Targeting of Vacuolar Membrane Localized Members of the TPK Channel Family
a Molecular Plant Physiology and Biophysics, Julius-von-Sachs-Institute, University of Würzburg, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany
b Heidelberger Institut für Pflanzenwissenschaften (HIP), University of Heidelberg, Im Neuenheimer Feld 230, 69120 Heidelberg, Germany (K.S.)
c Present address: Physiological Ecology of Plants, University of Tübingen, Auf der Morgenstelle 1, 72076 Tübingen, Germany
1 To whom correspondence should be addressed at the Department of Molecular Plant Physiology and Biophysics. E-mail hedrich{at}botanik.uni-wuerzburg.de, fax +49 (0)931/888-6157, tel. +49 (0)931/888-6100.
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Abstract |
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 TOP Abstract INTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Four members of the tandem-pore potassium channel family of Arabidopsis thaliana (TPK1, 2, 3, and 5) reside in the vacuolar membrane, whereas TPK4 is a plasma membrane K+-channel. By constructing chimeras between TPK1 and TPK4, we attempted to identify channel domains involved in the trafficking process and found that the TPK1 cytoplasmic C-terminal domain (CT) is critical for the ER- as well as Golgi-sorting steps. Following site-directed mutagenesis, we identified a diacidic motif (DLE) required for ER-export of TPK1. However, this diacidic motif in the C-terminus is not conserved among other members of the TPK family, and TPK3 sorting is independent of its CT. Moreover, the 14-3-3 binding site of TPK1, essential for channel activation, is not involved in channel sorting.
Received for publication May 29, 2008. Accepted for publication September 8, 2008.
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INTRODUCTION |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Plant ion channel activities have been recorded from plasma membrane and various internal membranes and organelles, such as ER (Klüsener et al., 1995), vacuole (Hedrich and Neher, 1987), mitochondria (Petrussa et al., 2001), and chloroplasts (Schönknecht et al., 1988). Among the different channel types identified, potassium channels have been mainly localized to the plasma membrane (Ache et al., 2000; Hirsch et al., 1998) and the vacuolar membrane (Czempinski et al., 2002; Gobert et al., 2007; Latz et al., 2007; Schönknecht et al., 2002; Voelker et al., 2006). This localization probably reflects the housekeeping function of these transport proteins in regulating potassium fluxes across the two membranes for ion homeostasis and turgor formation. K+-channel diversity in the plasma membrane is generated by homo- and heterotetramers of the nine Shaker-type channel subunits (Dreyer et al., 1997, 2004; Xicluna et al., 2007). In contrast, K+-channels of the vacuolar membrane seem to be constituted mostly of tandem-pore potassium channels of the TPK-family as well as the Kir-like KCO3 (Czempinski et al., 2002; Gobert et al., 2007; Latz et al., 2007; Schönknecht et al., 2002; Voelker et al., 2006). Contributions by heteromeric channel formation among TPKs to vacuolar channel diversity seem unlikely (Voelker et al., 2006). Both TPK1 and TPK4 have been shown to mediate instantaneous potassium currents, but, in contrast to the Ca2+-activated vacuolar channel TPK1 (Bihler et al., 2005; Gobert et al., 2007), TPK4 is localized in the plasma membrane (Becker et al., 2004).
Although differential membrane targeting of individual members is found in many other membrane protein families (AtVAMP-family (Uemura et al., 2005), AtCLC-family (De Angeli et al., 2006; Marmagne et al., 2007), ZmPIP-family (Zelazny et al., 2007) etc.), very little is known about the underlying sorting determinants. The synthesis of membrane proteins takes place at the ER, where the nascent membrane proteins are folded and integrated into the lipid bilayer co-translationally (Deutsch, 2003). As is the case for the animal K(ATP) channel, correct channel assembly seems to be a prerequisite for surface expression (Cartier et al., 2001; Zerangue et al., 1999), since folding and assembly of all ER proteins are under strict surveillance of ER quality-control mechanisms (Ellgaard and Helenius, 2003). It is generally assumed that membrane-bound cargo designated for the late secretory pathway is concentrated at highly motile ER export sites (daSilva et al., 2004), from which COP II-coated vectors bud and fuse with cis-Golgi (Andreeva et al., 2000; Bar-Peled and Raikhel, 1997; Barlowe, 2003; daSilva et al., 2004; Phillipson et al., 2001; Ritzenthaler et al., 2002; Takeuchi et al., 2000; Yang et al., 2005). In contrast to the bulk-flow export of soluble proteins, membrane proteins are often subjected to selective export via interaction with COPII coat proteins.
The interaction of the yeast membrane protein Sys1p with the COPII coat proteins and its subsequent ER-export has been shown to depend on diacidic amino acid motifs usually composed of two aspartates or glutamates separated by another amino acid [D/E]X[D/E] (Malkus et al., 2002; Votsmeier and Gallwitz, 2001). Related motifs are associated with ER-export of membrane proteins in mammals (Ma et al., 2001; Nishimura and Balch, 1997; Sevier et al., 2000; Wang et al., 2004). Recently, diacidic motifs involved in protein targeting have been identified in the plant Golgi proteins GONST1 and CASP as well as in the plant plasma membrane potassium channel KAT1 (Hanton et al., 2005; Mikosch et al., 2006). However, not all exported membrane proteins bear these diacidic motifs. There is evidence for an alternative export motif consisting of two hydrophobic/aromatic residues. This hydrophobic motif, like the diacidic one, mediates COPII coat protein interaction (Kappeler et al., 1997; Nufer et al., 2002); however, the binding site on COPII for each motif appears to be different (Miller et al., 2003). Finally, dibasic motifs as found in the plant membrane-anchored prolyl hydroxylase have been reported being involved in ER-export, too (Yuasa et al., 2005).
For mammalian ion channels, it is well known that surface expression often is regulated by ER retention/retrieval as well as release motifs within these proteins. Retention motifs can be composed of hydrophobic residues like the CVLF-motif in a splice variant of the MaxiK channel (hSlo) (Zarei et al., 2004). More commonly, arginine-based basic motifs have been identified (Scott et al., 2001; Yuan et al., 2003; Zerangue et al., 1999) as in the animal two-pore potassium channel KCNK3 (O'Kelly et al., 2002). Basic motifs bind to COP I coat-proteins responsible for retrograde Golgi–ER transport (Contreras et al., 2004; Nilsson et al., 1989; O'Kelly et al., 2002; Yuan et al., 2003; Zerangue et al., 1999). Thus, to overcome ER-retention, such basic motifs need to be masked. In the case of KCNK3, O'Kelly et al. (2002) have shown that binding of 14-3-3 proteins to the phosphorylated channel prevents β-COP acquisition, allowing forward trafficking of KCNK3.
In plants, trafficking of all Golgi leaving membrane proteins requires a further sorting step regulating transport to lytic vacuoles (LV), protein storage vacuoles (PSV), or to the plasma membrane. Transport to both vacuole types may involve an intermediate prevacuolar compartment (PVC) and specific vesicles such as dense vesicles (DV) and clathrin-coated vesicles (CCV) (Müntz, 2007). Only little is known about the mechanisms, which define the destination of plant membrane proteins along this sorting path. The N-terminal Longin-domain of SNARE proteins of the VAMP-family determines whether these type II transmembrane proteins localize to either the tonoplast or the plasma membrane (Uemura et al., 2005). The vacuolar sorting receptor AtELP/VSR1 interacts with CCV coat proteins (Sanderfoot et al., 1998; Song et al., 2006). Thus, vacuolar sorting is highly analogous to the sorting process of membrane proteins into COPII vesicles involving the interaction with coat-forming proteins through cytoplasmic located binding domains. Since the membrane thickness increases along the secretory pathway, the length of transmembrane segments has been proposed as an additional sorting parameter. Artificially generated transmembrane domains (TMDs) consisting of 17 or 20 aa accumulate in the ER or Golgi, respectively. Longer (23 aa) TMDs were targeted to the plasma membrane, which was thus considered as the default compartment of type I transmembrane proteins lacking any additional sorting information (Brandizzi et al., 2002). This hypothesis was confirmed for targeting of SNARE proteins, which locate to the ER if they contain a TMD comprising 17 aa, but reside in Golgi or post-Golgi compartments when possessing a 20-aa-long TMD (Uemura et al., 2005).
To gain insight into plant multitopic membrane protein sorting, we focused here on the targeting of vacuolar and plasma-membrane localized members of the Arabidopsis TPK channel family. To identify and investigate possible sorting signals, we generated channel chimeras between TPK1 and TPK4. In addition, site-directed mutagenesis in combination with deletion mutants was used to analyse potential sorting signals within the vacuolar TPK1 channel protein. Mutant channel cDNAs were transiently expressed in onion epidermal cells and fluorescent protein fusions were subsequently tracked and localized by confocal laser scanning microscopy. We show here that the vacuolar TPK channels are targeted in a Golgi-dependent fashion to the membrane of the large central vacuole, and that, for TPK1, this process involves selective ER as well as Golgi-export steps in which a C-terminal-located diacidic motif plays an essential role.
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RESULTS |
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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TPK1 Exhibits Golgi-Dependent Vacuolar Targeting
The Arabidopsis potassium channel TPK1 localizes to the vacuolar membrane (Czempinski et al., 2002; Schönknecht et al., 2002). Vacuolar targeting of TPK1 might rely on Golgi-dependent sorting steps or, as reported for the PSV trafficking of
-TIP, could occur along a Golgi-independent route (Jiang and Rogers, 1998). To distinguish between these routes, we applied the fungal toxin Brefeldin A, which inhibits secretion and causes redistribution of Golgi membranes (Driouich et al., 1992). Under control conditions, in cells co-expressing TPK1-mRFP1 and the Golgi-Marker (ST-GFP), in the absence of Brefeldin A, TPK1 channels were observed at the vacuolar membrane while ST-GFP exhibited Golgi staining (Figure 1A–1C). Upon incubation with Brefeldin A, the Golgi-marker redistributed into an ER-like compartment throughout the cell. Similarly, Golgi-apparatus disruption prevented vacuolar trafficking of TPK1 resulting in co-localization with the Golgi-marker (Figure 1D–1F). This indicates that vacuolar targeting of TPK1 occurs via Golgi-dependent sorting machinery.
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14-3-3 Binding Is Not Involved in ER Export
The animal TPK channel KCNK3 possesses a 14-3-3 binding and a basic motif, which are involved in ER-release and surface expression of this membrane protein. Similar motifs are found in Arabidopsis TPK1, both of which are located in close proximity within the cytoplasmic N-terminus (RKRRLRRSRSpAP: the type I 14-3-3-binding motif is underlined). To test whether 14-3-3-mediated ER-release might represent a mechanism allowing forward trafficking of TPK1 in planta, we silenced the respective phosphorylation site (Ser-42) within the TPK1 14-3-3 binding motif (RSRSpAP). Mutating Ser-42 to alanine (TPK1-S42A) prevented 14-3-3 binding in vitro (Latz et al., 2007), but not vacuolar targeting of TPK1 (Figure 2A). Likewise, mimicking phosphorylation by exchanging Ser-42 for glutamate (TPK1-S42E) did not affect vacuolar localization of TPK1 (Figure 2B). Thus, 14-3-3 binding, which regulates channel gating (Latz et al., 2007), seems not to be involved in targeting of TPK1 to the vacuole.
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Trafficking of Chimeric TPK Channels
The generation of chimeras between structurally and functionally related proteins exhibiting different sub-cellular localization has been used to study molecular determinants important for trafficking (Cervelli et al., 2004; Jiang and Rogers, 1998; Nothwehr et al., 1993, 1995; Nothwehr and Stevens, 1994; Oufattole et al., 2005). Based on individual channel fusions with GFP, we confirmed the localization of TPK1, 2, 3, and 5 at the vacuolar membrane as well as TPK4 expression in the plasma membrane (Latz et al., 2007). The number of TPK4 channels targeted to the plasma membrane seemed to be strictly controlled, however, since a large fraction of TPK4 was found to be localized in the ER. Longer incubation after transformation did not result in enhanced export to the plasma membrane.
TPK1 and TPK4 share 35% amino acid identity in the region of TMD 1–4. Their cytoplasmic domains, in contrast, show little sequence conservation (5% identity in the amino terminus). We exchanged the amino- (NT) and the carboxy-termini (CT) between TPK1 and TPK4 and determined the sub-cellular localization of the resulting chimeric proteins. Supporting our observation that the 14-3-3 motif of TPK1 is not required for vacuolar targeting, TPK1 possessing the NT of TPK4 still trafficked to the tonoplast (Figure 2C). We thus concluded that the NT of TPK1 is not necessary for vacuolar targeting of TPK1, nor is the TPK4 NT sufficient to redirect TPK1 targeting. Consequently, no dominant targeting motifs seem to be present in the NT of TPK1 and TPK4.
We then exchanged the CT of TPK1 with the CT of TPK4. TPK1-TPK4CT was found in the ER, but was no longer present in the tonoplast (Figure 2D). Conversely, the TPK1 CT did not cause redirection of TPK4-TPK1CT to the vacuole (Supplemental Figure 1). These observations indicate that the TPK1 CT might be required, but is not sufficient for vacuolar targeting.
In addition to switching cytoplasmic domains, we also generated TPK1/4 chimeras, which comprised the N-terminus, the first pore, and the internal cytoplasmic loop of one channel fused to the second pore and cytoplasmic C-terminus of the other channel. Following transient expression, both chimeras accumulated in the ER network (Figure 2E and 2F), confirming that the TPK1 CT is not sufficient for vacuolar localization. At this point, we cannot exclude the possibility that the TPK1/4 chimeric channels are retained in the ER by other means than the missing targeting information of the TPK1 CT. To test whether the TPK1 CT actually contains targeting signals and to identify them, we dissected the C-terminus of TPK1 by a series of truncations.
ER and Golgi Sorting Signals in TPK1 C-Terminus
According to the consensus transmembrane prediction for TPK channels (Schwacke et al., 2003), the cytoplasmic CT of TPK1 initiates at amino acid position 271, C-terminal to TMD4, and extents to amino acid position 363 (Figure 3A). TPK1 also contains two EF-hands in this region that exhibit structural homology to the neuronal Ca2+-binding protein calcineurin. Taking advantage of the crystal structures of calcineurin in complex with FKBP12 and FK506 (Griffith et al., 1995), we generated a homology model highlighting the helix-loop-helix structure (H-L-H) of this domain (Figure 3B). Based on this model, we generated deletion mutants, which were named according to the position of their end point relative to the helices and bridging loops (H1-L1-H2 for EF-hand I, L2 for the connecting loop, H3-L3-H4 for EF-hand II).
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The first construct investigated was the TPK1-
CT mutant lacking the entire cytoplasmic CT (Figure 4A–4C, Table 1, CT). This mutant as well as TPK1- H1 (Supplemental Figure 2 and Table 1) exhibited persistent ER retention. In a search for ER escape motifs, we gradually increased the length of TPK1 carboxy terminus (CT) relative to TMD4. When we extended the TPK1 CT to include helix H1 (TPK1-L1), TPK1 escaped from the ER 16 h following transformation and partially entered Golgi stacks (Figure 4D–4F). Compared to full-length TPK1, however, trafficking of TPK1-L1 was considerably slower (Table 1). After extending the CT to include H1-L1 (TPK1-H2), ER export appeared accelerated and, 24 h following transformation, mutant channels appeared in the vacuolar membrane (Figure 4G–4I, Table 1, H2). This suggests that either L1 contains vacuolar-sorting information or that vacuolar localization is simply a consequence of accelerated ER-to-Golgi transport. Interestingly, the TPK1 mutants L2 and H3, possessing the entire EF-hand I, seemed to have lost vacuolar targeting information again and appeared almost exclusively in the Golgi when recorded 24 h following transformation (Figure 4J–4O and Table 1). About 43 h later, however, those channels also reached the vacuole, indicating delayed Golgi sorting (Table 1). Finally, when appending helix H3 (TPK1-L3) or extending the TPK1 CT beyond this region (TPK1-H4), all truncated mutants showed wild-type-like targeting to the vacuolar membrane (Figure 4P–4R, Table 1, and Supplemental Figure 2).
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TPK1 ER-Export Uses a CT Diacidic Motif
Based on our findings on TPK1 targeting, the minimal requirement for ER-export of the channel is H1, and, for further export into the Golgi, L1 is necessary. Notably, both domains are missing in TPK4 (Figure 3A), which is largely retained in the ER. We therefore looked for known sorting motifs within the H1-L1 region and identified three diacidic motifs (292-ITNNDLEAADLDEDGVV-308, underlined). While the first diacidic motif (DAI) is located within H1 (Figure 3A), DAII and DAIII comprise part of the L1 loop in the C-terminus of TPK1. To test whether one of these diacidic motifs plays a role in ER-export of TPK1, we replaced the respective acidic amino acids by glycines. Mutations in DAII and III did not affect vacuolar trafficking of TPK1 (Figure 5B and Table 1, DAII+III). In contrast, the DAI mutation of TPK1 resulted in full ER-retention (Figure 5A and Table 1, DAI), supporting the idea that a diacidic motif is required for the ER export of TPK1.
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Interestingly, plant TPK1 orthologs from potato or rice, for example, carry a similar DLE-motif within an EF-hand, suggesting a common principle in the ER-export of these channels. On the contrary, the Arabidopsis paralogues of TPK1 lack such a diacidic motif in their respective C-terminal H1 domain (Figure 3A, vertical box) suggesting that these channels either use different signals for selective ER export or leave the ER by bulk-flow. In the course of these experiments, we tested the importance of the TPK3 CT in vacuolar trafficking of this ubiquitously expressed member of the TPK family. In these studies, we observed that TPK3
CT, in contrast to TPK1CT, could be effectively transported to the vacuolar membrane (Figure 6A–6C). Due to the lack of unambiguous CT sorting motifs in TPK3, we focused on the role of TMDs, since TMDs have already been shown to bear ER-retention motifs or physical sorting information (Brandizzi et al., 2002; Fiedler and Rothman, 1997; Jiang and Rogers, 1998; Sato et al., 2003; Wang et al., 2002). In order to test whether TMD-located signals are involved in vacuolar sorting, the last transmembrane domain of either TPK1 or TPK3 was fused to mRFP1. The TPK1-TMD4 construct, comprising aa 217–273, exhibited mainly localization in the ER up to 43 h post transformation. This is in contrast to the ER/Golgi-marker ERD2-GFP, which intensely stains the Golgi (Figure 6D–6F). Similar to ERD2, the TPK3-TMD4 construct, composed of aa 292–353, accumulated in the Golgi (Figure 6J–6L). To investigate whether the TPK1 CT possessing the diacidic motif would promote the ER-export of TPK1-TMD4, we extended this construct to cover aa 217–363. ER-export of the corresponding mRFP1 fusion protein, however, was still hampered resulting in ER retention of TPK1-TMD4-CT (Figure 6G–6I).
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Surface expression and thus channel density in target membranes can be regulated by ER retention (Arniges et al., 2006; O'Kelly et al., 2002; Yuan et al., 2003; Zerangue et al., 1999) and by cycling between endosomal and plasma membranes (e.g. Hurst et al., 2004). In this study, we have explored the mechanisms involved in trafficking of TPK ion channels to the vacuolar membrane. Our studies prove that the Arabidopsis tandem pore channel TPK1 targets to the central, lytic vacuole via a Golgi-dependent pathway. This is in contrast to the aquaporin
-TIP, which targets to the protein storage vacuole in a Golgi-independent manner (Jiang and Rogers, 1998; Park et al., 2004). However, we cannot exclude the possibility that TPK1 is also transported to smaller, probably neutral vacuoles or protein storage vacuoles, since microarray-hybridization data provide clear evidence for the expression of TPK1 in seeds (Zimmermann et al., 2004, genevestigator, https://www.genevestigator.ethz.ch). Localization to smaller vacuoles next to the central vacuole was also shown for the ectopically expressed vacuolar AtTPKs in Arabidopsis thaliana and tobacco cells (Voelker et al., 2006).
In addition to their physiological functions, 14-3-3 proteins also appear to regulate ion channel trafficking. O'Kelly and colleagues (2008) showed that 14-3-3 proteins control plasma membrane trafficking of the animal potassium channel KCNK3 by masking an ER-retention motif. Furthermore, chimeric ion channels carrying an unusual 14-3-3 binding motif (SWTY) exhibit an elevated plasma membrane density (Coblitz et al., 2005; Shikano et al., 2005). Recently, Sottocornola and colleagues (2008) proposed a trafficking-promoting effect of 14-3-3 proteins in addition to the gating effect on the plant plasma membrane potassium channel KAT1. Although TPK1 activity is enhanced by 14-3-3-induced channel opening (Latz et al., 2007), ER export and thus vacuolar trafficking of this channel do not involve 14-3-3-dependent mechanisms. On the contrary, our results demonstrate that the CT of TPK1, rather that the N-terminus encompassing the 14-3-3 binding motif, is necessary for vacuolar trafficking. Deletion studies within the TPK1 CT revealed a stretch of 24 amino acids as the minimal prerequisite for vacuolar targeting. The length of the deletion in the TPK1 carboxy-terminus correlated with the rate of vacuolar trafficking, consistent with previous studies suggesting a proposed gradual loss of essential domains involved in sorting (Cervelli et al., 2004; Hanton et al., 2005; Mason et al., 2006). TPK1 CT deletion mutants CT and H1 were retained in the ER while the truncation variants L3 and H3 were capable of ER escape but were delayed in Golgi-export. This indicates that active sorting into vesicles dominates over the so-called bulk-flow mode of transport. In the case of the TPK1-L1 mutant, which successfully escapes from the ER, but does not localize to the tonoplast, loss of a vacuolar sorting signal seems likely. On the other hand, trafficking of the majority of deletion mutants appeared much slower than that of the native channel, rendering judgement of targeting efficiency difficult. It should be noted that onion cells died within 2–3 d after particle bombardment, as assessd by examining cytoplasmic streaming or the movement of Golgi-stacks, leaving only a narrow time frame for the analysis of slowly targeting channels.
Although the TPK1 CT is required for vacuolar trafficking, it was not sufficient for targeting the half-channel chimeras TPK4-P1–TPK1-P2 or TPK4-1CT to the vacuole, indicating that additional channel domains participate in channel sorting. It is tempting to speculate that these chimeras fold improperly or fail to assemble correctly into the membrane, since TMDs are known to exert cooperative functions in translational membrane integration as shown for TMD3 and TMD4 of the Shaker channel AtKAT1 (Sato et al., 2002). In type I membrane proteins, the TMD alone can be sufficient for targeting (Jiang and Rogers, 1998). We therefore tested the last TMD of TPK1 as well as TPK3 in targeting mRFP1 to the compartments of the secretory pathway. The TPK1-TMD4 construct mainly resided in the ER, whereas TPK3-TMD4 accumulated in the Golgi and neither channel could be observed in the vacuolar membrane. These results confirm findings by Brandizzi et al. (2002) made with artificial TMDs of different length. In that study, a reporter fused to a 17-aa-long TMD localized to the ER, while a 20-aa TMD directed a reporter protein to the Golgi. A third, 23-aa-long TMD was responsible for reporter activity at the plasma membrane. The last TMDs of TPK1 and TPK3 are predicted to span 22 and 21 aa, respectively, while the other TMDs are between 19 and 22 aa. According to the data of Brandizzi, they might be sufficiently long for ER-escape but too short for exit into the Golgi. It is questionable whether a longer TPK TMD4 would thus lead to vacuolar localization, because the default destination for type I proteins seems to be the plasma membrane rather than the vacuolar membrane (Bednarek and Raikhel, 1992; Denecke et al., 1990).
In search of motifs that would direct ER export, we looked for sorting signals within the CT of TPK1 and found several diacidic motifs. Our analyses suggests that two of these (DAII and DAIII) are not involved in sorting, but rather coordinate/bind Ca2+ as part of the EF-hand I. The diacidic motif DAI (296-DLE-298) in Helix H1 of EF-hand I instead confers on the protein the ability to escape from ER. Upon co-expression with the wild-type TPK1, the corresponding mutant does not co-localize with wild-type channel anymore. This may argue for a function not only in ER-export, but additionally in channel assembly. Disruption of assembly-promoting domains often leads to ER-retention. On the other hand, two TPK1CT deletion mutants possessing an intact DAI, TPK1H2(d309–363)::mGFP4 and TPK1H3(d328–363)::mGFP4, also exhibit non-overlapping fluorescence when co-expressed with TPK1::mRFP1 (Figure 4G and 4M). Both mutants, however, escape from ER. Our results suggest that lack of co-localization of TPK1-DAI and wild-type TPK1 is due to the destruction of the ‘DLE’ ER-export signal. This ER-export signal is identical to those identified in the membrane proteins Sys1p, CASP, and GONST1 (Hanton et al., 2005; Votsmeier and Gallwitz, 2001), but is different from the diacidic motif of the potassium channel KAT1 (DLD) (Mikosch et al., 2006). Since CASP and GONST1 are Golgi-localized, KAT1 is at plasma membrane, and TPK1 is tonoplast localized, this ER-export motif seems not to be involved post-Golgi sorting. Instead, it is more likely that this motif mediates the binding of TPK1 to a COPII coat protein (Sec23/Sec24-complex) as shown for a similar diacidic motif in yeast (Votsmeier and Gallwitz, 2001). In this way, the TPK1 channel, like other diacidic motif-containing membrane proteins, could recruit the COPII coat promoting the de-novo formation of ER export sites (Hanton et al., 2007).
Notably, the diacidic ER-export motif found in TPK1 is conserved among its plant orthologs (Supplemental Figure 3), suggesting an evolutionarily conserved and common targeting mechanism. At the same time, our work shows that within Arabidopsis, members of the same family (TPKs) residing in the same (vacuolar) compartment employ different targeting strategies as judged from structural differences in their C-terminal domains. Since consecutive deletions within the C-terminus did not result in a gradual impairment of trafficking, the TPK1 vacuolar-sorting motif seems to be based on a more complex tertiary structure, rather than being comprised of a sequential motif. The detailed mechanisms underlying post-Golgi sorting of plant ion channels in general and vacuolar transport proteins in particular still have to be identified.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Cloning of AtTPK Deletion Mutants, Chimeras, and Point Mutants
Cloning of TPK1-TPK5 was described previously (Becker et al., 2004; Latz et al., 2007). TPK1 carboxy-terminal deletion-mutants were generated using PCR amplification with TPK1s and the following anti-sense primers:
CTas = aa 1–273; H1as = aa 1–284; L1as = aa 1–299; H2as = aa 1–308; L2as = aa 1–321; H3as = aa 1–327; L3as = aa 1–338; H4as = aa 1–343. All the oligonucleotide-sequences can be retrieved from Supplemental Data File 1. The PCR-products were then TOPO-ligated into pCRII-TOPO® (Invitrogen, Paisley, UK) and subsequently inserted N-terminally in frame with either mRFP1 (Campbell et al., 2002) or mGFP4 (Haseloff et al., 1997) into pPILY (Ferrando et al., 2000) using Cfr9I and Eco47III/KspAI. All site-directed mutagenesis reactions were performed using either Pfu-Turbo® (Stratagene, La Jolla, CA, USA) or PhusionTM (Finnzymes, Espoo, Finland) as DNA-polymerases. The NT-chimeras TPK1-TPK4NT::mGFP4/pPILY was generated by introducing an EcoRI recognition upstream of the sequence encoding the first trans-membrane domain of TPK1 and TPK4 using site-directed mutagenesis with TPK1::mGFP4/pPILY and TPK4::mGFP4/pPILY as template and TPK1-EcoRIs, TPK4-EcoRIs and the complementary oligonucleotides as primers. The TPK1 NT was then replaced by TPK4 NT using the restriction sites for Cfr9I and EcoRI.
The CT-Chimeras TPK1-TPK4CT::mGFP4/pPILY and TPK4-TPK1CT::mGFP4/pPILY were obtained by introducing KspAI recognition site C-terminally of the last TMD of TPK4 by site-directed mutagenesis of TPK4/pcDNA3.1D-V5-His-TOPO using TPK4-KspAIs and the complementary anti-sense primer. TPK1CT was then introduced via the restriction sites for BamHI and KspAI from TPK1CT/pCRII-TOPO. In order to introduce TPK1CT into TPK4-KspAI/pcDNA3.1D, the TPK1CT was PCR amplified using TPK1CTs and TPK1as, TOPO-ligated into pCRII-TOPO® and then transferred via KspAI and ApaI. Finally, these chimeric coding sequences were ligated into mGFP4/pPILY via Cfr9I and Eco47III.
The pore-chimeras, TPK4P1-TPK1P2 and TPK1P1–TPK4P2, were created by PCR amplification of either the channel part N-terminally or C-terminally of TMD3 using TPK1sense and TPK1-TMD3as (TPK1-P1), TPK1-TMD3s and TPK1as (TPK1-P2), TPK4s and TPK4-TMD3as (TPK4-P1), TPK4-TMD3s and TPK4as (TPK4-P2) as primers.
The P1-PCR products were TOPO-cloned into pcDNA3.1D/V5-His-TOPO® (Invitrogen, Paisley, UK) and P2-PCR products into pCRII-TOPO®. The channel fragments were combined using XhoI and EcoRI within TPKx-P1/pcDNA3.1D-V5-His-TOPO® and finally ligated into mGFP4/pPILY via BamHI and Eco47III.
The TPK1 S42A, S42E mutations were introduced as described elsewhere (Latz et al., 2007). The D296G-E298G (DAI) and D300G-D302G (DAII+III) mutants of TPK1 were generated by site-directed mutagenesis of TPK1::mGFP4/pPILY with D296G–E298Gs, D300G–D302Gs and the complementary anti-sense primers.
TPK3 without its CT was generated by PCR using TPK3s and TPK3CTas as primers. The PCR-product was TOPO-ligated into pCRII-TOPO® and then ligated via XbaI and KspAI into mRFP1/pPILY, which was opened with XbaI and Eco47III.
The vectors TPK1-TMD4/pSAT6A-RFP-N1, TPK1-TMD4-CT/pSAT6A-RFP-N1, and TPK3-TMD4/pSAT6A-RFP-N1 were obtained by PCR with TPK3P2ms and TPK3CTas for TPK3-TMD4 and TPK1P2ms and CTas/TPK1as for TPK1-TMD4/ TPK1-TMD4+CT. The PCR products were TOPO-ligated into pCRII-TOPO® and then recombined in frame with RFP in pSAT6A-RFP-N1 (Chung et al., 2005). TPK1-TMD4 and TPK1TMD4-CT were cut out with HindIII and KspAI/Eco47III, TPK3-TMD4 with HindIII and KspAI and pSAT6A-RFP-N1 was opened with SmaI and HindIII.
Plant Transformation via Particle Bombardment
Onion epidermal cells were transfected by biolistic delivery of tungsten particles (tungsten M-17, Bio-Rad Laboratories, Hercules, CA, USA) coated with the respective DNA. The procedure is described elsewhere (Becker et al., 2004). For co-bombardments of GFP and RFP vectors, equal amounts of DNA were used for precipitation. To confirm Golgi-dependent trafficking of AtTPK1, the onion epidermis was stripped off immediately after bombardment and incubated for 24 h on agar plates with and without 54 mM Brefeldin A (Sigma-Aldrich, St Louis, MO, USA).
Microscopy
After incubation, usually up to 43 h after bombardment, the epidermis was stripped off and placed in 0.3 M KNO3 on a microscope slice. Cells were imaged using a confocal laser scanning microscope (LSM 5 Pascal, Zeiss, Oberkochen, Germany) under a 40x Plan-Apochromat water immersion or a 63x Plan-Neofluar oil immersion objective. For co-localization of RFP and GFP constructs, the pinhole was adjusted to one arbitrary unit and scanning was performed in multi-track mode. GFP fluorescence was measured using a 488-nm excitation wavelength and emission bandpath 505–530 nm, while RFP was excited at 543 nm and fluorescence emission was detected using a longpass filter LP560 nm. Images of figure1 and images (D), (E), and (F) of Figure 2 are horizontal projections of images with different focal planes. Images were not subjected to any image processing.
Markers of the Secretory Pathway
As a marker for the endoplasmatic reticulum, we co-transfected an ER-retained GFP in the form of GFP::Err/pMG (by B.G. Pickard); to stain ER/Golgi compartments, we used the Arabidopsis K/HDEL-receptor analogue ERD2 in the form of ERD2::GFP/pVKH18En6; and for Golgi-stacks alone, we used a derivative of the rat sialyl transferase (ST) in the form of STtmd::RFP/pVKH18En6 (Boevink et al., 1998).
Alignment and Homology Modelling of TPK1 CT
Alignment and amino acid identity calculation was done using the free Multiple Sequence Alignment Editor and Shading Utility ‘Genedoc’ (www.psc.edu/biomed/genedoc). Homology modeling of TPK1 CT was performed as described elsewhere.
Accession Numbers
Sequence data from this article can be found in the EMBL/GenBank data libraries under accession numbers: TPK1(At5g55630) NM_124945
[GenBank]
; TPK2(At5g46370) NM_124007
[GenBank]
; TPK3(At4g18160) NM_117926
[GenBank]
; TPK4(At1g02510) NM_100132
[GenBank]
; TPK5(At4g01840) NM_116414
[GenBank]
.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Supplementary Data are available at Molecular Plant Online.
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TOPAbstractINTRODUCTIONRESULTSDISCUSSIONMETHODSSUPPLEMENTARY DATAFUNDING |
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Deutsche Forschungsgemeinschaft (SFB487 to R.H.).
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Acknowledgements |
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We thank Barbara G. Pickard (Washington University) and Chris Hawes (Oxford Brookes University) for providing us with markers of the secretory pathway. We gratefully acknowlegde L. Banta for correcting the manuscript. No conflict of interest declared.
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