN binding leads to the translocation of STING from
the ER to the Golgi with the COP-II complex, and
eventually to the activation of the STING-TBK1-IRF3
signalling pathway. The very recent study has even
casted doubt on the idea that the closed conformation
of STING dimer is critical for activation [34
]. In the
study, a linked amidobenzimidazoles-based compound
(diABZI-compound 2) was developed. This novel non-
nucleotide compound efficiently activated STING with
maintaining an open STING dimer conformation. So far,
only the conformation of the C-terminal cytoplasmic
domain of STING was analyzed. Structural elucidation
of full-length STING, which includes N-terminal

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 Innate immunity signalling and membrane trafficking Taguchi and Mukai 3

Figure 2

Transmembrane domain Cyclic dinucleotide-Binding Domain C-Terminal Tail

1 21 41 47 67 87 106 116 136 151 341 379

C88/C91 human: V147L/M human: N154S human: V155M human: C206Y human: R281Q human: R284G/S
(Palmitoylation) (mouse: V146L/M) (mouse: N153S) (mouse: V154M) (mouse: C205Y) (mouse: R280Q) (mouse: R283G/S)
Y. Liu et al., N Engl J Med 371, 507 (2014). I. Melki et al., J Allergy Clin Immunol 140, 543 (2017).
J. Munoz et al., JAMA Dermatol 151, 872 (2015). H. Konno et al., Cell Rep 23, 1112 (2018).

cGAMP

90°

cytosol

lumen
Current Opinion in Cell Biology

Schematic overview of STING and localization of mutations that cause SAVI.

V147/N154/V155 (shown in red), but not C206/R281/R284 (shown in blue), lie in the domain that is involved in STING dimerization.

transmembrane domains, will help understand the molec-
ular mechanism of STING activation with CDNs.
STING-associated disease
Gain-of-function mutations in STING have been asso-
ciated with an autoinflammatory syndrome termed
STING-associated vasculopathy with onset in infancy
(SAVI) (Figure 2) [35–38]. SAVI is characterized by
early-onset systemic inflammation with fever, a severe
skin vasculopathy that leads to extensive tissue loss in
some cases, and interstitial lung disease resulting in
pulmonary fibrosis and end-stage respiratory failure.
The SAVI mutations (V147L, N154S, V155M, C206Y,
R281Q, and R284G) are shown to be associated with
constitutive activation of STING without DNA and
cGAMP stimulation. These SAVI variants did not local-
ize at the ER, instead localized at the perinuclear com-
partments [26
]. The suppression of ER-to-Golgi traffic
abolished TBK1 binding to the SAVI variants [26
],
suggesting that even the constitutively active SAVI
variants required the exocytic membrane traffic from
the ER for their activity. In contrast to wild-type
STING, how SAVI variants can get access to the exo-
cytic membrane traffic without cGAMP stimulation is
not understood.
Interestingly, 4 variants (C206Y, R281Q, R284G, and
R284S) [38,39] that were recently identified did not lie
in the domain that is involved in STING dimerization. A
recent survey of STING mutations in the Catalogue of
Somatic Mutations in Cancer database led to the demon-
stration that R284M mutation was able to hyperactivate
STING [40]. Alternative mutation of R284K or R284T
also hyperactivates STING. Thus, multiple types of
amino acid substitution at 284 confer a gain-of-function
mutation, indicating a high degree of specificity for the
function of R284. Given that R284 is exposed at the
surface of STING, R284 may be involved in protein
interactions by which the ER localization of STING is
maintained.

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4 Membrane Trafficking
STING activation at the Golgi
As described above, the post-ER membrane compart-
ments are required for activation of STING. Recent
evidence suggested that palmitoylation of STING at
the Golgi is essential for activation of STING [23

].
Treatment with protein palmitoylation inhibitor 2-bro-
mopalmitate (2-BP) abolished STING-dependent type I
interferon response. Mutation of two membrane-proximal
Cys residues (C88/91) suppressed palmitoylation, and this
STING mutant could not induce STING-dependent
host defense genes. The response elicited by the SAVI
variants was also effectively inhibited by 2-BP or an
introduction of C88/91S mutation. The importance of
C88/91 in STING activation was further emphasized
by the recent findings of identification of STING inhi-
bitors that target C88/91 of STING [41

,42

].
Protein palmitoylation has been implicated in the clus-
tering of a number of proteins [43] such as H-ras and Fas
into lipid rafts (specific membrane domains enriched in
cholesterol and sphingomyelin (SM)). Cholesterol is sug-
gested to be enriched at the TGN, and cholesterol
together with SM generated by SM synthase 1 are
thought to form lipid rafts at the TGN [44]. Treatment
of cells with C6-ceramide disrupted lipid rafts at the Golgi
by generating short-chain SM that disturbs the lipid order
[44]. C6-ceramide inhibited the STING activity without
affecting the translocation of STING to the Golgi [23

].
Therefore, STING palmitoylation may allow clustering
of STING at the lipid rafts of the TGN, which then
facilitates the recruitment and interaction of downstream
signalling factors such as TBK1 and IRF3 on STING.
Post-Golgi trafficking of STING
After exit from the Golgi, STING moved to REs and then
to the p62-positive compartments/lysosomes [22,23

,45].
STING appears to be degraded eventually at lysosomes
because chloroquine or bafilomycin A1, which interferes
with lysosomal functions, prevented STING degradation
[24,46]. Bafilomycin A1 treatment enhanced STING-
dependent host defense genes [46], suggesting that deg-
radation of STING at lysosomes is required to terminate
the STING signalling. The palmitoylation of STING at
the Golgi was not required for the degradation process,
because the palmitoylation-deficient STING mutant
(C88/91S) underwent degradation like wild-type STING
[23

]. The exact molecular mechanism underlying
STING trafficking from endosomes to lysosomes is not
clear. There have been controversies regarding the con-
tribution of the autophagic process to degradation of
STING [45–47].
Membrane trafficking of TLR4
TLRs are type I transmembrane proteins that are com-
posed of three distinct structural domains: a leucine-rich
repeat containing ectodomain involved in PAMP binding,
a transmembrane domain, and a cytosolic domain that
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binds downstream adaptor proteins. Much of our current
knowledge of PAMP-induced PRR trafficking has been
derived from the study of TLR4. TLR4 binds extracel-
lular lipopolysaccharide (LPS), the major outer mem-
brane component of Gram-negative bacteria. After
binding to LPS at the PM, TLR4 interacts with the
PtdIns(4,5)P2-effector TIRAP and the associated
MyD88, which results in induction of the secretion of
proinflammatory cytokines [48]. After translocation to
early endosomes/phagosomes, the complex disassembles
most likely due to the low concentration of PtdIns(4,5)
P2. This allows other adaptors TRAM and TRIF to bind
to TLR4, which triggers secretion of the type I interfer-
ons [49].
A role of Rab11-positive REs in TLR4 signalling has
been demonstrated [50,51]. Endogenous TLR4 localizes
mostly in Rab11-positive REs in unstimulated human
monocytes. By stimulation with Escherichia coli, a Gram-
negative bacterium, TLR4 was transported from REs to
E. coli-positive phagosomes. Knockdown of Rab11
resulted in emptying of TLR4 in REs, reduction of
TLR4 around phagosomes, and inhibition of E. coli-
induced IRF3 activation. Signalling lymphocytic activa-
tion molecule family 1 (SLAMF1) is an Ig-like receptor
and a costimulatory molecule that initiates signal trans-
duction networks in a variety of immune cells. In resting
macrophages, SLAMF1 was localized to REs, but upon
addition of E. coli, it was trafficked together with TRAM
from REs to E. coli-positive phagosomes in a Rab11-
dependent manner. Thus, the membrane traffic from
REs helps fuel phagosomes with TLR4 and its effector
protein for maximal TLR4 signalling (Figure 3). Rab11
also functioned in maintaining proper subcellular locali-
zation of endosomal TLR9 [52].
TLR4 degradation at lysosomes
VPS33B is the homolog of yeast class C vacuolar protein
sorting (vps) protein Vps33p. Class C vps proteins func-
tion in at least two multiprotein complexes, class C core
vacuole/endosome tethering (CORVET) and homotypic
fusion and vacuole protein sorting (HOPS), which inter-
act with yeast orthologs of Rab5 and Rab7 GTPases and
are crucial for several steps in vesicular trafficking path-
ways leading to lysosomes. In a recent study, Drosophila
and mammalian Vps33B proteins were shown to play
critical roles in the degradation of TLR4 after LPS
stimulation [53
]. TLR4 accumulated at Rab5-positive,
Rab7-positive, or Rab11-positive endosomes in Vps33B-
silenced macrophages. The prolonged residency of TLR4
in these endosomes paralleled well with the enhanced
expression of proinflammatory cytokines. Lack of Vps33B
had no effect on trafficking of non-microbial cargo.
Arthrogryposis, renal dysfunction, and cholestasis syn-
drome (ARC) is a multisystem disorder associated with
abnormalities in polarized liver and kidney cells.
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 Innate immunity signalling and membrane trafficking Taguchi and Mukai 5

Figure 3

Gram-negative bacteria

LPS TLR4

PI(4,5)P2
TIRAP Phagocytosis
MyD88
Endocytosis

Rab11

TRAM Early TRAM Phagosomes Recycling

TRIF endosomes TRIF endosomes

Induction of type I interferons

Induction of proinflammatory cytokines

Current Opinion in Cell Biology

Regulation of the TLR4 signalling pathway by membrane trafficking.

Persistent infections and sepsis are recurring symptoms in
ARC patients. Germline mutations in VPS33B are found
in approximately 75% of individuals with ARC [54].
Exaggerated inflammatory responses induced by persis-
tence of PRRs in aberrant endosomal compartments may
contribute to symptoms of ARC syndrome.
PRRs trigger distinct signalling pathways, and the molec-
ular mechanism of how PRR signals are terminated.
These studies are anticipated to make broad conceptual
contributions, beyond the innate immunity field, to the
understanding of essentials of membrane traffic and cell
biology.

Concluding remarks
The last few decades have witnessed substantial progress
in the field of innate immunity since the identification of
the first mammalian TLRs in the late 1990s. Technical
advances in cell biology, biochemistry, and molecular
biology have facilitated our understanding of the regula-
tion of PRR signalling. The importance of understanding
cell biological aspects of innate immunity has been
emphasized by the fact that some autoinflammatory
and autoimmune diseases are caused by mislocalization
of PRRs.
Despite these advances, many questions remain unan-
swered regarding the regulators of membrane trafficking
of PRRs, the nature of various endosomes from which
Conflict of interest statement
Nothing declared.
Author contribution statement
Draft and funding acquisition: T.T. and K.M.; Concep-
tualization and writing: T.T.
Acknowledgements
K.M. is supported by Japan Society for the Promotion of Science (JSPS)
KAKENHI Grant JP17K15445, Mochida Memorial Foundation for Medical
and Pharmaceutical Research, and MSD Life Science Foundation, Public
Interest Incorporated Foundation. T.T. is supported by JSPS KAKENHI
Grants JP16H04782, JP15H05903, and AMED-PRIME.

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6 Membrane Trafficking
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 Innate immunity signalling and membrane trafficking Taguchi and Mukai 7
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