A di-acidic sequence motif enhances the surface expression of the potassium channel TASK-3

We have characterized a sequence motif, EDE, in the proximal C-terminus of the acid-sensitive potassium channel TASK-3. Human TASK-3 channels were expressed in Xenopus oocytes, and the density of the channels at the surface membrane was studied with two complementary techniques: a luminometric surface expression assay of hemagglutinin epitope-tagged TASK-3 channels and voltage-clamp measurements of the acid-sensitive potassium current. Both approaches showed that mutation of the two glutamate residues of the EDE motif to alanine (ADA mutant) markedly reduced the transport of TASK-3 channels to the cell surface. Mutation of the central aspartate of the EDE motif had no effect on surface expression. The functional role of the EDE motif was further characterized in chimaeric constructs consisting of truncated Kir2.1 channels to which the C-terminus of TASK-3 was attached. In these constructs, too, replacement of the EDE motif by ADA strongly reduced surface expression. Live-cell imaging of enhanced green fluorescent protein-tagged channels expressed in COS-7 cells showed that 24 h after transfection wild-type TASK-3 was mainly localized to the cell surface whereas the ADA mutant was largely retained in the endoplasmic reticulum (ER). Mutation of a second di-acidic motif in the C-terminus of TASK-3 (DAE) had no effect on surface expression. Coexpression of TASK-3 with a GTP-restricted mutant of the coat recruitment GTPase Sar1 (Sar1H79G) resulted in ER retention of the channel. Our data suggest that the di-acidic motif, EDE, in human TASK-3 is a major determinant of the rate of ER export and is required for efficient surface expression of the channel.     

p11, an annexin II subunit,an auxiliary protein associated with the background K+ channel,TASK-1

TASK-1 belongs to the 2P domain K+ channel family and is the prototype of background K+ channels that set the resting membrane potential and tune action potential duration. Its activity is highly regulated by hormones and neurotransmitters. Although numerous auxiliary proteins have been described to modify biophysical, pharmacological and expression properties of different voltage- and Ca2+-sensitive K+ channels, none of them is known to modulate 2P domain K+ channel activity. We show here that p11 interacts specifically with the TASK-1 K+ channel. p11 is a subunit of annexin II, a cytoplasmic protein thought to bind and organize specialized membrane cytoskeleton compartments. This association with p11 requires the integrity of the last three C-terminal amino acids, Ser-Ser-Val, in TASK-1. Using series of C-terminal TASK-1 deletion mutants and several TASK-1-GFP chimeras, we demonstrate that association with p11 is essential for trafficking of TASK-1 to the plasma membrane. p11 association with the TASK-1 channel masks an endoplasmic reticulum retention signal identified as Lys-Arg-Arg that precedes the Ser-Ser-Val sequence.     

The stop transfer sequence of the human UDP-glucuronosyltransferase 1A determines localization to the endoplasmic reticulum by both static retention and retrieval mechanisms

Human UDP-glucuronosyltransferase 1A (UGT1A) isoforms are endoplasmic reticulum (ER)-resident type I membrane proteins responsible for the detoxification of a broad range of toxic phenolic compounds. These proteins contain a C-terminal stop transfer sequence with a transmembrane domain (TMD), which anchors the protein into the membrane, followed by a short cytosolic tail (CT). Here, we investigated the mechanism of ER residency of UGT1A mediated by the stop transfer sequence by analysing the subcellular localization and sensitivity to endoglycosidases of chimeric proteins formed by fusion of UGT1A stop transfer sequence (TMD/CT) with the ectodomain of the plasma membrane CD4 reporter protein. We showed that the stop transfer sequence, when attached to C-terminus of the CD4 ectodomain was able to prevent it from being transported to the cell surface. The protein was retained in the ER indicating that this sequence functions as an ER localization signal. Furthermore, we demonstrated that ER localization conferred by the stop transfer sequence was mediated in part by the KSKTH retrieval signal located on the CT. Interestingly, our data indicated that UGT1A TMD alone was sufficient to retain the protein in ER without recycling from Golgi compartment, and brought evidence that organelle localization conferred by UGT1A TMD was determined by the length of its hydrophobic core. We conclude that both retrieval mechanism and static retention mediated by the stop transfer sequence contribute to ER residency of UGT1A proteins.     

Forward Transport of K2p3.1: mediation by 14-3-3 and COPI, modulation by p11.

Surface expression of the K(2P)3.1 two-pore domain potassium channel is regulated by phosphorylation-dependent binding of 14-3-3, leading to suppression of coatomer coat protein I (COPI)-mediated retention in endoplasmic reticulum (ER). Here, we investigate the nature of the macromolecular regulatory complexes that mediate forward and retrograde transport. We demonstrate that (i) the channel employs two separate but interacting COPI binding sites on the N- and C-termini; (ii) disrupting COPI binding to either site interferes with the ER retention; (iii) p11 and 14-3-3 do not interact on their own; (iv) p11 binding to the C-terminal retention motif is dependent on 14-3-3; and (v) p11 is coexpressed in only a subset of tissues with K(2P)3.1, while 14-3-3 expression is ubiquitous. We conclude that K(2P)3.1 forward transport requires 14-3-3 suppression of COPI binding, whereas p11 serves a modulatory role.     

Subclass-specific localization and trafficking of Arabidopsis p24 proteins in the ER-Golgi interface

We describe a comprehensive analysis of the subcellular localization and in vivo trafficking of Arabidopsis p24 proteins. In Arabidopsis, there are 11 p24 proteins, which fall into only δ and β subfamilies. Interestingly, the δ subfamily of p24 proteins in Arabidopsis is elaborated spectacularly in evolution, which can be grouped into two subclasses: p24δ1 and p24δ2. We found that, although all p24δ proteins possess classic COPII/COPI binding motifs in their cytosolic C-termini, p24δ1 proteins are localized to the endoplasmic reticulum (ER), p24δ2 proteins are localized to both ER and Golgi. Two p24β proteins reside largely in Golgi. Similar to Atp24 (termed p24δ1c in this study), p24δ2d also cycles between the ER and Golgi. Interestingly, coexpression with p24β1 could retain p24δ2d, but not p24δ1d in Golgi. We revealed that the lumenal coiled-coil domain of p24δ2d is required for its steady-state localization in Golgi, probably through its interaction with p24β1. In p24β1, there is no classic COPII or COPI binding motif in its C-terminus. However, the protein also cycles between the ER and Golgi. We found that a conserved RV motif located at the extreme end of the C-terminus of p24β1 plays an important role in its Golgi target.     

14-3-3 dimers probe the assembly status of multimeric membrane proteins

BACKGROUND: Arginine-based endoplasmic reticulum (ER) localization signals are involved in the heteromultimeric assembly of membrane protein complexes like ATP-sensitive potassium channels (K(ATP)) or GABA(B) G protein-coupled receptors. They constitute a trafficking checkpoint that prevents ER exit of unassembled subunits or partially assembled complexes. For K(ATP) channels, the mechanism that leads to masking of the ER localization signals in the fully assembled octameric complex is unknown. RESULTS: By employing a tetrameric affinity construct of the C terminus of the K(ATP) channel alpha subunit, Kir6.2, we found that 14-3-3 isoforms epsilon and zeta specifically recognize the arginine-based ER localization signal present in this cytosolic tail. The interaction was reconstituted by using purified 14-3-3 proteins. Competition with a nonphosphorylated 14-3-3 high-affinity binding peptide implies that the canonical substrate binding groove of 14-3-3 is involved. Comparison of monomeric CD4, dimeric CD8, and artificially tetramerized CD4 fusions correlates the copy number of the tail containing the arginine-based signal with 14-3-3 binding, resulting in the surface expression of the membrane protein. Binding experiments revealed that the COPI vesicle coat can specifically recognize the arginine-based ER localization signal and competes with 14-3-3 for the binding site. CONCLUSIONS: The COPI vesicle coat and proteins of the 14-3-3 family recognize arginine-based ER localization signals on multimeric membrane proteins. The equilibrium between these two competing reactions depends on the valency and spatial arrangement of the signal-containing tails. We propose a mechanism in which 14-3-3 bound to the correctly assembled multimer mediates release of the complex from the ER.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

(Arg)3 within the N-terminal domain of glucosidase I contains ER targeting information but is not required absolutely for ER localization

Glucosidase I is an endoplasmic reticulum (ER) type II membrane enzyme that cleaves the distal alpha1,2-glucose of the asparagine-linked GlcNAc2-Man9-Glc3 precursor. To identify sequence motifs responsible for ER localization, we prepared a protein chimera by transferring the cytosolic and transmembrane domain of glucosidase I to the luminal domain of Golgi-Man9-mannosidase. The GIM9 hybrid was overexpressed in COS 1 cells as an ER-resident protein that displayed alpha1,2-mannosidase activity, excluding the possibility that the glucosidase I-specific domains interfere with folding of the Man9-mannosidase catalytic domain. After substitution of the Args in position 7, 8, or 9 relative to the N-terminus by leucine, the GIM9 mutants were transported to the cell surface indicating that the (Arg)3 sequence functions as an ER-targeting motif. Cell surface expression was also observed after substitution of Arg-7 or Arg-8 but not Arg-9 in GIM9 by either lysine or histidine. Thus the side chain structure, including its positive charge, appears to be essential for signal function. Analysis of the N-linked glycans suggests that the (Arg)3 sequence mediates ER localization through Golgi-to-ER retrograde transport. Glucosidase I remained localized in the ER after truncation or mutation of the N-terminal (Arg)3 signal, in contrast to comparable GIM9 mutants. ER localization was also observed with an M9GI chimera consisting of the cytosolic and transmembrane domain of Man9-mannosidase and the glucosidase I catalytic domain. ER-specific targeting information must therefore be provided by sequence motifs contained within the glucosidase I luminal domain. This structural information appears to direct ER localization by retention rather than by retrieval, as concluded from N-linked Man9-GlcNAc2 being the major glycan released from the wild-type enzyme.     

双孔钾通道TASK-1的研究进展

双孔钾离子通道是一种背景钾离子通道,广泛分布于各种兴奋和非兴奋细胞中,并具有许多重要的生理功能。TASK-1是双孔钾离子通道家族的重要一员,它对缺氧和细胞外酸化敏感,参与形成心肌动作电住平台期,调节呼吸、肺动脉平滑肌收缩和醛固酮的分泌,并且是麻醉剂的作用靶点,人们不断对其进行研究并取得了很多重要结果,本文将概述双孔钾通道TASK-1的研究进展。     

Di-arginine signals and the K-rich domain retain the Ca²⁺ sensor STIM1 in the endoplasmic reticulum

STIM1 is a core component of the store‐operated Ca²⁺‐entry channel involved in Ca²⁺‐signaling with an important role in the activation of immune cells and many other cell types. In response to cell activation, STIM1 protein senses low Ca²⁺ concentration in the lumen of the endoplasmic reticulum (ER) and activates the channel protein Orai1 in the plasma membrane by direct physical contact. The related protein STIM2 functions similar but its physiological role is less well defined. We found that STIM2, but not STIM1, contains a di‐lysine ER‐retention signal. This restricts the function of STIM2 as Ca²⁺ sensor to the ER while STIM1 can reach the plasma membrane. The intracellular distribution of STIM1 is regulated in a cell‐cycle‐dependent manner with cell surface expression of STIM1 during mitosis. Efficient retention of STIM1 in the ER during interphase depends on its lysine‐rich domain and a di‐arginine ER retention signal. Store‐operated Ca²⁺‐entry enhanced ER retention, suggesting that trafficking of STIM1 is regulated and this regulation contributes to STIM1s role as multifunctional component in Ca²⁺‐signaling.     

Topology and endoplasmic reticulum retention signals of the lysosomal storage disease-related membrane protein CLN6

CLN6 is a polytopic membrane protein of unknown function resident in the endoplasmic reticulum (ER). Mutant CLN6 causes the lysosomal storage disorder neuronal ceroid lipofuscinosis. Defining the topology of CLN6, and the structural domains and motifs required for interaction with cytosolic and luminal proteins may allow insights into its function. In this study we analysed the topology, ER retention and oligomerization of CLN6. We demonstrated, by differential membrane permeabilization of transfected BHK cells using specific detergents and two distinct antibodies, that CLN6 contains an N-terminal cytoplasmic domain, seven transmembrane domains, and a luminal C terminus. Mutational analyses and confocal immunofluorescence microscopy showed that changes of potential ER localization signals in the N- or C-terminal domain (a triple arginine cluster, and a dileucine motif) did not alter the subcellular localization of CLN6. The deletion of a dilysine motif impaired partially the ER localization of CLN6. Furthermore, expression analyses of fusion and deletion constructs in non-neuronal and neuronal cells suggested that two portions of CLN6 contributed to its retention within the ER. We showed that the N-terminal domain was necessary but not sufficient for ER retention of CLN6 and that deletion of transmembrane domains 6 and 7 was accompanied with the loss of ER localization and, in some instances, trafficking to the cisGolgi. From these data we concluded that CLN6 maintains its ER localization by expressing retention signals present in both the N-terminal cytosolic domain and in the carboxy-proximal transmembrane domains 6 and 7. Additionally, the ability of CLN6 to homodimerize may also prevent exit from the ER via an interaction with membrane-associated factors.     

Expression of TASK-1, a pH-sensitive twin-pore domain K(+) channel,in rat myocytes

We have investigated the expression of TASK-1, a pH-sensitive, twin-pore domain K(+) channel in the rat heart. A mammalian cell line of Chinese hamster ovary cells (CHO), transfected with a plasmid containing mouse TASK-1, demonstrated the specificity of the anti-TASK-1 antibody. TASK-1 expression in cardiac tissue was initially demonstrated by Western blot and then localized by immunofluorescence. In single rat ventricular myocytes, strong staining of the TASK-1 protein was located at the intercalated disks and across the cell in a striated pattern, corresponding to the transverse axial tubular network (T tubules). In contrast, single rat atrial myocytes were stained at the intercalated disks with a weak punctate, striated pattern corresponding to underdeveloped T tubules. Also, formamide was used to induce the detubulation of ventricular myocytes, which enabled confirmation that TASK-1 protein expression occurs in T tubules. Consistent with this, RT-PCR revealed the expression of TASK-1 mRNA in total RNA from both the ventricles and atria. In this study, we conclusively demonstrated that TASK-1 protein and mRNA were expressed in rat atrial and ventricular tissue. The extensive distribution of TASK-1 shown to exist within myocyte membranes may provide a potential future target for antiarrhythmic drugs.     

Identification and functional characterization of cereblon as a binding protein for large-conductance calcium-activated potassium channel in rat brain

Large-conductance Ca2+-activated K+ (BK(Ca)) channels are activated by membrane depolarization and modulated by intracellular Ca2+. Here, we report the direct interaction of cereblon (CRBN) with the cytosolic carboxy-terminus of the BK(Ca) channel alpha subunit (Slo). Rat CRBN contained the N-terminal domain of the Lon protease, a 'regulators of G protein-signaling' (RGS)-like domain, a leucine zipper (LZ) motif, and four putative protein kinase C (PKC) phosphorylation sites. RNA messages of rat cereblon (rCRBN) were widely distributed in different tissues with especially high-levels of expression in the brain. Direct association of rCRBN with the BK(Ca) channel was confirmed by immunoprecipitation in brain lysate, and the two proteins were co-localized in cultured rat hippocampal neurons. Ionic currents evoked by the rSlo channel were dramatically suppressed upon coexpression of rCRBN. rCRBN decreased the formation of the tetrameric rSlo complex thus reducing the surface expression of functional channels. Therefore, we suggest that CRBN may play an important role in assembly and surface expression of functional BK(Ca) channels by direct interaction with the cytosolic C-terminus of its alpha-subunit.     

The correlation between expression and localization of a foreign gene product in rice endosperm

Glucagon-like peptide 1 (GLP-1) is a 30 amino acid peptide hormone involved in insulin stimulation that is dependent upon blood glucose levels. We have previously reported that when this short peptide gene was directly expressed under the control of a glutelin promoter and its signal peptide, it was not accumulated in transgenic rice seed due to gene silencing. However, when the modified GLP-1 (mGLP-1) gene was enlarged to 5xmGLP-1 (mGLPx5) by tandem repeat, no silencing was observed. The mGLPx5 peptide could be accumulated in rice seed and its localization was mainly limited to the endoplasmic reticulum (ER). We also investigated alternative cellular localization sites that would increase accumulation. The relationship between the expression level and localization was examined by attaching the chitinase signal peptide to mGLPx5 to direct it into the intercellular space (apoplast), or by expression as a fusion protein with glutelin by insertion into a variable region of the acidic subunit, thus directing the peptide to protein body II (PB II). Attachment of the KDEL ER retention signal to the 6xmGLP-1 (mGLPx6) or its fusion to the C-terminus of the 13 kDa prolamin directed the peptide to the ER or PB I, respectively. Unexpectedly, these results indicated that mGLPx5 without any signal except for the glutelin signal peptide was accumulated to the greatest extent in rice endosperm. It can thus be concluded that the ER is a suitable intracellular organelle for accumulation of mGLPx5 peptide.     

双孔钾通道TASK-1 的研究进展

双孔钾离子通道是一种背景钾离子通道,广泛分布于各种兴奋和非兴奋细胞中,并具有许多重要的生理功能。TASK-1是双孔钾离子通道家族的重要一员,它对缺氧和细胞外酸化敏感,参与形成心肌动作电位平台期,调节呼吸、肺动脉平滑肌收缩和醛固酮的分泌,并且是麻醉剂的作用靶点,人们不断对其进行研究并取得了很多重要结果,本文将概述双孔钾通道TASK-1的研究进展。     

TASK-3 dominates the background potassium conductance in rat adrenal glomerulosa cells

In a preceding study we showed that the highly negative resting membrane potential of rat adrenal glomerulosa cells is related to background potassium channel(s), which belong to the two-pore domain channel family. TWIK-related acid-sensitive K+ channel (TASK-1) expression was found in glomerulosa tissue, and the currents elicited by injection of glomerulosa mRNA (I(glom)) or TASK-1 cRNA (I(TASK-1)) showed remarkable similarity in Xenopus laevis oocytes. However, based on the different sensitivity of these currents to acidification, we concluded that TASK-1 may be responsible for a maximum of 25% of the weakly pH-dependent glomerulosa background K+ current. Here we demonstrate that TASK-3, a close relative of TASK-1, is expressed abundantly in glomerulosa cells. Northern blot detected TASK-3 message in adrenal glomerulosa, but not in other tissues. Quantitative RT-PCR experiments indicated even higher mRNA expression of TASK-3 than TASK-1 in glomerulosa tissue. Similarly to the glomerulosa background current, the current expressed by injection of TASK-3 cRNA (I(TASK-3)) was less acid-sensitive than I(TASK-1). Ruthenium red in the micromolar range inhibited I(glom) and I(TASK-3), but not I(TASK-1). Like I(TASK-1), I(TASK-3) was inhibited by stimulation of AT1a angiotensin II receptor coexpressed with the potassium channel. The high level of expression and its pharmacological properties suggest that TASK-3 dominates the resting potassium conductance of glomerulosa cells.     

In vivo trafficking and localization of p24 proteins in plant cells

p24 proteins constitute a family of putative cargo receptors that traffic in the early secretory pathway. p24 proteins can be divided into four subfamilies (p23, p24, p25 and p26) by sequence homology. In contrast to mammals and yeast, most plant p24 proteins contain in their cytosolic C-terminus both a dilysine motif in the −3, −4 position and a diaromatic motif in the −7, −8 position. We have previously shown that the cytosolic tail of Arabidopsis p24 proteins has the ability to interact with ARF1 and coatomer (through the dilysine motif) and with COPII subunits (through the diaromatic motif). Here, we establish the localization and trafficking properties of an Arabidopsis thaliana p24 protein ( At p24) and have investigated the contribution of the sorting motifs in its cytosolic tail to its in vivo localization. At p24-red fluorescent protein localizes exclusively to the endoplasmic reticulum (ER), in contrast with the localization of p24 proteins in other eukaryotes, and the dilysine motif is necessary and sufficient for ER localization. In contrast, At p24 mutants lacking the dilysine motif are transported along the secretory pathway to the prevacuolar compartment and the vacuole, although a significant fraction is also found at the plasma membrane. Finally, we have found that ER export of At p24 is COPII dependent, while its ER localization requires COPI function, presumably for efficient Golgi to ER recycling.     

The C-terminal C1 cassette of the N -methyl-D-aspartate receptor 1 subunit contains a bi-partite nuclear localization sequence

The N -methyl-D-aspartate receptor (NMDAR) is a multimeric transmembrane protein composed of at least two subunits. One subunit, NR1, is derived from a single gene and can be subdivided into three regions: the N-terminal extracellular domain, the transmembrane regions, and the C-terminal intracellular domain. The N-terminal domain is responsible for Mg2+ metal ion binding and channel activity, while the transmembrane domains are important for ion channel formation. The intracellular C-terminal domain is involved in regulating receptor activity and subcellular localization. Our recent experiments indicated that the intracellular C-terminal domain, when expressed independently, localizes almost exclusively in the nucleus. An examination of the amino acid sequence reveals the presence of a putative nuclear localization sequence (NLS) in the C1 cassette of the NR1 intracellular C-terminus. Using an expression vector designed to test whether a putative NLS sequence is a valid, functional NLS, we have demonstrated that a bi-partite NLS does in fact exist within the NR1-1 C-terminus. Computer algorithms identified a putative helix-loop-helix motif that spanned the C0C1 cassettes of the C-terminus. These data suggest that the NR1 subunit may represent another member of a family of transmembrane proteins that undergo intramembrane proteolysis, releasing a cytosolic peptide that is actively translocated to the nucleus leading to alterations in gene regulation.