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Plant Physiol, September 2001, Vol. 127, pp. 10-13

SCIENTIFIC CORRESPONDENCE

Ion Transporters in the Nucleus?1


Marjori Matzke,* Werner Aufsatz, Wolfgang Gregor, Johannes van der Winden, István Papp, and Antonius J.M. Matzke

Institute of Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A-5020 Salzburg, Austria


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The nucleus of eukaryotic cells is bounded by a double membrane system perforated by the nuclear pores, which provide an aqueous channel for bidirectional transport of ions and molecules between the nucleus and the cytoplasm (Smith and Raikhel, 1999). The presence of these large pores, which have an average open diameter of approximately 10 nm, has tended to divert attention from the possible ion transport properties of the inner and outer nuclear membranes (INM and ONM, respectively). In principle, ions could be translocated across either membrane into or out of the perinuclear space, which is continuous with the lumen of the endoplasmic reticulum (Fig. 1). Ion transport across the entire nuclear envelope (NE) or the INM is particularly intriguing because of the potential for independently regulated ion fluxes in the nucleus to influence various aspects of nuclear physiology. Although the traditional view holds that the nuclear pores are static aqueous channels that offer no resistance to the flow of inorganic ions, there is growing evidence, particularly from electrophysiological studies and experiments using the calcium reporter aequorin, which suggests that they can present a barrier to ions in various cell types (Mazzanti et al., 2001). Given the contentious nature of NE ion permeability (Brini and Carafoli, 2000; Mazzanti et al., 2001) and the dearth of information about ion transport proteins in the INM or ONM, additional studies in this area are clearly warranted. The identification of potential ion transporters in the nucleus would permit functional analyses to be carried out with the possible pathways of nuclear ion transport in mind.



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  		Figure 1.   Possible ion transporters in the nucleus. Screens of ion transport proteins for putative bipartite NLSs identified possible nuclear variants of the transporters that are shown in the INM. Potential routes of ion transport across the INM, ONM, and NE are shown by the three sets of small bidirectional arrows. Whether a transport protein resides in the nucleus or the plasma membrane (or other cytoplasmic membrane) might depend on the presence of an NLS (heavy arrows). PM, Plasma membrane; ER, endoplasmic reticulum; PNS, perinuclear space.

The uncertainty about ion transport proteins in the INM or ONM is due in part to difficulties in obtaining nuclear membrane preparations free of contamination from other cellular membranes. Electrophysiological approaches for studying ion channels, such as patch clamp analyses, can be performed on isolated nuclei, but this technique does not guarantee access to INM channels. Patch clamp studies performed to date have identified various nuclear ion channels in animal and plant cells (Mazzanti et al., 2001). The exact location of these nuclear channels is not always specified, although some have been reported to be present in the ONM (Franco-Obregón et al., 2000) and others in the INM (Rousseau et al., 1996). Several nuclear ion channels detected so far might represent conductance substates of the nuclear pores (Mazzanti et al., 2001).

With respect to molecular analyses of nuclear ion channels, the only nuclear membrane channel to be cloned up until now is nuclear chloride channel-27/chloride intracellular channel (NCC27/CLIC1), which is located predominantly in the nuclei of some types of animal cells (Valenzuela et al., 1997; Tonini et al., 2000; Tulk et al., 2000). Disposition in the INM would imply that a protein contains one or more nuclear localization signals (NLSs) to facilitate transport through the nuclear pores from the site of synthesis in the cytoplasm. Indeed, NCC27/CLIC1 was reported to have two monopartite NLSs (Valenzuela et al., 1997), which are defined as a cluster of basic amino acid residues (Hodel et al., 2001).

We searched the Arabidopsis database for NCC27/CLIC1 homologs and found four proteins (accession nos. AAF79440, AAG12679, AAG24946, and BAB09367) that have been annotated as putative glutathione-dependent dehydroascorbate reductases (DHAR). The four Arabidopsis DHARs have only weak overall homology to NCC27/CLIC1 and other CLIC family members (ranging from 21%-25% amino acid identity), and the only common motif identified through the INTERPRO domain database (http://www.ebi.ac.uk/interpro/) is a glutathione-S-transferase domain. NCC27/CLIC1 has several motifs characteristic of chloride channels that are not present in Arabidopsis DHARs, which also do not contain NLSs. Therefore, despite the sequence similarity to NCC27/CLIC1, Arabidopsis DHARs do not appear to be chloride channels, although they might modulate channel activity similarly to some animal glutathione-S-transferases (Dulhunty et al., 2001).

To get a handle on potential INM ion transporters in plants, we have screened various families of recognized and putative ion transport proteins in Arabidopsis (http://www-biology.ucsd.edu/~ipaulsen/transport/atha.html) for potential bipartite NLSs using the INTERPRO domain database. Bipartite NLSs consist of short runs of basic amino acids separated by approximately 10 amino acids. The prototypical bipartite NLS is present in Xenopus laevis nucleoplasmin and has the sequence KR-10 amino acids-KKKL (Hodel et al., 2001). Only a few NLSs have been functionally characterized in plants, but they follow the general pattern observed for animal and yeast NLSs (Jans et al., 2000). Although we must emphasize that the presence of a bipartite NLS-like sequence does not guarantee that a protein is indeed nuclear, the analysis permits a preselection of possible INM ion transporters, which can then be analyzed in more detail using other methods. It should also be mentioned that the INTERPRO domain database only detects putative bipartite and not monopartite or other classes of NLSs (Jans et al., 2000). Therefore, in addition to picking up potential false positives, it is also possible that bona fide nuclear proteins would not be identified using this database.

Our survey has revealed that a number of established or predicted transport proteins contain one or more putative bipartite NLSs. These include some calcium ATPases and some members listed in the voltage-gated ion channel family (Table I). The following families of ion transporter proteins were screened for bipartite NLSs.


                              
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  		Table I.   Ion transporter proteins screened for possible bipartite NLSs

Nomenclature corresponds to the Arabidopsis Genome Initiative names, which are used in the Transport Proteins in Arabidopsis database (http://www-biology.ucsd.edu/~ipaulsen/transport/atha.html). Putative NLSs, identified through the INTERPRO domain data base (http://www.ebi.ac.uk/interpro) are shown in bold. Underlined regions show conserved amino acids in related proteins. The numbers in parentheses indicate the amino acid numbers flanking the NLSs.

(1) V-type ATPases are H+- or Na+-translocating ATPases present in vacuoles. Of 30 nonredundant entries, none contained an NLS. Members of this family offer good controls for the reliability of using an NLS as a predictor of nuclear localization, as none is expected to be nuclear based on previously known functions.

(2) P-type ATPases catalyze cation uptake and/or efflux by ATP hydrolysis. We screened recognized and putative Ca2+ and H+ ATPases for NLSs. Of 18 nonredundant entries for Ca2+ ATPases, six had one or two putative NLSs (Table I), suggesting that the NE actively transports Ca2+. Indeed, the Ca2+ transport properties of the NE have been studied intensively and there is considerable evidence that the Ca2+ concentration can be regulated independently in the nucleus and the cytoplasm in both plant and animal cells (Bootman et al., 2000; Brini and Carafoli, 2000; Pauly et al., 2000). The possible presence of Ca2+ ATPases in the INM would imply that calcium is pumped into and/or out of the perinuclear space (Fig. 1). This makes sense in view of the probable continuity of this compartment with the lumen of the endoplasmic reticulum, which accumulates Ca2+ by a pump and releases it via gated channels (Brini and Carafoli, 2000). None of the 12 nonredundant entries for H+ ATPases had an NLS, suggesting that H+ transport is not a property of the NE or INM.

(3) Voltage-gated ion channel family members annotated as probable cyclic nucleotide gated cation channels and K+ channels were screened for NLSs. Three of 19 in the former category and two of 15 in the latter category contained one or more NLSs (Table I).

(4) Chloride channel family members were screened and one out of seven was found to contain a possible NLS (Table I).

The presence of putative NLSs in these transport proteins supports the idea that the NE and/or INM actively transports Ca2+ and that it contains voltage- and cyclic nucleotide-gated cation channels (Fig. 1). The candidates for NE/INM transporter proteins preselected by the presence of a putative bipartite NLS can be further analyzed by (1) making GFP protein fusions to confirm nuclear localization, (2) carrying out functional analyses using various gene silencing techniques, and (3) performing electrophysiological experiments to determine channel characteristics in transfected cells (Tonini et al., 2000) or liposomes (Guihard et al., 2000).

Three of the transport proteins identified as potentially nuclear have been studied previously, although the presence of NLSs has not been reported. Indeed, one protein has been reported to be a plasma membrane Ca2+ ATPase (At5g57110; Bonza et al., 2000). Although this result might indicate the unreliability of using the presence of putative NLSs to predict subcellular localization of proteins, it could also simply reflect the biochemical similarity between plant plasma membrane and endomembrane Ca2+ ATPases. Another factor might be that only one bipartite NLS is present in this large protein, which might lead to a less strict partitioning between the nucleus and plasma membrane or other cytoplasmic membranes compared with homologs containing more than one NLS (Table I). Finally, high levels of expression could lead to deposition of a transport protein in both plasma and nuclear membranes, similar to that observed for NCC27 in Chinese hamster ovary cells (Tonini et al., 2000).

A second transport protein that we have identified as potentially nuclear has been reported previously as the DND1 "defense, no death" gene (Köhler et al., 1999; Leng et al., 1999; Clough et al., 2000). This cyclic nucleotide-gated cation channel (At5g15410) is involved in broad-spectrum disease resistance and the hypersensitive response (Clough et al., 2000), and it contains three putative NLSs (Table I). Given the striking phenotype of dnd1 mutants, it will be interesting to ascertain whether it is indeed a nuclear protein. Finally, a K+ channel containing a putative NLS (At3g02850) has been described previously as a Shaker-like channel involved in the plant response to water stress (Gaymard et al., 1998). Although it is premature to speculate on possible physiological functions of putative nuclear ion transporters, these potential examples suggest that they could play important roles in signal transduction and stress response pathways.

Whether the INM is "simple or complex" (Georgatos, 2001) is a crucial question in cell biology that deserves further investigation in both plant and animal systems. Five INM integral membrane proteins have been characterized so far in animal cells: the lamin B receptor, the lamina-associated polypeptide-1, lamina-associated polypeptide-2, emerin, and MAN1 (Georgatos, 2001). Although none of these proteins is an ion transporter protein, it is likely that some will eventually be identified. This prediction is supported by the recent report of an atypical P-Type ATPase that is present in the INM of mammalian cells and that binds to a SWI/SNF-like protein (Mansharamani et al., 2001), a finding which reveals a fascinating link between INM pumps, chromatin regulatory proteins, and possibly gene expression. The tools available for studying protein function and subcellular location in Arabidopsis will be extremely useful for delving further into the ion transport properties of plant nuclear membranes and determining the roles of these transport processes in nuclear physiology and plant development.

    FOOTNOTES

Received June 19, 2001; accepted July 1, 2001.

1 This work was supported by the Austrian Fonds zur Förderung der wissenschaftlichen Forschung (grant no. Z21-MED).

* Corresponding author; e-mail mmatzke{at}imb.oeaw.ac.at; fax 43-662-63961-29.


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© 2001 American Society of Plant Physiologists



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