Download The role of protein–protein interactions in the intracellular traffic of

Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
DOI 10.1007/s00424-014-1672-2
INVITED REVIEW
The role of protein–protein interactions in the intracellular traffic
of the potassium channels TASK-1 and TASK-3
Markus Kilisch & Olga Lytovchenko &
Blanche Schwappach & Vijay Renigunta & Jürgen Daut
Received: 20 November 2014 / Revised: 10 December 2014 / Accepted: 11 December 2014 / Published online: 7 January 2015
# Springer-Verlag Berlin Heidelberg 2015
Abstract The intracellular transport of membrane proteins is
controlled by trafficking signals: Short peptide motifs that
mediate the contact with COPI, COPII or various clathrinassociated coat proteins. In addition, many membrane proteins
interact with accessory proteins that are involved in the sorting
of these proteins to different intracellular compartments. In the
K2P channels, TASK-1 and TASK-3, the influence of protein–
protein interactions on sorting decisions has been studied in
some detail. Both TASK paralogues interact with the adaptor
protein 14-3-3; TASK-1 interacts, in addition, with the adaptor
protein p11 (S100A10) and the endosomal SNARE protein
syntaxin-8. The role of these interacting proteins in controlling
the intracellular traffic of the channels and the underlying
molecular mechanisms are summarised in this review. In the
case of 14-3-3, the interacting protein masks a retention signal
in the C-terminus of the channel; in the case of p11, the
interacting protein carries a retention signal that localises the
channel to the endoplasmic reticulum; and in the case of
syntaxin-8, the interacting protein carries an endocytosis signal that complements an endocytosis signal of the channel.
These examples illustrate some of the mechanisms by which
interacting proteins may determine the itinerary of a membrane protein within a cell and suggest that the intracellular
Markus Kilisch and Olga Lytovchenko contributed equally.
This article is published as part of the special issue on K2P-channels.
V. Renigunta (*) : J. Daut (*)
Institute of Physiology and Pathophysiology, Marburg University,
35037 Marburg, Germany
e-mail: [email protected]
e-mail: [email protected]
M. Kilisch : O. Lytovchenko : B. Schwappach
Institute of Molecular Biology, Göttingen University,
37073 Göttingen, Germany
traffic of membrane proteins may be adapted to the specific
functions of that protein by multiple protein–protein
interactions.
Keywords K2P-channels . Retention signal . Intracellular
traffic . Endocytosis
Introduction
In trying to understand the function of a protein, it is necessary
to study its interaction with other proteins. Many large proteins are highly dynamic and intrinsically unstable macromolecules that have surfaces capable of interacting with other
proteins. In principle, interaction with other proteins can have
the following effects on the function of a protein: (1) The
folding and the stability of the protein can be enhanced by
protein–protein interactions (PPI); the cytoplasm and the lumen of the endoplasmic reticulum (ER), for example, contain
multiple molecular chaperones that enable or accelerate folding to the ‘native’ state of the protein [36, 37], but are not
present in their final structure. (2) The assembly of multimeric
proteins can be assisted by PPI [23, 24]. (3) The function of
the protein may require the permanent interaction with other
proteins; many proteins form homomeric or heteromeric complexes with obligatory accessory subunits. (4) The function of
the protein may be temporarily suspended by interaction with
another protein, for example, by association with inhibitory
subunits. (5) The subcellular localisation and the itinerary of
intracellular traffic of the protein may be altered by interaction
with other proteins [40]. The proteins that influence intracellular traffic share some properties with chaperones; for example, they interact only transiently and are not present in the
‘final’ or ‘native’ form of the protein. To emphasise functional
similarities between chaperones and adaptor proteins involved
in trafficking, the concept of the ‘proteostasis trafficking
1106
network’ has been developed recently [37, 40]. (6) The binding of some interacting proteins depends on prior posttranslational modification of the target protein [99], e.g., phosphorylation [32, 91, 108]; this mechanism allows PPI to be regulated, e.g., via protein kinases. (7) Finally, it should be mentioned in this context that PPI have recently been recognised
as a promising target for small-molecule drugs [1, 70, 81].
Ion channels are particularly suitable proteins for studying
PPI because their biophysical properties (indicating their conformational state) and their copy number at the cell surface
can be studied with high resolution using the patch-clamp
technique [35]. Focusing on ion channels, numerous examples
can be found where PPI are essential for their physiological
function: First, many ion channels require additional regulatory subunits to acquire their typical biophysical properties
such as gating and inactivation kinetics; this applies for sodium channels [15], potassium channels [84, 118], calcium
channels [21] and chloride channels [102]. Second, some
ion channels associate with inhibitory subunits that cause a
reduction in the current [96, 106]. Third, many ion channels
require the association with accessory proteins for their
targeting to the correct subcellular compartments or for their
efficient transport to the surface membrane.
There are several principal mechanisms by which PPI can
modulate the intracellular traffic: (1) An interacting protein
can mask a trafficking signal of the channel, for example, an
ER-localisation signal, and thus promote forward trafficking
to the surface membrane [117]. (2) An interacting protein can
carry an ER-localisation signal and thus impede forward trafficking of the channel to the surface membrane [93]. (3) An
interacting protein can carry an endocytosis signal and thus
promote internalisation of the channel [92]. (4) An interacting
protein can stabilise a trafficking-competent conformation of
the channel and in this way indirectly promote its surface
expression [120]. In this review, we will focus on the intracellular traffic of a particular subfamily of two-pore-domain
potassium channels (K2P-channels), namely the acid-sensitive
K+ channels TASK-1 and TASK-3, in which the role of PPI in
regulating intracellular has been studied in some detail, and
we will give concrete examples of the four mechanisms mentioned above.
The human genome contains 15 genes coding for two-pore
domain potassium channels (K2P-channels). The structure of
K2P-channels differs from that of the 63 other [33, 116] human
potassium channels known: K2P-channel subunits have four
transmembrane domains (M1–M4), two pore domains (P1
and P2) and two helical cap domains (C1 and C2) [13, 14,
66], as illustrated in Fig. 1a. Functional K2P-channels are
usually homo-dimers with a twofold symmetry. The inner
wall of the pore is formed by the two M2 and M4 domains.
The selectivity filter of K2P-channels is formed by the two P1
and the two P2 domains; it is very similar to that of tetrameric
potassium channels showing fourfold symmetry. The acid-
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
sensitive potassium channels TASK-1 [22], TASK-3 [45, 90]
and TASK-5 [4, 43, 44] represent one of the five subfamilies
of K2P-channels. The three TASK channels are highly homologous with >50 % identical amino acids. TASK-1 and TASK3 play an important role in the central and peripheral nervous
system, in the heart, in the adrenal gland and in many other
tissues [5, 25, 46]. The functional characteristics of the other
member of the TASK subfamily, TASK-5, are still unknown.
None of the groups investigating TASK-5 has been able to
measure TASK-5 currents, either in heterologous expression
systems or in native cells. TASK-5 is almost exclusively
expressed in neurons of the auditory system [43]. Its subcellular localisation and its possible contribution to the electrical
activity of the cells are still unclear.
One of the characteristics that distinguishes TASK-1 and
TASK-3 from other potassium channels is their pronounced
inhibition by extracellular acidification. The channels are only
moderately voltage dependent; their open probability increases with depolarisation [90]. Although the TASK-1 and
TASK-3 currents measured in expression systems are very
similar, the subcellular expression pattern and the singlechannel properties of TASK-1 and TASK-3 differ substantially. When expressed in mammalian cell lines, TASK-3 channels are predominantly localised to the surface membrane,
whereas TASK-1 channels are largely retained in intracellular
compartments [93]. Heterologous expression in Xenopus oocytes requires about 30 times higher amounts of complementary RNA (cRNA) for TASK-1 than for TASK-3. Injection of
a small quantity of cRNA (50 pg) in Xenopus oocytes gives
rise to large TASK-3 currents, but under the same conditions
no TASK-1 currents can be recorded [93], as illustrated in
Fig. 1b. Injecting the same amount of cRNA coding for a
chimeric TASK-1/TASK-3 construct in which the C-terminus
of TASK-3 was attached to the M4 domain of TASK-1 (T3/T1
construct) gives rise to nearly the same currents as injecting
cRNA for TASK-3. Similar currents are also found with the
reverse construct, in which the C-terminus of TASK-3 was
attached to the M4 domain of TASK-1 (T1/T3 construct).
These observations can be explained by the fact that (1) the
single-channel conductance and the open probability of
TASK-3 are higher than those of TASK-1 [22, 73, 90], and
(2) the intracellular traffic of TASK-1 along the biosynthetic
pathway is slower (and perhaps more tightly regulated) than
that of TASK-3 [93].
In recent years, the concept has emerged that in many cell
types the density of ion channels at the surface membrane, and
thus their functional role, can be regulated posttranslationally:
The bidirectional transport between endoplasmic reticulum
(ER) and Golgi complex, i.e., ER export and retrieval, the
export from and retrieval to the Golgi complex, the endocytosis and the recycling as well as the rate of degradation of the
channels can be modulated by interaction with coat proteins,
adaptor proteins and other interacting proteins [17, 48, 50, 51,
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
1107
Fig. 1 Functional topology of TASK channel subunits; two subunits
assemble in an antiparallel fashion to form a functional channel. a The
topology of TASK-1; the four transmembrane α-helices (M1-M4), the
two pore helices (P1 and P2), the two cap helices (C1 and C2) and the
cytosolic regions involved in regulating intracellular traffic are shown;
blue line, selectivity filter; red spheres, permeating K+ ions. The 14-3-3
binding domains at the extreme C-terminus of TASK-, TASK-3 and
TASK-5 are compared. b Comparison of the relative current amplitudes
of TASK-1, TASK-3 (and chimeras thereof) expressed in Xenopus oocytes; T3/T1 represents a TASK-3 channel whose cytosolic C-terminus
was replaced by the C-terminus of TASK-1; T1/T3 represents the TASK1 channel whose cytosolic C-terminus was replaced by the C-terminus of
TASK-3. The same very low amount of cRNA (50 pg/oocyte) was
injected into the oocytes for each construct. The current amplitude was
determined as the acid-sensitive current that could be inhibited by decreasing the extracellular pH to 6.0. The current was recorded in the same
batches of oocytes; the current measured with the T1/T3 chimera was
taken as a reference point (from Renigunta et al. [93]). It should be noted
that large TASK-1 currents can be recorded after injection of higher
quantities of TASK-1 cRNA (0.75-3 ng/oocyte [92, 93, 120])
61, 83, 109, 110]. In this review, we will summarise our
current knowledge on the intracellular transport of K2P-channels of the TASK subfamily, and we will use these channels to
illustrate the different mechanisms by which PPI can modulate
intracellular traffic. The endocytosis of K2P-channels [75] and
the intracellular transport of TWIK-1 and THIK-2 channels
[8] are reviewed in two other articles of this special issue on
K2P-channels.
seven isoforms of 14-3-3 proteins. They have no enzymatic
activity or cellular function of their own, but they can alter the
function of numerous other proteins (their ‘clients’) by binding to one of the conserved 14-3-3 binding domains [28, 78,
79, 91, 101]. In most cases, the binding of 14-3-3 proteins is
dependent on the phosphorylation of the serine present in the
14-3-3 binding motifs of their clients. Thus, 14-3-3 proteins
may be regarded as universal switch proteins whose functional role depends entirely on the properties of their clients.
TASK channels were the first ion channels identified as clients
of 14-3-3 proteins [88, 89]. Yeast-two-hybrid screens using
the entire C-terminus of rat TASK-1, TASK-3 and TASK-5 as
bait identified 14-3-3 proteins as interacting partners of
TASK-1, TASK-3 and TASK-5 [88, 89]. Using three different
complementary DNA (cDNA) libraries from rat brain, four
A tri-basic retention signal in the C-terminus of TASK-1
and TASK-3: the role of 14-3-3 proteins
The 14-3-3 proteins are small acidic proteins which are highly
expressed in all eukaryotic cells. The human genome contains
1108
a
Curren
nt (μA)
3
2
TASK-3
1
∆C1
0
-120 -100 -80 -60 -40 -20
0
20
Membrane potential (mV)
b
Current ((μA)
6
4
TASK-1
2
∆C1
0
-120 -100 -80 -60 -40 -20
0
20
Membrane potential (mV)
c
1.2
Normalised current amplitude
N
different isoforms of 14-3-3 (σ, β, θ and ζ) were found to
interact with TASK-1 [88]. Screening of a human heart cDNA
library with the last 16 amino acids of human TASK-1 independently yielded 14-3-3β as an interacting protein [76]. Progressive truncation of the C-terminus from the proximal side
showed that the last 40 amino acids of TASK-1 contributed to
the binding of 14-3-3 and that the last five amino acids of
TASK-1 (RRSSV-COOH, Fig. 1) were essential for interaction
with 14-3-3 proteins [88]. The consensus motif for binding of
14-3-3 was found to be RRxSx-COOH [88], a variant of the socalled mode-3 binding motif for 14-3-3 proteins [100, 101].
Mutants of TASK-1 or TASK-3 that are incapable of
interacting with 14-3-3 proteins produce only very small
currents or no measurable current at all [76, 88]. For example,
deletion of the ultimate amino acid at the C-terminus (ΔC1
mutation) abolished interaction between TASK channels and
14-3-3 proteins in the yeast-two hybrid assay, caused the
accumulation of TASK channels in the Golgi complex [120]
and reduced TASK-1 and TASK-3 currents in heterologous
expression systems to very low levels [76, 88, 120], as illustrated in Fig. 2. The effects of the C-terminal deletions on the
TASK currents measured in heterologous expression systems
are entirely attributable to changes in surface expression of
TASK-1 and TASK-3. The biophysical properties of the channels are unaffected by the C-terminal deletions [88]. The
striking effects of the ΔC1 mutations on the TASK current
amplitude could be reproduced using a reporter construct
based on the membrane protein CD8, a co-receptor expressed
in cytotoxic T-cells. The cytosolic C-terminus of TASK-1 or
TASK-3, or the corresponding ΔC1 or ΔC5 mutants, was
attached to the intracellular C-terminus of CD8, and the
changes in the subcellular localisation of this fusion protein
were visualised in COS-7 cells using fluorescence imaging.
Figure 3 shows that the wild-type CD8 construct is localised
to the cell surface (as indicated by the homogeneous staining
of the entire surface of the COS-7 cells). The ΔC5 mutant also
shows cell surface staining (albeit less than the CD8 fusion
protein containing the wild-type C-terminus), with some of
the protein being retained in the Golgi complex. In contrast,
the ΔC1 mutant shows striking intracellular retention.
The role of the last six amino acids of C-terminus of TASK1 and TASK-3 in the intracellular traffic of the channels was
studied by systematic mutagenesis of another set of CD8TASK fusion proteins in which only the last 44 amino acids
of TASK-1 or TASK-3 were attached to the C-terminus of
CD8. The surface expression of these reporter constructs was
measured using a luminometric technique. Figure 4 shows
that the ‘control’ reporter constructs (containing the last 44
amino acids of TASK-1 and TASK-3) showed a large surface
expression, whereas the ΔC1 mutants showed virtually no
surface expression at all. The similarity of the effects of the
ΔC1 mutants observed in the current measurements (Fig. 2)
and the surface expression of the CD8 constructs (Fig. 4)
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
1.0
0.8
0.6
TASK-3
TASK-1
0.4
0.2
0
wt ∆C1 ∆C5
wt ∆C1 ∆C5
Fig. 2 Comparison of the currents measured with wild-type and ΔC1
and ΔC5 mutants of TASK-3 and TASK-1 in Xenopus oocytes. a Typical
current–voltage relation of human TASK-3 (black curve) and human
TASK-3ΔC1 (red curve). b Typical current–voltage relation of human
TASK-1 (black curve) and human TASK-1ΔC1 (red curve). c Statistics
of the relative current amplitudes measured with TASK-3, TASK-3ΔC1
and TASK-3ΔC5 (red) and with TASK-1, TASK-1ΔC1 and TASK1ΔC5 (from Zuzarte et al. [120])
suggests that 14-3-3 proteins do also interact with the last 44
amino acids of TASK-1 or TASK-3 in the CD8 reporter
construct. It should be noted in this context that CD8, 14-3-3
and TASK channels are all dimeric proteins.
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
TASK-1 CT
TASK-3 CT
1109
TASK-1 ∆C1
TASK-3 ∆C1
TASK-1 ∆C5
TASK-3 ∆C5
Fig. 3 Indirect immune-staining of CD8-TASK fusion proteins (original
data). The entire cytosolic C-terminus of TASK-1 (upper row) or TASK-3
(lower row) was attached to the cytosolic C-terminus of CD8. The middle
column shows images of the fusion proteins from which the amino acid at
the extreme C-terminus was removed (ΔC1 mutants); the right column
shows images of the fusion proteins from which the last five amino acids
at the extreme C-terminus were removed (ΔC5 mutants). Cells were
transfected using a Fugene6 (Roche), fixed with formaldehyde, permeabilized (0.05 % SDS, 0.3 % Triton X-100 in PBS) and stained with antiCD8 antibody (3030, Diatec, Norway, dilution 1:100) followed by a
fluorescent secondary antibody. The scale bars are 15 μm
The reason for the intracellular retention of the ΔC1 mutant
was elucidated by mutagenesis on the basis of the ΔC1
mutant [120], both in TASK-1 and TASK-3 channels
expressed in Xenopus oocytes, and in CD8 reporter proteins
expressed in COS-7 cells. For example, the cell surface expression of the ΔC1 mutant of the CD8 construct could be
rescued by mutating the two arginine residues of the
MKRRKSV motif of TASK-3 to alanine [120], as illustrated
in Fig. 4. Systematic mutagenesis of the C-terminal amino
acids of the ΔC1 mutants revealed that the extreme Cterminus of TASK-1 and TASK-3 contains a retention signal,
KRR (positions 389–391 in TASK-1 and positions 369–371
in TASK-3). Both the current measurements in oocytes and
the surface expression measurements with the CD8 reporter
protein constructs showed that the lysine residues of the KRR
motif could not be substituted by arginine (in other words,
RRR was less effective as a retention signal than the KRR
signal) and none of the two arginine residues could be
substituted by lysine (KKR and KRK were less effective than
KRR). Thus, KRR represents a novel retention signal which
differs from the canonical di-basic (RxR) retention signal and
from the canonical C-terminal di-lysine (KxKxx-COOH)
retention signal [120]. Since the retained ΔC1 constructs colocalised with coat protein complex COPI [120], it may be
assumed that the KRR signal causes intracellular retention by
activating COPI-mediated retrograde transport of TASK-1
and TASK-3.
The most likely explanation for the correlation between
binding of 14-3-3 proteins and TASK current density or
cell surface expression is that the binding of 14-3-3 proteins masks the KRR retention signal (Fig. 1a) and in this
way disinhibits the forward transport of the channel from
the Golgi complex to the surface membrane. In other
words, the binding motifs for 14-3-3 (RRxSx) and COPI
(KRR) overlap, which makes the binding of 14-3-3 and
COPI at the C-terminus of TASK-1 and TASK-3 mutually
exclusive. Several crystal structures of 14-3-3 proteins are
available [49, 114, 115]. The 14-3-3 dimer displays a
typical W-shape with each monomer forming a central
binding groove that accommodates the phosphorylated
interaction motif of the TASK-1 or TASK-3 C-terminus
[1]. The 14-3-3 proteins have a much lower affinity for the
ΔC1 mutants [1], and the lack of 14-3-3 binding to ΔC1
constructs exposes the KRR retention signal.
1110
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
Fig. 4 The surface expression of
CD8-TASK fusion proteins
measured with a luminometric
technique in COS-1 cells. The
fusion proteins and the amino
acids at the extreme C-terminus
are shown schematically on top.
T3C44 represents the last 44
amino acids of TASK-3; T1C44
represents the last 44 amino acids
of TASK-1. The normalised
surface expression measured with
the CD8-TASK-3 fusion protein
(red) and the CD8-TASK-1
fusion protein (blue) and the ΔC1
and ΔC5 mutants thereof is
shown in the bar graph (from
Zuzarte et al. [120])
Normalised surface expressio
on
1.2
1.0
0.8
0.6
0.4
0.2
0
TASK channel constructs in which the last five amino acids
at the C-terminus were removed (ΔC5 mutants) were also
found to be useful for understanding the mechanism of action
of 14-3-3 proteins. The removal of the last five amino acids of
TASK-1 or TASK-3 has the following consequences: (1) the
binding of 14-3-3 proteins is abolished because the mutant does
not fit into the binding groove of 14-3-3, and (2) the binding to
COPI is abolished because the two arginine residues of the
KRR motif are deleted. This causes a higher surface expression
of the ΔC5 mutants as compared to the ΔC1 mutants. There is,
however, an interesting difference in the effects of the ΔC5
mutation on the currents produced by full-length TASK-1 and
TASK-3 channels on the one hand (Fig. 2) and on the surface
expression of the CD8 reporter protein construct (which
contained only the last 44 amino acids of the channel) on the
other hand (Fig. 4): The surface expression of the ΔC5 mutant
of the CD8 fusion protein was just as large as in the ‘wild-type’
construct, whereas the current amplitude produced by the ΔC5
mutants of TASK-1 and TASK-3 was reduced as compared to
wild-type channels. This discrepancy may be attributable to the
fact that in the intact channel the binding of 14-3-3 proteins not
only masks the KRR retention signal but has additional effects
on the cytosolic domains of the channel. For example, 14-3-3
protein may ‘clamp’ the cytosolic domains of the channel
protein in a conformation that facilitates forward transport
[98, 101]. Consistent with this interpretation, the CD8 fusion
proteins used for studying subcellular localisation (which
contained the entire C-terminus of TASK-1 or TASK-3) also
showed a clear difference between the ‘wild-type’ construct
and the ΔC5 mutant (Fig. 3).
Mutation of the penultimate serine residue (S393 in human
TASK-1 and S373 in human TASK-3) to alanine, mimicking
the dephosphorylated state, abolished the binding of 14-3-3
and reduced surface expression and current amplitude of
TASK-1 and TASK-3 in a similar way as the ΔC1 mutant
[58, 76, 88, 120]. This can be explained by electrostatic
interactions between the phosphate moiety of each TASK
channel and positively charged side chains in the 14-3-3
binding grooves [1]. Thus, phosphorylation of the serine
residue at the penultimate position is required for highaffinity binding of 14-3-3 proteins. The phosphorylation of
S393 in TASK-1 and S373 in TASK-3 can be directly demonstrated by ‘phos-tag’ (NARD institute) electrophoresis [58],
as illustrated in Fig. 5. O’Kelly and co-workers have shown
that cAMP-dependent protein kinase and ribosomal S6 kinase
(RSK2) can phosphorylate S393 of TASK-1 in vitro. These
findings provide strong evidence for the notion that activation
of protein kinase A via cAMP can regulate the intracellular
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
1111
Fig. 5 In vitro phosphorylation of TASK C-termini by PKA (original
data). GST fusion proteins displaying the last 15 amino acids of either
channel were incubated with recombinant PKA and ATP as indicated.
After incubation for one hour, samples were resolved on an SDS-PAGE
gel containing 100 μM Phostag reagent and 100 μM MnCl2, which
retards the migration of phosphorylated residues. Constructs with both
(TASK-1) or one (TASK-3) serine residue present in the distal C-terminus
were analysed to demonstrate phosphorylation on the serine residues of
the 14-3-3-binding motif. Mutation of these serines to alanine residues
abolished phosphorylation
transport of TASK-1 and TASK-3 channels to the surface
membrane. Thus, the density of TASK-1 and TASK-3 currents in the cells is regulated in a very complex way (1) at the
molecular level, by reduction of their open probability via Gprotein coupled receptors [65, 87, 97, 112], (2) at the transcriptional level, for example, by upregulation of transcription
in cancer cells [72, 82, 113] and (3) at the posttranslational
level, for example, by modulation of their intracellular traffic
via production of second messengers such as cAMP [58],
activation of protein kinases and subsequent binding of the
switch protein 14-3-3.
It should be noted here that the KRR motif is not the only
retention signal identified in TASK-1 and TASK-3. Both
channels have a very short N-terminus that starts with the
sequence MKR (Fig. 1). It was found that replacement of
the di-basic KR motif by other amino acids (e.g. NQ) markedly increased the currents produced by ‘wild-type’ TASK-1
and TASK-3 channels (and their Δ5 mutants) expressed in
Xenopus oocytes and that the effect of this putative di-basic
retention signal could be transferred to the reporter protein
CD74 [120]. Thus, the MKR motif is a bona fide retention
signal. In earlier work, it had been proposed that the binding of
COPI to the N-terminal KR motif of TASK-1 or TASK-3 and
the binding of 14-3-3 to the extreme C-terminus were mutually exclusive [76, 77]. This hypothesis was based on the
observation that mutation of the N-terminal KR motif rescued
the currents of the ΔC1 mutant. In later work, this observation
could not be reproduced [120]; instead, it was found that the
intracellular retention of the ΔC1 mutants was mediated by a
motif (KRR) at the C-terminus of TASK-1 and TASK-3, and
could not be rescued by mutation of the N-terminal KR motif
[120]. The latter observations provide further support for the
idea that the C-terminal retention signal (KRR), which can
bind COPI, overlaps with the C-terminal 14-3-3 binding site
(KRRxSx-COOH) and that this is the reason why the binding
of 14-3-3 and COPI is mutually exclusive.
The extreme C-terminus of TASK-5 channels also has a
putative 14-3-3 binding domain (Fig. 1a) and was found to
interact with 14-3-3ζ and 14-3-3ε in a yeast-two-hybrid
screen [88]. Since TASK-5 channels could not be functionally
expressed so far, the functional consequences of their interaction with 14-3-3 are still unknown. A puzzling finding is that
TASK-5 has a polymorphism (G95E) [43, 44] that is expected
to make the channel non-functional or to profoundly alter its
ion selectivity. About 50 % of the tested individuals were
found to be heterozygous for this mutation, and about 25 %
were found to be homozygous [43]. The following observations suggest that TASK-5, despite its obstinate ‘silence’ in
heterologous expression systems, may be a functional channel
in vivo: (1) Chimeras of TASK-3 and TASK-5 channels
produced measurable potassium currents [43]. (2) TASK-5
orthologs in various species are highly conserved. (3)
TASK-5 is rather selectively and highly expressed in the
auditory brain stem neurons [43]. (4) When deafness was
induced in rats by ablation of the cochlea, the expression of
TASK-5 in the cochlear nucleus decreased to ∼16 % of control
after 3 days and to <1 % after 3 weeks [18]. Further experiments are needed to clarify the function and the subcellular
localisation of TASK-5 and the possible role of 14-3-3 in
regulating its intracellular traffic.
A retention factor that can bind to TASK-1: the role of p11
(S100A10)
The K2P-channel TASK-1 was found to interact with p11, also
known as S100A10. The protein p11 is a member of the
1112
b
P1
M2
M1
C2
N
P1
M1
xxxKxK
i20
p11
M2
P2
M4
C1
M3
C1
C2
a
P2
family of S100 proteins [59]. This protein family, which has at
least 25 members, consists of small, acidic proteins which are
mainly localised to the cytoplasm and have no enzymatic
function of their own [34]. Like 14-3-3 proteins, S100 proteins
assemble as homo- or heterodimers and mainly act as switch
proteins that modulate the function of client proteins [94,
103]. A common feature of all S100 proteins is that each
monomer possesses two EF-hand motifs that bind calcium;
calcium binding produces a conformational change which
alters the function of the protein [34]. The only exception in
this regard is p11 (S100A10); in this paralogue, the calciumbinding domain is not conserved, which results in a much
lower calcium sensitivity. p11 is highly expressed in the brain,
and its expression level is modulated by neurotransmitters,
growth factors, cytokines and glucocorticoids [104]. A major
interacting partner of p11 is the phospholipid-binding protein
annexin A2; in addition, p11 has been shown to interact with
serotonin receptors and to promote their surface expression.
Knockout of p11 gave rise to a depression-like behavioural
phenotype in mice, and it has been convincingly argued that
alterations in the expression of p11 may play a role in the
genesis of depression [104] and drug addiction [3]. The structure of p11 has been resolved [95]: each monomer possesses
four alpha helices (HI to HIV); p11 forms an antiparallel dimer
mainly through hydrophobic interactions between HI and HIV,
as indicated in the schematic drawing of Fig. 6a (for simplicity, HII and HIII have been omitted).
The interaction between TASK-1 and p11 was detected in a
yeast-two-hybrid screen using the C-terminus of TASK-1 as
bait and a human heart cDNA library as prey [31]. Initially, it
was proposed that p11 binds to the extreme C-terminus of
TASK-1 and promotes surface expression of the channel by
masking the retention signal KRR [31]. This hypothesis was
based, among other observations, on mutagenesis experiments
which showed that the ΔC1, ΔC2 and ΔC3 mutants of TASK1 (where one, two or three residues at the extreme C-terminus,
SSV, are removed) were unable to produce any currents. Later,
it was found that the inability of these mutants to produce any
surface expression or TASK-1 current was most likely related
to the lack of binding of 14-3-3 proteins to the extreme Cterminus of TASK-1 [76, 88, 93, 120]. Thus, at least some of
the results of the initial study on the effects of p11 on TASK-1
channels [31] can be explained by the effects of 14-3-3 on
TASK-1 channels, which were not known at the time of
publication. The idea that p11 does not bind directly to the
extreme C-terminus of TASK-1 is also supported by in vitro
peptide interaction experiments that suggested that the binding of p11 to TASK-1 may require prior binding of 14-3-3
proteins to the C-terminus of TASK-1 [77].
Four years after the initial study on the effects of p11 [31], it
was shown that p11 binds to a region in the proximal Cterminus of TASK-1 [93], which was denoted the i20 domain
because it comprises 20 amino acids. The i20 domain is not
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
M4
M3
N
HIV
HI
HI
p11 HIV
KxKxxx
i20
Fig. 6 Hypothetical structure of the TASK-1-p11 heterotetramer. a Schematic drawing of the antiparallel arrangement of the TASK-1 homodimer
and the (hypothetical) position of the antiparallel p11 homodimer; for
clarity, the two protomers of the channel have been moved apart and
helices II and III of p11 have been left out. It was assumed that the TASK1-p11 heterotetramer shows a twofold symmetry. The domain on p11 to
which the i20 domain of TASK-1 binds is not yet known. b The effect of
removing the p11-binding domain on TASK-1 currents measured in
Xenopus oocytes; in the Δi20 mutant, the p11 binding domain of
TASK-1 was excised (from Renigunta et al. [93])
present in TASK-3 channels (which do not interact with p11).
Removal of the i20 domain abolished the interaction between
in TASK-1 and p11. Surprisingly, expression of TASK-1
channels in which the i20 domain was deleted (TASK-1Δi20
mutant) produced a much larger potassium current than wildtype TASK-1 channels (Fig. 6b). Mutagenesis experiments
showed that the dependence of the amplitude of TASK-1
currents on the binding of p11 is attributable to a conserved
di-lysine retention signal in the C-terminus of p11 (KxKxx(x)
or HxKxx(x) [93] (Fig. 7a), which retrieves the channel to the
endoplasmic reticulum as long as p11 binds to the channel.
The functionality of this putative retention signal was tested
using CD8-p11 fusion proteins, similar to the ones depicted in
Fig. 4. The last 36 amino acids of the C-terminus of p11 were
attached to CD8 (Fig. 7b), and the surface expression of this
reporter construct was measured with a luminometric technique. Attachment of the last 36 amino acids of p11 abolished
surface expression of the reporter construct (Fig. 7c). The
surface expression could be rescued by deleting the last four
amino acids of p11 and thus invalidating the retention signal
(Fig. 7a, b, c).
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
a
Human
Rat
Canis
Xenopus
C-terminus of p11 orthologs
PLAVDKIMKDLDQCRDGKVGFQSFFSLIAGLTIACNDYFVVHMKQKGKK
PLAVDKIMKDLDQCRDGKVGFQSFLSLVAGLIIACNDYFVVHMKQKK
PLAVDKIMKDLDQCRDGKVGFQSFFSLIAGLTIACNDYFVIHMKQKGKK
PMTVDKIMKDLDDCRKGQVNFRSYCSLIAGLLIACNEYYVKHMKKR
b
a
TASK-1
stx8
linke
r
A tyrosine-based endocytosis signal in the C-terminus
of TASK-1: the role of syntaxin-8
The SNARE protein syntaxin-8 (stx8) was found to interact
with TASK-1 in a variant of the yeast-two-hybrid screen that
preferentially detects interactions with integral membrane
proteins and membrane-associated protein. When full-length
TASK-1 was used as a bait with a human brain cDNA library,
the endosomal SNARE protein syntaxin-8 was detected as a
prey [92]. This interaction was confirmed by coimmunoprecipitation of the two proteins from lysates of a
mammalian cell line, without overexpression of the SNARE
protein or the channel. The R-SNARE VAMP8 and the three
Q-SNAREs syntaxin-8, syntaxin-7 and vti1b form the
endosomal SNARE complex which is involved in fusion
events in the early and late endosomal and lysosomal compartments [41]. The topology of stx8 is shown in Fig. 8a. Stx8
is a tail-anchored protein that has a transmembrane domain, a
b
C
TMD
It can be concluded from these data that binding of p11 to
the proximal C-terminus of TASK-1 causes retention of the
channel in the endoplasmic reticulum. Thus, p11 acts as a
‘retention factor’, and dissociation of p11 from the channel is
required for efficient surface expression of the channel. Since
both p11 and TASK-1 assemble as dimers, it is not unlikely
that the quaternary structure of the TASK-1/p11 complex is a
tetramer with twofold symmetry, as indicated schematically in
the ‘functional topology’ drawing of Fig. 6a.
c
SNA
RE m
otif
Fig. 7 The retention signal at the
extreme C-terminus of p11. a
Alignment of the C-terminus of
some orthologs of p11. The
histidine and lysine residues of
the lysine-based retention signal,
[K/H]xKxx(x), are highlighted in
red. b The CD8-p11 fusion
proteins used to test the
functionality of the retention
signal at the extreme C-terminus
of p11; CD8 forms stable dimers;
for simplicity only one protomer
is shown. The last 36 residues
acids of p11 were attached to the
cytosolic C-terminus of CD8; in
the mutant, the last four residues
were deleted to invalidate the
retention signal. c The surface
expression of the two constructs
shown in panel (b), as measured
with a luminometric assay (from
Renigunta et al. [93])
1113
overlay
Hc
Hb
Ha
N
DRRQNLL
Fig. 8 The secondary structure and the subcellular localisation of
syntaxin-8. a The secondary structure of stx8 including the retention
signal in the linker between the Hb and Hc domains. b Co-localisation
of TASK-1 (tagged with EGFP) and stx8 (tagged with DsRed monomer)
in COS-7 cells 48 h after transfection (original data). The punctuate
structures probably represent the early endosome
1114
a
b
Ra
ate of endocyttosis
Q-type SNARE motif [41] and three N-terminal helices Ha,
Hb and Hc (a so-called Habc domain). The SNARE domain of
stx8 interacts with the SNARE domains of the other three
endosomal SNAREs to form the tetrahelical bundle (the transSNARE complex) that mediates fusion of the donor and
acceptor membranes in the endosomal compartments. The
three alpha-helices of the Habc domain interact with each
other to form a ternary helical bundle which may be involved
in the regulation of tethering and fusion of transport vesicles.
The linker between the Hc and the SNARE domain is a
flexible, unstructured region that is essential for the interaction
with TASK-1 [92]. The linker between the Hb and the Hc
domain contains an (atypical) lysine-based endocytosis signal:
77
DRRQNLL83 (Fig. 8a). It should be noted here that the Cterminus of TASK-1, too, contains a trafficking signal in its Cterminus: the tyrosine-based endocytosis signal 300YAEV303
(Fig. 1a).
When expressed in mammalian cell lines, fluorescencelabelled TASK-1 and stx8 showed a striking co-localisation
in the early endosomal compartment (Fig. 8b) [92].
Coexpression of TASK-1 channels with stx8 caused a marked
reduction in TASK-1 currents, both in Xenopus oocytes and in
mammalian cell lines (Fig. 9a). When both the lysine-based
endocytosis signal in stx8 and the tyrosine-based endocytosis
signal in TASK-1 were invalidated by mutations, the effect of
stx8 on the TASK-1 current was abolished (Fig. 9a). When
only one of these motifs was mutated, the effect of stx8 on
TASK-1 currents was diminished [92]. The rate of endocytosis of TASK-1 channels, as measured with an antibody uptake
assay, was substantially increased by co-transfection with stx8
(Fig. 9b). However, when the endocytosis signals on stx8 and
TASK-1 were mutated, the rate of endocytosis was the same
as under control conditions (transfection of TASK-1 alone)
(Fig. 9b). A mutant of stx8 that is incapable of forming a
SNARE complex had very much the same effects as wild-type
stx8. Systematic mutagenesis experiments showed that the
linker between the Ha helix and the SNARE motif of stx8 is
essential for the interaction with TASK-1, and that the Cterminus of TASK-1 is essential for the interaction with stx8
[92]. These (and other) observations suggest that TASK-1
channels and the endosomal SNARE protein stx8 are
endocytosed in a cooperative manner [92], as illustrated schematically in Fig. 10: The two proteins may bind to adjacent
sites in AP-2, and endocytosis may be stimulated by simultaneous occupancy of both sites. Such a cooperative endocytosis would have a number of interesting functional implications: (1) The endosomal SNARE protein stx8 reaches its
destination (the endosomal compartment) via the surface
membrane. (2) Unassembled SNARE proteins can have effects unrelated to membrane fusion. (3) The endocytosis of
stx8 may be modulated by TASK-1. (4) The endocytosis of
TASK-1 may be modulated by stx8. (5) The cooperative
endocytosis of TASK-1 and the unassembled SNARE protein
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
Fig. 9 Cooperative endocytosis of TASK-1 and stx8. a The amplitude of
TASK-1 currents measured in Xenopus oocytes after expression of
TASK-1 alone and after coexpression of TASK-1 and stx8. In the Y→
A mutant, the tyrosine-based endocytosis signal on TASK-1 (see Fig. 1a)
was invalidated; in the LL→AA mutant, the leucine-based endocytosis
signal in stx8 was invalidated. When both endocytosis signals were
invalidated the effect of stx8 on TASK-1 current amplitude was
abolished. b The effect of co-transfection of stx8 on the rate of endocytosis of TASK-1 channels measured in COS-7 cells using an antibody
uptake assay. Co-transfection of stx8 increased the net rate of endocytosis
about ninefold. When the TASK-1Y→A mutant and the stx8LL→AA mutant
were co-transfected, the rate of endocytosis was no larger than under
control conditions (transfection of wild-type TASK-1 alone); data from
Renigunta et al. [92])
stx8 may contribute to the sorting of the channel to specific
intracellular compartments.
It has been suggested by several groups that the surface expression of some membrane proteins may be
regulated by activation of protein kinase C [27, 39, 42].
Recently, it has been reported that activation of protein
kinase C may promote endocytosis of TASK-1 channels
within 15 min [29], and it has been suggested that a
trafficking motif immediately downstream of the i20
domain (Fig. 1), 317SREKLQYSIP326, may play a role
in this process. A similar putative endocytic motif,
FREKLAYA, has been found in the neurotransmitter
transporters of the SLC6 family [38]. However, the
mechanisms responsible for the effect on PKC on the
endocytosis of SLC6 transporters and TASK-1 and the
functional role of the putative novel endocytic signal [12,
29] need further clarification.
1115
C2
C1
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
M4
M3
otif
M2
TMD
P1
M1
P2
C
SNA
linke
r
C
RE m
N
Hc
Hb
Ha
N
YAEV
DRRQNLL
AP-2 complex
Fig. 10 Schematic diagram of the cooperative binding of the tyrosinebased endocytosis signal on TASK-1 and the leucine-based endocytosis
signal on stx8 to the adaptor protein AP-2
A di-acidic ER-export signal in TASK-3: the role of Sec24
The cytosolic domains of several membrane proteins, including ion channels and G-protein coupled receptors, contain diacidic ER export motifs [7, 52–54, 56, 63, 74, 80, 107] with
the consensus sequence [D/E] x [D/E], where x can be any
amino acid, although in most cases analysed the export ability
of D cannot be fully substituted by E and vice versa [119]. In
plants, a tri-acidic ER-export motif, DxDxE, has been found
[64]. The di- or tri-acidic motifs interact with the so-called Bsite of the cargo receptor Sec24 [9]. The heterodimeric complex of Sec23 and Sec24 forms the inner layer of the COPII
coat, and Sec 24 serves as a receptor for cargo transported
from the ER to the Golgi complex [67, 68, 71]. The interaction
of proteins carrying the ER-export signals with Sec24 occurs
at specific ER-export sites, where the cargo proteins are concentrated [71, 119] and the budding of transport vesicles is
initiated. In a precisely orchestrated sequence of events, triggered by the small GTPase Sar1, the coat proteins Sec24,
Sec23, Sec 13 and Sec31 associate to form the COPII coat
complex, which exclusively mediates the vesicular transport
of newly synthesised membrane proteins to the Golgi complex. In many cases, the export from the ER is rate-limiting for
the transport of cargo proteins from the ER to the surface
membrane.
At the proximal end of the cytosolic C-terminus of human
TASK-3, there is a motif consisting of eight amino acids,
252
EDERRDAE259, that may contain one or even two diacidic ER-export signals. This octapeptide motif is highly conserved in TASK-3 orthologs from insects to mammals [121].
Mutation of the proximal di-acidic motif, EDE, to ADA greatly
reduced the current density and surface expression of TASK-3
in heterologous expression systems [121]. Mutation of the
distal di-acidic motif, DAE, to AAA had no effect. When the
C-terminus of TASK-3 was transplanted to the C-terminus of a
different potassium channel, Kir2.1, the function of the EDE
motif was preserved, i.e., mutation of the EDE motif to ADA
reduced current amplitude and surface expression of the Kir2.1/
TASK-3 chimera [121], as illustrated in Figure 11. From these
results, it was concluded that 252EDE255 is a functional ERexport signal that facilitates the export of TASK-3 from the ER.
The human genome contains four isoforms of Sec24, which
display differential selectivity in the export of proteins from the
ER [56, 111]. The various isoforms have differential selectivity
towards different variants of the [D/E]x[D/E] ER-export signal.
Thus, the interaction of a specific Sec24 isoform with the EDE
motif may contribute to defining the intracellular route and
destination of TASK-3 channels.
A very similar di-acidic motif is also found in human
TASK-1 (252EDEKRDAE259) (Fig. 1a), but mutations of the
acidic amino acids had no effect on TASK-1 current or surface
expression (Zuzarte, Rinné, Preisig-Müller and Daut, unpublished results). This finding may be attributable to the fact that
ER-export is rate-limiting for the transport of TASK-3 from
the ER to the cell surface, whereas in the transport of TASK-1
to the cell surface other mechanisms are rate-limiting; the
visualisation of the function of the di-acidic (EDE) motif in
TASK-1 may require experiments in which the slower transport steps of TASK-1 are sidestepped.
Functional implications
The data presented above suggest that the intracellular traffic
of TASK-3 and especially of TASK-1 channels is highly
regulated. TASK-1 interacts with at least three accessory
proteins: 14-3-3, p11 and syntaxin-8. A membrane yeasttwo-hybrid screen using the entire TASK-1 channels as bait
1116
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
take place in vivo and are they physiologically relevant? In the
following, we discuss this question with regard to the three
interacting proteins investigated so far.
HA
a
N
ED
E
D
AE
C
Relative surface exp
R
pression
b
Kir2.1-T3ct Kir2.1-T3ct Kir2.1-T3ct
EDERRDAE ADARRDAE EDERRAAA
Ba2+ sensitive
e current
c
Kir2.1-T3ct
Kir2
1-T3ct Kir2.1-T3ct
Kir2 1-T3ct Kir2
Kir2.1-T3ct
1-T3ct
EDERRDAE ADARRDAE EDERRAAA
Fig. 11 The effect of the di-acidic ER-export signal of TASK-1 on
inward rectifier currents. The C-terminus of TASK-3 (T3ct) was
transplanted to the C-terminus of the inward rectifier K+ channel Kir2.1
(which had been truncated at position 373 to eliminate the endogenous
ER-export signal of Kir2.1). a Schematic diagram of the Kir2.1/TASK-1
fusion protein with the position of the extracellular HA-tag indicated. b
The surface expression of the Kir2.1/T3ct fusion protein measured in
Xenopus oocytes with a luminometric technique. Mutation of the EDE
motif to ADA reduced surface expression. c The potassium current
produced by the Kir2.1/T3ct fusion protein expressed in Xenopus oocytes. The Kir2.1 channel blocker Ba2+ was used to define the current
flowing through the Kir2.1/TASK-1 chimeric channel protein. Mutation
of the EDE motif to ADA reduced current amplitude; data from Zuzarte
et al. [120]
yielded a large number of potential interacting proteins (n=
63), the majority of which are suspected to play a role in
intracellular trafficking (Renigunta and Daut, unpublished
results). There is no reason to assume that TASK-1 channels
are exceptional in this respect, and it is likely that many
membrane proteins have a large number of interacting proteins that influence their intracellular traffic. The major question that arises from the data presented above is: Do the
interactions of TASK-1 and TASK-3 with accessory proteins
The adaptor protein 14-3-3 The 14-3-3 proteins are switch
proteins that have many ‘clients’ to whom they can bind
depending on the phosphorylation of serine residues in the
canonical 14-3-3 binding sites, and for a growing number of
client proteins it has been shown that this interaction can cause
profound alterations of the state of the cells [19, 30, 91, 101].
Therefore, especially in view of the large effect of 14-3-3
binding on the surface expression of TASK-1 and TASK-3,
it is very likely that this interaction is functionally relevant.
Further corroboration of this tenet will depend on antibodies
capable of detecting and purifying native TASK channels. For
native ATP-sensitive potassium channels (KATP-channels), a
correlation between their cell surface expression and the
abundance of 14-3-3 proteins has been demonstrated in primary cardiac myocytes [2]. Given that 14-3-3 proteins are
bound to TASK channels with a much higher affinity than to
KATP-channels, TASK-1 channels represent an attractive system to study the effects of 14-3-3 on membrane protein
trafficking in native cardiomyocytes. A major obstacle for
our understanding of the action of 14-3-3 in vivo is the fact
that these proteins interact with a multitude of clients and that
the precise affinity and abundance of the interaction partners is
usually unknown. The situation calls for a more quantitative
investigation of 14-3-3-client interactions so that correlations
between binding parameters and biological effect can be
analysed in detail. In addition, drugs that perturb 14-3-3client interaction such as derivatives of the fungal toxin
fusicoccin A [1, 69] will be invaluable to query the relevance
of such as interactions in vivo.
The adaptor protein p11 The functional role of the interaction
between p11 and TASK-1 is not yet clear; the affinity of the
binding of p11 to TASK-1 has not been determined and the
free cytosolic concentrations of p11 are not known. However,
it has been shown that the surface expression of TASK-1
channels is markedly increased in the TASK-1Δi20 mutant
(in which the p11 binding domain has been deleted), both in
mammalian cell lines and in Xenopus oocytes. This suggests
that endogenous p11 is present in the cytosol at sufficient
concentration to trap a fraction of the TASK-1 channels in
the endoplasmic reticulum. TASK-1 is strongly expressed in
neurons in many areas of the brain [105]. The expression of
p11 (S100A10) is regulated in various brain regions by
growth factors such as brain-derived neurotrophic factor
(BDNF) and cytokines such as interferon γ and tumour necrosis factor β (TGFβ), and by glucocorticoids. Furthermore,
the mRNA coding for p11 is upregulated by treatment with
various antidepressant drugs [104], and p11 knockout mice
show a depression-like behaviour. Therefore, p11 has been
Pflugers Arch - Eur J Physiol (2015) 467:1105–1120
proposed to be a potential target for antidepressant therapy
[104]. It is tempting to speculate that some of the antidepressant effects of p11 might be mediated by its interaction with
TASK-1, causing a reduction of TASK-1 current density in
neurons.
The SNARE protein syntaxin-8 The interaction between
syntaxin-8 and TASK-1 has been demonstrated by coimmunoprecipitation in a cell line endogenously expressing
both proteins [92]. This finding suggests that the interaction
may also take place in vivo and is not a mere consequence of
the overexpression (which was required to elucidate the mechanisms of action of stx8). SNARE proteins, together with Rab
GTPases and phosphoinositides, play an important role in
defining the identity of intracellular compartments [6, 20,
55, 62, 86]. They are involved in specific membrane fusion
processes and thus contribute to shaping the intracellular
itinerary of membrane proteins. The notion that unassembled
SNARE proteins have functions unrelated to membrane fusion is relatively new and needs further confirmation. There
are indications that SNARE proteins may also modulate the
intracellular traffic of ion channels other than TASK-1 [10, 11,
16, 26, 47], but in most cases the mechanisms by which the
SNARE proteins affect vesicular transport have remained
unclear. The cooperative endocytosis of syntaxin-8 and
TASK-1 links the itinerary of a SNARE protein to that of a
cargo molecule. In other words, the presence of stx8 might
influence the sorting decisions of TASK-1 and vice versa.
This may turn out to be a prototypic protein–protein interaction that provides specificity to the itinerary and fate of membrane proteins. It should be noted here that only few SNARE
proteins, including syntaxin-8 and syntaxin-6, have a classical
linear endocytosis signal. However, several monomeric
SNARE proteins have unconventional binding sites linking
them to coat components or coat adaptor proteins involved in
intracellular transport (including Sec24, AP180, CALM and
Hrb) [56, 57, 60, 85]; these binding sites are distinct from the
canonical linear motifs and are defined by the tertiary structure
of the SNARE proteins. In these cases, too, the binding of
disassembled SNARE proteins to specific vesicles may provide a mechanism for pre-determining subsequent transport
steps [60].
Conclusions
The K2P-channels TASK-1 and TASK-3 provide an illustrative example of how protein–protein interactions may control
the life cycle of membrane proteins. It has been proposed that
all cells possess a trafficking proteostasis network (TPN) that
manages the folding of proteins and their transport between
intracellular compartments [40]. It is likely that membrane
1117
proteins encounter many interacting proteins during their passage through different compartments and that some of these
interactions influence subsequent sorting decisions. Membrane proteins obviously have multiple binding sites for
interacting proteins, both in their unstructured regions and in
structured domains [51, 57]. Interacting proteins may modulate the intracellular traffic of their ‘clients’ by three types of
mechanisms: (1) The binding of the interacting protein may
mask canonical trafficking signals (whose number keeps
expanding). (2) The interacting proteins may themselves carry
trafficking signals by which they modulate the sorting decisions of their clients [93]. (3) The interacting proteins may
influence the folding of their clients, which again might expose or mask trafficking signals or influence intracellular
traffic of their clients in some other way [40]. In conclusion,
interacting proteins may adapt the complex itinerary of a
protein within the cell to the specific functions of that protein;
thus, the binding sites for specific interacting proteins might
be regarded as trafficking motifs in the wider sense.
Acknowledgments This study was supported by the Deutsche
Forschungsgemeinschaft (FOR 1086, TP7 and TP9; SFB 593, TP4).
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